Pore properties and pharmacological features of the P2X receptor channel in airway ciliated cells
1 Department of Chemistry
2 The Zlotowski Center for Neuroscience, and the
4 Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
3 Faculty of Life Sciences, and the Leslie and Susan Gonda Interdisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan 52900, Israel
Abstract
Airway ciliated cells express an ATP-gated P2X receptor channel of unknown subunit composition (P2Xcilia) which is modulated by Na+ and by long exposures to ATP. P2Xcilia was investigated by recording currents from freshly dissociated rabbit airway ciliated cells with the patch-clamp technique in the whole-cell configuration. During the initial continuous exposure to extracellular ATP, P2Xcilia currents gradually increase in magnitude (priming), yet the permeability to N-methyl-D-glucamine (NMDG) does not change, indicating that priming does not arise from a progressive change in pore diameter. Na+, which readily permeates P2Xcilia receptor channels, was found to inhibit the channel extracellular to the electric field. The rank order of permeability to various monovalent cations is: Li+, Na+, K+, Rb+, Cs+, NMDG+ and TEA+, with a relative permeability of 1.35, 1.0, 0.99, 0.91, 0.79, 0.19 and 0.10, respectively. The rank order for the alkali cations follows an Eisenman series XI for a high-strength field site. Ca2+ has been estimated to be 7-fold more permeant than Na+. The rise in [Ca2+]i in ciliated cells, induced by the activation of P2Xcilia, is largely inhibited by either Brilliant Blue G or KN-62, indicating that P2X7 may be a part of P2Xcilia. P2Xcilia is augmented by Zn2+ and by ivermectin, and P2X4 receptor protein is detected by immunolabelling at the basal half of the cilia, strongly suggesting that P2X4 is a component of P2Xcilia receptor channels. Taken together, these results suggest that P2Xcilia is either assembled from P2X4 and P2X7 subunits, or formed from modified P2X4 subunits.
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Introduction
Cation-selective ion channels activated by extracellular ATP (P2X receptor channels) are widely distributed in electrically excitable cells such as neurones, and in non-excitable cells such as immune cells and epithelial cells (North, 2002). Seven subunits of the mammalian P2X receptor family (P2X1 to P2X7) have been cloned, and all of them can form functional homomeric receptor channels in heterologous expression systems (North, 2002; Jones et al. 2004). Six functional heteromeric receptor channels (P2X1/P2X2, P2X1/P2X4, P2X1/P2X5, P2X2/P2X3, P2X2/P2X6 and P2X4/P2X6) have also been characterized (Torres et al. 1998; Le et al. 1998, 1999; King et al. 2000; Brown et al. 2002; Nicke et al. 2005). Co-immunoprecipitation assays performed on HEK293 cells co-expressing different P2X subunits suggest that additional hetero-oligomeric assemblies are possible among the different P2X receptor subunits (Torres et al. 1999). The pharmacological and biophysical properties of native P2X receptor channels often deviate from those of heterologously expressed P2X receptor channels. This suggests that native P2X receptor channels might be processed differently or that they are heteromeric channels of yet uncharacterized combinations of P2X subunits.
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Airway ciliated cells express a P2X receptor channel (P2Xcilia) of unknown composition. This channel is interesting both from a physiological and from a biophysical standpoint because of its modulation by both ATP and Na+. P2Xcilia receptor currents gradually increase over tens of seconds during the first exposure to ATP whereas subsequent applications of ATP activate the current significantly faster (Korngreen et al. 1998). This priming phenomenon can be observed for several minutes after the initial application of ATP, and thus ATP can be considered both an agonist and a long-term modulator of the P2Xcilia receptor channel. P2Xcilia receptor channels are also modulated by Na+ (Ma et al. 1999). The effect of Na+ can be overcome by raising the concentration of ATP, suggesting an allosteric mechanism of regulation. While the physiological role of P2Xcilia receptor channels has not yet been resolved, comparing the effects of Na+ on cilia activation induced by different concentrations of ATP suggests that P2Xcilia receptor channels contribute to the mechanisms sustaining the cilia at a high level of activation during continued exposure to ATP (Silberberg et al. 2001).
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The unusual properties of P2Xcilia receptor channels, and their potential physiological significance, prompted us to investigate the relative permeability of the channel to various cations and the pharmacological properties of the channel in order to address four specific questions. First, does the gradual increase in membrane current observed during long exposures to ATP result from a change in ion selectivity, as has been proposed for P2X2, P2X4 and P2X7 receptor channels Second, if Na+ can permeate P2Xcilia receptor channels, is the binding site for inhibition by Na+ located on the extracellular or intracellular side of the channel The answer to this question could help guide the development of strategies to exploit P2Xcilia receptor channels to enhance mucociliary clearance. Third, are P2Xcilia receptor channels highly permeable to Ca2+, as might be expected if the channel contributes to cilia activation by ATP (Korngreen & Priel, 1994, 1996) Fourth, is P2Xcilia composed of P2X7 receptor subunits, as suggested by some of its properties Here we show that the channels do not undergo pore dilatation, that the binding site for Na+ is extracellular, and that the channels are highly permeable to Ca2+. In addition, the results suggest that the pore of P2Xcilia receptor channels contains a high-strength field site, and put constrains on the diameter of the narrow part of the ion conduction pore. Pharmacological and immunohistochemical experiments suggest that P2Xcilia receptor channels may contain both P2X4 and P2X7 subunits.
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Methods
Isolation of single ciliated cells
Male New Zealand white rabbits (2–3 months old) were killed as previously described (Korngreen et al. 1998), according to the guidelines laid down by the animal welfare committee of Ben-Gurion University, by gradual exposure to carbon dioxide followed by exsanguination. Care was taken to slowly increase the gas flow over several minutes to prevent any visual signs of distress. The trachea was removed and the epithelium surgically separated from the cartilage and cut into pieces of approximately 0.3 cm x 0.3 cm. The tissue was maintained in physiological extracellular solution (DPBS) containing (mM): NaCl 137, KCl 2.7, CaCl2 0.9, MgCl2 0.5, Na2HPO4 8, KH2PO4 1.47 and D-glucose 5; pH 7.4. The epithelium was incubated for 25 min, at 37°C, in DPBS supplemented with 13 U ml–1 papain (Sigma), 1 mg ml–1 bovine serum albumin (BSA) (Sigma, Fraction V, essentially fatty acid free) and 1 mg ml–1 1,4-dithiothreitol (Merck, Darmstadt, Germany). Cells were then dispersed by repeated aspiration with a fire-polished Pasteur pipette, concentrated by centrifugation 800 g for 10 min, and re-suspended in DPBS. The isolated cells were transferred to 35-mm tissue culture dishes and used immediately. Single ciliated cells remained active for several hours after isolation and increased cilia beating in response to ATP. Part of the tissue was maintained overnight in DPBS at 4°C and used on the next day. No obvious differences in the experimental results, in cell viability or in sensitivity to ATP, were observed when cells were used on the next day (Ma et al. 2002).
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Solutions and drugs
Solutions were made with highly purified water (NANOpure, Barnstead, Debuque, IA, USA), using chemicals of analytical grade. The standard extracellular solution contained (mM): CsCl 151, CaCl2 1.58, Tes 5, D-glucose 10 and 3,4-diaminopyridine (DAP) 1; pH adjusted to 7.4 with CsOH (295–300 mosmol l–1). The standard pipette solution contained (mM): CsCl 155, Tes 5, EGTA 0.1, Mg-ATP 1, Na2GTP 0.1 and CaCl2 0.096 (0.1 μM Ca2+, estimated by Max Chelator 6.0, Chris Patton, Stanford University, CA, USA); pH adjusted to 7.2 with CsOH (285–290 mosmol l–1). Deviations from these solutions are noted in the text. K2ATP, Mg-ATP, 2'-3'-O-(4-benzoylbenzoyl) ATP triethylammonium salt (BzATP), Brilliant Blue G (BBG), 1-[N,O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62) and ivermectin (IVM) were from Sigma. Na2GTP was from Boehringer Mannheim. DAP was from Fluka. TEA was from Fluka and from Sigma. Solutions containing ATP or other nucleotides were freshly prepared each day and the pH of the solution was readjusted.
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Whole-cell current recording
Membrane currents were recorded using the standard whole-cell configuration of the patch-clamp technique (Hamill et al. 1981), as previously described (Korngreen et al. 1998). Briefly, patch pipettes (2–3 M, when filled with intracellular solution) were fabricated from borosilicate glass (GC150F-7.5, Clark Electromedical Instruments, Reading, UK), coated with Sylgard 184 (Dow Corning, Midland, MI) and fire-polished shortly before use. Capacitive currents were electronically compensated. Series resistance was not compensated in most experiments, as the currents activated by ATP were small and slow. Once the whole-cell configuration was established, the cell was continuously superfused with the desired extracellular solutions via a gravity-fed perfusion system. The bathing solution was changed using a Teflon rotary valve, which selected among one of six reservoirs. The continuous perfusion of the cell prevented fluctuations in the concentration of ATP due to degradation of ATP by ecto-nucleotidases, and prevented the possible build-up of degradation products such as ADP. Liquid junction potentials were determined by adjusting the bath potential to zero with a patch pipette containing internal solution placed in internal solution, and then measuring the bath potential in each of the external solutions (Neher & Sakmann, 1992). Liquid junction potentials were at most 1.5 mV and hence were not corrected. All measurements were at room temperature (18–22°C).
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Data acquisition and analysis
Experiments were initiated at least 2 min after establishing the whole-cell configuration to allow equilibration of the cytosol with the pipette solution. Membrane currents were recorded under voltage clamp using an Axopatch 200A patch-clamp amplifier (Axon Instruments, Union City, CA, USA). Stable recordings were typically maintained for 20–30 min. Membrane currents were low-pass filtered using an 8-pole Bessel filter (Frequency Devices, Haverhill, MA, USA) in series with the integral 4-pole Bessel filter of the patch-clamp amplifier and stored on video tape for subsequent analysis (VR-10B, Instrutech). Membrane currents were also digitized on-line using a Digidata 1200 interface board and pClamp 6.03 software (Axon Instruments). The sampling frequency was set to at least two times the corner frequency of the low-pass filter. SigmaPlot scientific software was used for non-linear curve fitting. Experimental results were consistently observed in cells from two or more animals; hence, all the results for a particular experiment were pooled and displayed as means ±S.E.M.; groups were compared with Student's t test.
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The permeability ratio, PX/PCs, was quantified by fitting the reversal potentials (Vrev) measured experimentally with the Goldman–Hodgkin–Katz (GHK) equation:
(1)
where R is the universal gas constant, T is absolute temperature, F is Faraday's constant, PX and PCs are the permeability (P) to any monovalent cation X and Cs+, respectively, X and Cs are the activity coefficients () of X+ and Cs+, respectively, and [X]0 and [Cs]i are the concentrations of X+ and Cs+ in the extracellular and intracellular solutions, respectively.
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When the extracellular solution contained Cs+ and Na+, the permeability ratio, PNa/PCs, was estimated using the equation:
(2)
Calculating the permeability of Ca2+ over Cs+
As P2Xcilia receptor channels are modulated by divalent cations (Korngreen et al. 1998), the relative permeability to Ca2+ was estimated by measuring the effects of 0.1–2 mM extracellular Ca2+ on the reversal potential of the ATP-activated currents. The relationship between Vrev and the relative ion permeability predicted by the GHK equation is described by the equation (Korngreen et al. 1998):
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(3)
The estimated permeability ratio PCa/PCs depends on the choice of activity coefficients for these ions (Ca and Cs). In mixed electrolyte solutions, each salt influences the activity coefficient of the other electrolytes in the solution. However, in mixed solutions of total ionic strength up to 0.2 M, it can be assumed that the activity of an individual ion is determined solely by the ionic strength of the solution, and not by the chemical nature of the other ions in the solution (Shatkay, 1968). Hence, the activity coefficients for CsCl and CaCl2 (Robinson & Stokes, 1959) and the activity coefficient for TEA-Cl (Lindenbaum & Boyd, 1964) were adjusted for total ionic strength, and used to estimate the activity coefficient of Cs+, Ca2+ and TEA+, respectively. For example, we used activity coefficients of 0.515 and 0.48 for 0.1 M and 0.2 M CaCl2, respectively. Assuming a linear relationship between ionic strength and activity coefficient in this range of concentrations, the activity coefficient of CaCl2 for a solution with an ionic strength of 0.15 M would be 0.4975. Various assumptions have been made in order to estimate the activity coefficient of a divalent cation (++) from the mean activity coefficient of the salt (±) in mixed solutions (Butler, 1968). In the present study, two conceptually different assumptions were used, ++=± (Shatkay, 1968) and ++= (±)2 (Butler, 1968). No difference in the estimate of PCa/PCs was observed.
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Measurement of intracellular calcium
Experiments were carried out on explants of epithelium from rabbit trachea using the procedure previously described (Korngreen & Priel, 1996). Briefly, the ciliary epithelium was peeled off the cartilage and cut into small pieces. Two or three pieces of epithelium were placed on a glass coverslip and incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 20 units ml–1 penicillin, 2.5 units ml–1 nystatin and 20 μg ml–1 streptomycin at 37°C with 5% CO2. The medium was replaced every 3 days. Measurements were performed after 6–19 days in culture. Measurements of intracellular calcium were performed as previously described (Korngreen & Priel, 1994). Briefly, the cells were preloaded with fura-2 by incubating the tissue in serum-free growth medium, containing 5 μM of the acetoxymethyl ester (AM) form of fura-2 (fura-2AM) and 500 μM probenecid, for 60 min at 32°C in a rotating water bath, followed by washing for 30 min in Ringer solution containing (mM): NaCl 150, KCl 2.5, CaCl2 1.5, MgCl2 1.5, D-glucose 5 and Hepes 5. Probenecid, an inhibitor of membrane organic anion transporters, is widely used to prevent extrusion of loaded fura-2 from the cells. The dye-loaded cells were epi-illuminated with light from a 75-W xenon lamp (Ushio Inc., Japan) filtered through 340 and 380 nm interference filters (Oriel Corp., Stamford, CT, USA) mounted on a four-position, rotating, filter wheel. The fluorescence, emitted at 510 nm, was detected by a photon-counting photomultiplier (H3460-53, Hamamatsu, Japan). The 340/380 nm fluorescence ratio, averaged over a period of 1 s, was stored in a computer. A calibration curve of the calcium concentration was created by titrating an external calibration solution with a solution of the same composition containing 10 mM CaCl2 (Grynkiewicz et al. 1985; Korngreen & Priel, 1994). To account for the difference between the fura-2 fluorescence signal in the intracellular medium and in the calibration solution, the maximal and minimal values of the 340/380 nm fluorescence ratio were measured from the cells. The maximal value was obtained by adding 5 μM ionomycin to the cells, which flooded the cells with Ca2+. The minimal value was obtained by adding ionomycin to the cells in a Ca2+-free medium. The calibration curve was corrected according to the obtained results. The non-specific signal was estimated by adding ionomycin to the cells in the presence of 1 mM Mn2+, which leads to quenching of fura-2 fluorescence. The calcium concentration was calculated directly from the corrected calibration curve by interpolation using an algorithm table. Prior to any treatment, the Ringer solution over the explants was changed twice. The basal [Ca2+]i from a single cell was measured for 1–5 min in the appropriate solution, and the results of this measurement taken as the reference value. Then, the solution was changed to the test solution and [Ca2+]i monitored from the same cell for an additional 10 min.
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Immunohistochemistry
Immunohistochemistry was carried out on paraffin embedded sections as previously described (Humason, 1979). The trachea was cleaned and washed with cold phosphate-buffered solution (PBS) containing (mM): Na2HPO4 77.5, NaH2PO4 22.5 and NaCl 150; pH 7.4. Slices (width, 5 mm) were obtained from each cleaned trachea, fixed in 4% formaldehyde in PBS overnight at 4°C, and embedded in paraffin blocks. The blocks were sliced (10 μm thick), mounted on SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany) and stored at –20°C. To remove paraffin, sections were washed twice for 20 min each in xylene followed by gradual re-hydration with ethanol (100%, 95%, 80% and 70%, 5 min in each) and finally in PBS. The slides were transferred to 50 mM sodium citrate solution (pH 6.0) and submitted to antigen retrieval by boiling in a microwave for 10 min. Non-specific binding sites were blocked by 30-min incubation at room temperature with 5% BSA and 5% normal goat serum (NGS) in PBS. The slides were incubated overnight at 4°C in PBS containing 1% NGS, 0.1% Triton X-100 and either 1: 400 primary antibody or primary antibody pre-absorbed overnight with a 10-fold excess of the homologous peptide antigen. Finally, the slides were exposed in PBS to the secondary goat anti-rabbit antibody 1: 500 Alexa flour 594 in the dark for 1 h. The slides were washed and covered by coverslips using Fluramount-G (Southern Biothechnology Associates, Inc. Birmingham, AL, USA). Immunoreactivity was visualized using a Zeiss LSM510 confocal microscope.
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Results
Long applications of ATP gradually activate a membrane conductance in ciliated cells
P2Xcilia receptor channels exhibit a time-dependent increase in current during the initial long exposure to ATP (Korngreen et al. 1998). This phenomenon was called ‘priming’ because the response to a subsequent application of ATP applied up to several minutes later activates more rapidly (Korngreen et al. 1998). Figure 1A is an example of such priming, showing a whole-cell current trace recorded in the standard intracellular and extracellular CsCl solutions, at a holding potential of –40 mV, from a ciliated cell freshly dissociated from rabbit airway epithelium. Exposing the cell to 200 μM ATP activated an inward current, which slowly increased in the presence of ATP to a maximal level of –65 pA. The ATP-activated current reached half of the maximal level after 87 s and deactivated to half the maximal current level within 5 s upon removal of the ATP. During a second application of ATP, the current activated significantly faster, reaching half maximal activation within 3 s. This gradual increase in the magnitude of the current during the initial sustained application of ATP and the much faster response to subsequent exposures to ATP was seen in all ciliated cells tested (n > 150).
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A, continuous whole-cell current trace recorded at a holding potential of –40 mV, from a ciliated cell not previously exposed to ATP. ATP (200 μM) was applied twice (indicated by the bars). The first application gradually activated an inward current, which opened significantly faster upon the second application of ATP. Standard intracellular and extracellular solutions. B and C, net ATP-activated current responses to voltage ramps measured during priming. The extracellular solution contained (mM): NMDG-Cl 151, Tes 5, D-glucose 10, DAP 1 and either CaCl2 1.58 (B) or BaCl2 0.1 (C). The pipette contained the standard CsCl intracellular solution. In both cases, the reversal potential did not change during priming, indicating that P2XCilia receptor channels do not exhibit a time-dependent change in ion selectivity during long exposures to ATP.
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The gradual increase in membrane conductance does not result from a time-dependent change in ion selectivity of the P2Xcilia receptor channel
An enhancement in membrane conductance can result from an increase in single-channel conductance, a rise in channel open probability (by increasing ATP affinity or efficacy) and/or from recruitment of silent channels. If the priming phenomenon described above results from changes in ion channel conductance, the selectivity of the channel would be likely to change with time. It is interesting that cells heterologously expressing rat P2X2, P2X4, P2X7 and heteromeric P2X2/P2X3 receptor channels, bullfrog P2X5 or Schistosoma mansoni P2X receptor channels exhibit time-dependent changes in ion selectivity with continuous application of agonist, which has led to the suggestion that some of the P2X receptor channels undergo pore dilatation during activation (Surprenant et al. 1996; Khakh et al. 1999a; Virginio et al. 1999; Jensik et al. 2001; Raouf et al. 2005; for review see Khakh & Lester, 1999,). We therefore investigated whether the gradual increase in membrane current observed in the ciliated cells during long applications of ATP (Fig. 1A) is also accompanied by a change in ion selectivity. In these experiments, whole-cell currents were recorded with the standard intracellular Cs+ solution and with Cs+ replaced by NMDG+ in the extracellular solution. Current–voltage relationships were constructed by applying voltage ramps throughout the priming process, and the net current activated by ATP was assessed by subtracting the current recorded under control conditions from the current recorded in the presence of ATP. Figure 1B shows current–voltage relationship measured at different times throughout the priming by ATP, as indicated by each current trace. Despite the progressive increase in current with time, the reversal potential of the current remained at –37 mV, indicating that the permeability to NMDG+ relative to Cs+ did not change during priming. A lack of change in the reversal potential during priming was observed in 7 out of 7 experiments. When the extracellular solution contained a low concentration of divalent cations, the reversal potential also remained constant during priming (Fig. 1C). The difference between the reversal potentials shown in Fig. 1B and C arises from permeability to Ca2+, as discussed later. These results show that during the priming process P2Xcilia receptor channels do not undergo pore dilatation, as suggested for some P2X receptors (Khakh & Lester, 1999,). As the permeability of P2Xcilia receptor channels does not change during priming, for the remainder of the experiments, measurements were conducted on fully primed channels.
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Na+ both inhibits and permeates P2Xcilia receptor channels
We have previously reported that extracellular Na+ directly inhibits P2Xcilia receptor channels, and that the degree of inhibition by Na+ depends on the interplay between the effective concentrations of ATP and Na+ (Ma et al. 1999). To establish whether P2Xcilia receptor channels are permeable to Na+, net whole-cell currents activated by ATP (200 μM) in response to voltage ramps were recorded in extracellular solutions containing different concentrations of NaCl. The ionic strength of the extracellular solution was maintained constant by replacing CsCl with equimolar amounts of NaCl. Figure 2A shows examples of membrane currents recorded in the different extracellular solutions as indicated by each current trace. The rightward shifts in the zero-current potential induced by increasing the concentration of Na+ relative to Cs+ indicate that Na+ is more permeable than Cs+. Figure 2B shows a graph of the mean (±S.E.M.) reversal potential of the current activated by ATP in extracellular solutions containing different concentrations of NaCl (, averaged from nine experiments of the type shown in Fig. 2A). The permeability ratio, PNa/PCs, was quantified by fitting Vrev measured experimentally using the GHK equation (eqn (2)), and found to be 1.27: 1 (continuous line). Permeability ratios of 1.4: 1 and 1.2: 1 are also shown for comparison (dashed lines). Clearly, in addition to inhibiting P2Xcilia receptor channels, Na+ permeates the channels.
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A, mean net whole-cell currents activated by ATP (200 μM) in response to voltage ramps in extracellular solutions containing either 15, 30, 75 or 120 mM NaCl (the total alkali cation concentration was adjusted to 151 mM with CsCl). The extracellular solution also contained (mM): Tes 5, D-glucose 10, and BaCl2 0.1. Standard intracellular solution. Five to 10 consecutive current traces were averaged for each experimental condition. B, mean (±S.E.M.) reversal potentials of the current activated by ATP in extracellular solutions containing different concentrations of NaCl (, averaged from nine experiments of the type shown in A). The relative permeability of Na+ and Cs+ (PNa/PCs) was estimated to be 1.27: 1 using eqn (2) (continuous line). Permeability ratios of 1.4: 1 and 1.2: 1 are also shown (dashed lines).
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The binding site for Na+ is extracellular
The permeability of P2Xcilia receptor channels to Na+ (Fig. 2) raises the possibility that Na+ enters the cell during channel activation and inhibits the channel from the inside. To explore this possibility, the effects of intracellular Na+ on the current activated by ATP were investigated by replacing CsCl in the pipette solution with an equimolar amount of NaCl (30 mM). Although it is possible to change the composition of the solution in the pipette during whole-cell recording, this is a rather slow process and it is difficult to determine when complete solution exchange has taken place. We therefore measured the currents activated by ATP under a single experimental condition in each cell, and made comparisons between cells by estimating the current density (pA/pF). The cells were exposed to ATP at least 2 min after establishing the whole-cell configuration to allow equilibration of the cytosol with the pipette solution. We chose a concentration of 30 mM Na+ because application of 30 mM Na+ to the extracellular solution reversibly inhibited the ATP-activated current by 90% (Fig. 3A and B). Thus, if Na+ inhibits the channel from outside the cell, significantly less inhibition would be expected when the Na+ is included in the pipette solution. This was indeed the case. Examples of current density traces recorded under control conditions and in the presence of 30 mM Na+ in the pipette solution are shown in Fig. 3C. The highly significant (P < 0.0001) greater inhibition by extracellular Na+ (Fig. 3B) suggests that the binding site for inhibition by Na+ is more readily accessible to extracellular Na+.
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A, continuous whole-cell trace recorded at a holding potential of –40 mV, from a cell exposed to ATP (300 μM) for the duration indicated by the top bar. At the time indicated by the lower bar, 30 mM of the extracellular CsCl was replaced by 30 mM NaCl. Standard intracellular solution. The cell was primed with ATP before the start of the experiment. B, mean (±S.E.M.) current in the presence of either extracellular (n= 17) or intracellular (n= 17) NaCl (30 mM), normalized to the current in the absence of NaCl (n= 24). C, continuous whole-cell current traces recorded at a holding potential of –40 mV, from cells exposed to ATP (300 μM) for the duration indicated by the bars. Standard extracellular solution. The intracellular solution contained either 155 mM CsCl (left-hand trace) or 30 mM NaCl and 125 mM CsCl (right-hand trace). The intracellular solution also contained (mM): Tes 5, EGTA 0.1, Mg-ATP 1, Na2GTP 0.1 and CaCl2 0.096.
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In order to determine whether inhibition by Na+ is voltage dependent, the effect of 15 mM extracellular NaCl on the current activated by ATP (200 μM) was measured at different holding potentials. On average, the current activated by ATP (200 μM) at –80, –60, –40 or +40 mV was attenuated by 15 mM NaCl by 68.4 ± 2.3% (n= 11), 68.5 ± 2.9% (n= 17), 69.4 ± 3.8% (n= 10) and 59.6 ± 4.4% (n= 9), respectively. The absence of significant voltage dependence of Na+ inhibition, suggests that the binding site for Na+ is not within the electric field.
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P2Xcilia receptor channels exhibit little discrimination between the alkali cations
The relative permeability of P2Xcilia receptor channels to the other alkali cations was estimated under bi-ionic conditions using the same experimental approach described above. The reversal potential of the net current activated by ATP was measured using the standard intracellular solution and an extracellular solution containing (mM): XCl 151, BaCl2 0.1, Tes 5, D-glucose 10, DAP 1; pH 7.4, where X represents a monovalent cation. Figure 4A shows an example of net ATP-activated current responses to voltage ramps measured from a cell in extracellular solutions containing Cs+, K+ or Li+, as indicated. As the reversal potentials of K+ and Li+ are positive, it can be concluded that these cations are more permeable than Cs+. An illustration of the relative permeability of the P2Xcilia receptor channel to the various cations is presented in Fig. 4B, which shows representative net ATP-activated current responses to voltage ramps assembled from different cells. The reversal potential in each extracellular solution served to calculate the relative permeability of different monovalent cations using eqn (1). The permeability (relative to Cs+) of Li+, K+, Rb+, NMDG+ and TEA+ were estimated to be 1.71, 1.26, 1.16, 0.24 and 0.13, respectively (Table 1). It is interesting that, despite the relatively weak discrimination between the alkali cations, the permeability of P2Xcilia receptor channels is greatest for Li+ and decreases with atomic number (Li+ > Na+ > K+ > Rb+ > Cs+), consistent with a site of high electrostatic field strength in the permeation pathway (Eisenman & Horn, 1983). The TEA+ permeability ratio reported here (PTEA/PNa, 0.1) is similar (within a factor of two) to that of the P2X receptor channel of rat parasympathetic ganglia (PTEA/PNa, < 0.1; Liu & Adams, 2001), and to that of rat P2X2 receptor channel (PTEA/PNa, 0.04; Evans et al. 1996; Ding & Sachs, 1999). The measurable permeability to TEA+ indicates that the diameter of the narrowest part of the pore of P2Xcilia receptor channels is somewhat greater than 0.66 nm (i.e. the mean diameter of TEA+). A similar estimate for the minimum pore diameter (< 0.7 nm) was obtained for the native P2X receptor channel in neurones from rat parasympathetic ganglia (Liu & Adams, 2001).
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A, net ATP-activated current responses to voltage ramps measured in extracellular solutions containing 151 mM of the indicated cation. Five to 10 consecutive current traces were averaged in each experimental condition. B, representative net ATP-activated current responses to voltage ramps measured in extracellular solutions containing the indicated monovalent cation. The currents were assembled from different cells. Calibration bar: 2 pA for NMDG+, Cs+ and TEA+, 1 pA for K+ and Li+, and 4 pA for Rb+.
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P2Xcilia receptor channels are 9-fold more permeable to Ca2+ than to Cs+
P2Xcilia receptor channel currents are strongly attenuated by extracellular divalent cations (Korngreen et al. 1998). This is demonstrated in Fig. 5A, which shows a continuous whole-cell current trace recorded at a holding potential of –40 mV, in the standard CsCl solutions. The inward current activated by ATP (200 μM) was completely inhibited by replacing the extracellular 151 mM CsCl solution by a solution containing 136 mM CsCl and 10 mM CaCl2. Switching back to the 151 mM CsCl solution then restored the current. The permeability of the channel to Ca2+ was therefore estimated by measuring the effects of 0.1–2 mM extracellular Ca2+ on the reversal potential of the ATP-activated current. To this end, TEA+ was used as the major extracellular cation, because the permeability of P2Xcilia receptor channels to TEA+ is 10-fold lower than to Cs+ (Table 1). The ATP-activated (difference) currents for one such experiment are shown in Fig. 5B. The reversal potential in 0.1, 0.3, 1.0 and 2.0 mM extracellular Ca2+ was –53, –49, –40 and –33 mV, respectively. When the extracellular solution contained a highly permeable cation (Cs+) in place of TEA+ (Fig. 5B, inset), Ca2+ (0.1–2 mM) did not shift the reversal potential, indicating that the observed Ca2+-dependent shifts in reversal potential are not due to surface charge effects. Figure 5C summarizes the dependence of the reversal potential on the concentration of extracellular Ca2+, determined in experiments of the type shown in Fig. 5B. A permeability ratio for PCa/PCs of 9: 1 was estimated by fitting the reversal potential data with eqn (3) (continuous line in Fig. 5C). Predicted reversal potentials assuming permeability ratios for PCa/PCs of 15: 1 and 6: 1 are also shown for comparison (dashed lines). The results shown in Fig. 5C clearly indicate that the conductance activated by ATP is more permeable to Ca2+ than to Cs+, and suggest that Ca2+ permeates the ATP-activated conductance even better than Ba2+, given that PBa/PCs was reported to be 4: 1 (Korngreen et al. 1998). To confirm this point, the relative permeability of Ca2+ and Ba2+ was compared in the same cell (Fig. 5D). Replacing Ba2+ (1 mM) by Ca2+ (1 mM) shifted the reversal potential from –44 to –36 mV. On average, the reversal potential in 1 mM Ca2+ and in 1 mM Ba2+ was –35.8 ± 1.4 and –42.5 ± 1.2 mV, respectively (n= 5), confirming that P2Xcilia receptor channels are more permeable to Ca2+ than to Ba2+.
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A, continuous whole-cell current trace recorded at a holding potential of –40 mV, from a cell exposed to ATP (200 μM) for the duration indicated by the top bar. The extracellular solutions contained either 1.58 mM CaCl2 and 151 mM CsCl or 10 mM CaCl2 and 136 mM CsCl (lower bar). The extracellular solutions also contained (mM): Tes 5, D-glucose 10 and DAP 1. The cell was primed with ATP before the start of the experiment. B, mean whole-cell currents activated by ATP (200 μM) in response to voltage ramps in extracellular solutions containing 0.1, 0.3, 1 or 2 mM CaCl2, as indicated by each current trace. The extracellular solution also contained (mM): Tes 5, D-glucose 10, DAP 1 and TEA-Cl (or CsCl in the inset) at either 151, 151, 149, or 147, respectively. Standard CsCl intracellular solution. Five to 10 consecutive current traces were averaged in each experimental condition. C, mean (±S.E.M.) reversal potentials of the current activated by ATP in extracellular solutions containing different concentrations of CaCl2 (). Each point is the average of 20–28 experiments of the type shown in A. The relative permeability of Ca2+ and Cs+ (PCa/PCs) was estimated from eqn (3) to be 9: 1 (continuous line). Permeability ratios of 15: 1 and 6: 1 are also shown (dashed lines). D, net ATP-activated current responses to voltage ramps measured in extracellular TEA-Cl solution containing either 1 mM CaCl2 or 1 mM BaCl2. Both extracellular solutions contained in addition (mM): TEA-Cl 149, Tes 5, D-glucose 10 and DAP 1; pH was adjusted to 7.4 with TEA-OH.
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P2Xcilia receptor channels share properties in common with P2X7 receptor channels
P2Xcilia receptor channels display properties that are commonly attributed to P2X7 receptor channels including: low sensitivity to ATP, significant attenuation by physiological concentrations of extracellular divalent cations, and modulation by extracellular Na+ (Korngreen et al. 1998; Ma et al. 1999). As with P2X7 receptor channels, P2Xcilia receptor channels are also more sensitive to BzATP than to ATP (the EC50 for BzATP and for ATP in a CsCl extracellular solution containing 1 mM CaCl2 was 100 μM and 530 μM, respectively). However, a greater sensitivity to BzATP is not a property unique to P2X7 receptor channels (Bianchi et al. 1999). Could P2Xcilia receptor channels be composed exclusively of P2X7 receptor subunits To further examine this possibility we measured the effects of known P2X7 receptor channel inhibitors on the changes in [Ca2+]i induced by BzATP in explants of rabbit airway epithelium.
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Average time course of changes in [Ca2+]i in ciliated cells induced by BzATP (100 μM) in Ringer solution (), in low Na+-containing Ringer solution (), and in low Na+-containing Ringer solution plus 10 μM BBG (&;). Reducing the Na+ concentration in the Ringer solution from 150 to 10 mM (replaced by NMDG+) did not influenced the basal [Ca2+]i or the initial fast rise in [Ca2+]i, while the sustained rise in [Ca2+]i was dramatically increased. The strong elevation in [Ca2+]i in low Na+-containing Ringer solution induced by BzATP was largely abolished by 10-min preincubation of the explants with 10 μM BBG. Each data point is the average of eight to nine experiments from four rabbits. The data points are at 10-s intervals, and every fifth S.E.M. is shown. Each experiment was performed on a single ciliated cell.
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P2Xcilia receptor channels also share properties in common with P2X4 receptor channels
P2X7 receptor channels are inhibited by 1 μM Zn2+ (Virginio et al. 1997). In contrast, P2Xcilia receptor channels were found to be augmented by extracellular Zn2+ (1 μM), as demonstrated in Fig. 7A. On average, Zn2+ augmented the P2Xcilia receptor channel currents 1.5-fold (Fig. 7B). This finding prompted us to investigate the possibility that P2Xcilia receptor channels share properties in common with the P2X4 receptor channel, which is also augmented by Zn2+ (Soto et al. 1996; North, 2002). Therefore, airway ciliated cells were exposed to ivermectin (IVM), a semisynthetic derivative of the natural fermentation products of Streptomyces avermitilis, which augments the current of P2X4 but not of P2X7 receptor channels (Khakh et al. 1999b). Figure 7C shows current traces recorded from a ciliated cell prior to (left-hand trace) and after exposing the cell to 10 μM IVM for 5 min (right-hand trace). We found, that IVM increased the current of P2Xcilia receptor channels in all tested cells (Fig. 7D), raising the possibility that P2Xcilia receptor channels contain both P2X4 and P2X7 subunits.
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A, continuous whole-cell current trace recorded at a holding potential of –40 mV, from a ciliated cell exposed to ATP (200 μM) for the duration indicated by the top bar. The addition of 1 μM Zn2+ to the extracellular solution (indicated by lower bar) reversibly increased the current. B, mean (±S.E.M.) ATP-activated current under control conditions (left) and in the presence of 1 μM Zn2+ (right) averaged from five cells. C, whole-cell current trace recorded at a holding potential of –40 mV, from a ciliated cell exposed to ATP (200 μM) before (left) and after (right) 5-min exposure to 10 μM IVM. D, mean (±S.E.M.) ATP-activated current under control conditions (left) and after 5 min exposure to 10 μM IVM (right) averaged from six cells. Statistical significance in B and D was determined with a paired Students t test with P < 0.05. Standard intracellular and extracellular solutions.
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Airway ciliated cells display P2X4 immunoreactivity
Immunohistochemical techniques were used to test the hypothesis that P2Xcilia receptor channels contain both P2X4 and P2X7 subunits. Robust immunohistochemical labelling localized primarily to the basal portion of the cilia was detected with the anti-P2X4 receptor antibody in rabbit airway epithelium (Fig. 8A). The specificity of the immunoreaction was verified by absorbing the primary antibody with an excess of the homologous peptide antigen, followed by the second antibody (Fig. 8B). In contrast to the pronounced labelling with anti-P2X4 antibodies, no conclusive specific labelling was detected with either of two anti-P2X7 receptor antibodies.
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A, immunolocalization of P2X4 receptors in rabbit airway epithelium. P2X4 immunostaining is evident in the proximal half of the cilia. Calibration bar, 10 μm. B, P2X4 immunostaining of the cilia is absent when the primary antibody is blocked by the antigen. GC, goblet cell; BM, basal membrane; Ci, cilia.
Discussion
The aim of the present study was to further characterize the P2X receptor channel expressed in airway ciliated cells. We have shown that in contrast to homomeric mammalian P2X2, P2X4 and P2X7 receptor channels, the gradual increase in membrane conductance observed with P2Xcilia receptor channels during long exposures to ATP is not associated with a time-dependent change in ion selectivity (Fig. 1). This made it possible to conduct reliable selectivity measurements, which indicate that Na+ both inhibits and permeates P2Xcilia receptor channels (Figs 2 and 3), and that the rank order of relative permeability for the alkali cations follows an Eisenman series XI for a site of high field strength (Fig. 4). In addition, we have shown that P2Xcilia receptor channels are highly permeable to Ca2+ (Fig. 5), consistent with their proposed role in regulating cilia activity (Ma et al. 1999; Silberberg et al. 2001). Finally, results have been presented which suggest that the P2Xcilia receptor channel is either an unusual variant of P2X4 or a heteromeric channel containing both P2X4 and P2X7 subunits.
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Priming
An intriguing property of P2Xcilia receptor channels is the time-dependent increase in current (priming) during the first long exposure to ATP (Korngreen et al. 1998; Fig. 1). The mechanism by which ATP primes the P2Xcilia receptor channels is not known. Nevertheless, priming is unlikely to be mediated by the P2Y pathway because priming persists when the G proteins are inhibited by GDP-S (Korngreen et al. 1998). Also, ambient release of ATP is not likely to play a significant role in priming because the total concentration of nucleotides released from airway epithelial cells is less than 200 nM, of which ATP is a small fraction (Lazarowski et al. 2004), whereas in dissociated airway ciliated cells, P2Xcilia receptor channels are relatively insensitive to ATP (EC50 is >200 μM in an extracellular solution containing 1.5 mM CaCl2; Korngreen et al. 1998). Moreover, all the experiments were performed with continuous perfusion of ATP to circumvent the possible effects of ambient ATP and/or ectoATPases. Priming appears to be distinct from the mechanism underlying current growth reported for P2X7 receptor channels. Repeated or prolonged activation of P2X7 receptor channels, in low extracellular divalent cation solutions (but not in the presence of physiological concentrations of divalent cations), leads to an increase in membrane permeability to large cations such as NMDG+ (Surprenant et al. 1996; Jiang et al. 2005). In contrast, priming of P2Xcilia receptor channels is observed at physiological levels of extracellular divalent cations, and the permeability of P2Xcilia receptor channels to NMDG+ does not change throughout the priming process (Fig. 1B and C). Fujiwara & Kubo (2004) have reported that changes in ion selectivity of P2X2 receptor channels are seen only when channel density is high. It is not known whether a higher density of P2Xcilia receptor channels would lead to changes in ion selectivity during long exposures to ATP. Nonetheless, it is clear that the native density of channels is sufficient to induce current growth which is not accompanied by increased permeability to large cations (Fig. 1). It was recently shown that the ATP-induced increase in permeability to NMDG+ and to the fluorescent dye YO-PRO-1 in P2X7-expressing cells arises from two separate permeation pathways, and they proposed that dilatation of the P2X7 receptor channel gives rise to the increase in permeability to NMDG+ (Jiang et al. 2005). A similar mechanism cannot account for the priming of P2Xcilia receptor channels because the relative permeability of NMDG+ and Cs+ is the same before and after priming. Thus, priming could result either from the recruitment of silent P2Xcilia receptor channels, from a progressive increase in sensitivity to ATP, a mechanism that may contribute to current growth of P2X7 receptor channels (Hibell et al. 2000), or from a mode shift of the channels to a conformation that supports high open probabilities. In either case, priming provides an additional dimension to the regulation of P2Xcilia receptor channel activity, and thus may have an important physiological function.
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Modulation by Na+
Extracellular Na+ inhibits P2Xcilia receptor channel activity (Ma et al. 1999; Silberberg et al. 2001). In the present study it was found that Na+ also permeates the channel (Fig. 2), which led us to investigate whether Na+ inhibits the channel from the extracellular or intracellular side of the membrane. Inhibition by 30 mM Na+ was significantly less when Na+ was applied from the inside, suggesting that the binding site for Na+ is more readily accessible from the extracellular side (Fig. 3). Furthermore, inhibition by Na+ is insensitive to voltage indicating that the Na+-binding site is not within the electric field. In N-type calcium channels, extracellular Na+ inhibits the channels by competing with Ca2+ for a binding site within the permeation pathway, but outside the electric field (Polo-Parada & Korn, 1997). It is unlikely that a similar mechanism of block of the ion permeation pathway also accounts for the effects of Na+ on the P2Xcilia receptor channel because Na+ inhibition can be overcome by raising the concentration of ATP (Ma et al. 1999). It is more likely that Na+ either directly reduces the affinity of the P2Xcilia receptor channel to ATP or acts as an allosteric modulator of channel gating. It is therefore tempting to predict that analogues of ATP with significantly greater efficacy could help improve mucociliary clearance in chronic obstruction airway disorders.
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Permeation properties of the pore
P2Xcilia receptor channels discriminate poorly between the alkali cations and are permeable to large cations such as TEA+ and NMDG+ (Fig. 4 and Table 1). These findings suggest that the selectivity filter of P2Xcilia receptor channels may not be as well defined in comparison to other more selective channels such as the voltage-dependent Na+, K+ and Ca2+ channels. It is surprising that, despite the relatively poor discrimination between the alkali cations, the permeability of P2Xcilia receptor channels is greatest for Li+ and decreases with atomic number (Li+ > Na+ > K+ > Rb+ > Cs+), consistent with a site of high field strength in the permeation pathway (Eisenman & Horn, 1983). The slightly greater relative permeability to NMDG+ in comparison to TEA+ is inconsistent with their respective mean diameters: 0.73 nm (Artigas & Gadsby, 2004) and 0.66 nm (Liu & Adams, 2001). This is probably due to the elongated shape of NMDG+. The dimensions of NMDG+ are 0.50 x 0.64 x 1.20 nm (Burnashev et al. 1996), and hence NMDG+ might fit through the pore better than TEA+. Judging from the relative permeability to TEA+, the diameter of the narrow part of the pore of the P2Xcilia receptor channel is slightly greater than 0.66 nm. Similarly, the minimum pore diameter of rat P2X1, P2X2 and undilated P2X7 receptor channels has been estimated to be less than 0.8 nm (Evans et al. 1996; Surprenant et al. 1996). It is noteworthy that human P2X5 receptor channels exhibit a time-independent high permeability to NMDG+ (PNMDG/PNa, 0.4), suggesting that the pore of these channels might be larger than that of other P2X receptor channels (Bo et al. 2003a).
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Reversal potential measurements were used to estimate PCa/PNa in rat P2X1, P2X2, P2X3 and P2X4 receptor channels and found to be 4 (Evans et al. 1996), 2.5 (Virginio et al. 1998), 1.2 (Virginio et al. 1998), and 4.2 (Buell et al. 1996; Soto et al. 1996), respectively. Although it is difficult to make comparisons between studies due to different experimental conditions and assumptions, it is interesting to note that direct measurements of fractional Ca2+ currents show that in P2X1, P2X2, P2X3 and P2X4 receptor channels approximately 12%, 6%, 3% and 11%, respectively, of the current is carried by Ca2+ (Egan & Khakh, 2004). Thus, in P2X receptor channels there appears to be a correlation between relative permeability to Ca2+ and the fractional Ca2+ current. From this it can be inferred that more than 10% of the current through P2Xcilia receptor channels is carried by Ca2+ under physiological conditions, consistent with their proposed role in regulating cilia activity (Ma et al. 1999; Silberberg et al. 2001).
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Possible subunit composition of P2Xcilia receptor channels
Relating the diverse physiological responses mediated by native P2X receptor channels to the cloned P2X receptor subtypes is a major challenge. ATP-activated currents with characteristics that do not coincide with any of the cloned channels could arise from splice variations, post translational modification, regulation by auxiliary proteins, or the expression of a mixed population of channels. The ATP-activated currents in airway ciliated cells have properties that are unique to P2X4 receptor channels and properties that are unique to P2X7 receptor channels (see below). Nevertheless, it is unlikely that P2Xcilia receptor channel currents arise from a mixed population of P2X4 and P2X7 homomeric receptor channels because these channels have very different sensitivities to ATP in the presence of physiological concentrations of divalent cations (North, 2002), yet the dose–response relationship for activation of P2Xcilia receptor currents by ATP is well described by a simple Hill equation (Korngreen et al. 1998). Furthermore, P2Xcilia receptor currents can be fully inhibited by extracellular Na+, whereas P2X4 receptor channels are largely insensitive to variations in the extracellular concentration of Na+. It might be that during the priming process the population of P2X4 receptor channels are desensitized by the relatively high concentrations of ATP (200 μM) and that these channels can be rescued from the desensitized state by either Zn2+ or IVM. However, this is unlikely because 1 μM Zn2+ is unable to activate desensitized rat P2X4 receptor channels (S.D. Silberberg, unpublished observations). Based on experiments in cell lines derived from airway epithelium it was recently proposed that the P2Xcilia receptor channel is a hetero-multimer of P2X4 and P2X6 subunits (P2X4+6; Liang et al. 2005). However, this is unlikely to be the case because P2X4+6 receptor channels appear to be relatively insensitive to extracellular Na+ (Le et al. 1998), and because P2X4+6 receptor channels have a sensitivity to ATP similar to that of homomeric P2X4 receptor channels (EC5010 μM; Le et al. 1998), whereas P2Xcilia receptor channels have a low sensitivity to ATP (Korngreen et al. 1998).
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P2Xcilia receptor channels and homomeric P2X7 receptor channels share a low sensitivity to ATP (EC50, 200 μM) at physiological concentrations of divalent cations (North, 2002). In addition, calcium influx induced by ATP in human lymphocytes is inhibited by extracellular Na+ (Pizzo et al. 1991; Wiley et al. 1992), extracellular Na+ reduces the potency of BzATP in HEK293 cells expressing the human P2X7 receptor channel (Michel et al. 1999), and Na+ prevents the changes in NMDG+ permeability induced by BzATP in HEK293 cells expressing the rat P2X7 receptor channel (Jiang et al. 2005). All these results suggest that P2X7 receptor channels are also directly modulated by extracellular Na+. Furthermore, P2Xcilia and P2X7 receptor channels are both significantly attenuated by physiological concentrations of extracellular divalent cations (Surprenant et al. 1996; Korngreen et al. 1998; Fig. 5) and are inhibited by BBG and by KN-62 (Humphreys et al. 1998; Jiang et al. 2000; Fig. 6). These properties are not shared by P2X4 receptor channels. On the other hand, P2X7 receptor channels are insensitive to IVM and are inhibited by 1 μM Zn2+ (Virginio et al. 1997), whereas both IVM and Zn2+ augment P2Xcilia receptor channel currents (Fig. 7). Of the non-desensitizing cloned P2X receptors, both P2X2 and P2X4 are augmented by Zn2+; however, only P2X4 is sensitive to IVM, indicating a special relationship between P2Xcilia and P2X4 receptor channels. Could P2Xcilia receptor channels be a hetero-oligomeric assembly containing P2X4 and P2X7 receptor subunits
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Co-immunoprecipitation assays performed on HEK293 cells co-expressing different P2X subunits suggest that P2X7 does not form channels with other P2X receptor channel subunits (Torres et al. 1999). However, it is conceivable that such interactions do occur in native tissue. P2X7 receptor channel protein was strongly detected in mouse lung (Sim et al. 2004), whereas P2X4 protein was identified by immunoreactivity in rat bronchus epithelium (Bo et al. 2003b). Furthermore, both P2X4 and P2X7 mRNA were detected by RT-PCR in epithelial cells freshly isolated from rat trachea (Marino et al. 1999), and P2X4 and P2X7 protein was detected by Western blot in pig airway epithelium (E. Ben-Tal Cohen, S. Weil and S.D. Silberberg, unpublished observations). None of these studies, however, specifically targeted ciliated cells nor did they demonstrate colocalization of these subunits. Thus, we used specific antibodies in combination with fluorescence microscopy to try to determine whether P2Xcilia receptor channels are composed of P2X4 and P2X7 subunits. The results for P2X4 are compelling. In intact trachea, specific binding of the anti-P2X4 antibody was detected in the proximal half of the cilia (Fig. 8). In contrast, we were unable to reliably detect P2X7 immunoreactivity using two different antibodies (see Methods). This finding does not exclude the possibility that P2X7 is expressed by these cells, as P2X7 receptor channels interact with several other proteins which could mask the binding sites for the antibodies (Kim et al. 2001). Taken together, these results suggest that P2Xcilia receptor channels contain at least one P2X4 subunit. Whether the inhibition of P2Xcilia receptor channels by extracellular Na+, extracellular divalent cations, KN-62 and BBG, as well as their low sensitivity to ATP arise from the contribution of P2X7 subunits or from modification/modulation of P2X4 subunits in a homomeric structure remains to be determined.
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The targeting of P2Xcilia receptor channels to the proximal half of the cilia suggests that they are poised to modulate cilia function. The restricted volume of the cilium combined with a relatively high permeability to Ca2+ for P2Xcilia receptor channels suggest that even at low open probabilities (i.e. high extracellular Na+ concentration) these channels could influence cilia activity. In view of the location of the channels, it is tempting to speculate that in addition to providing a pathway for ions, these proteins may have other functions in regulating cilia activity, analogous to the role of calcium channels in excitation–contraction coupling in skeletal muscle.
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2 The Zlotowski Center for Neuroscience, and the
4 Department of Life Sciences, Ben-Gurion University of the Negev, Beer-Sheva 84105, Israel
3 Faculty of Life Sciences, and the Leslie and Susan Gonda Interdisciplinary Brain Research Center, Bar-Ilan University, Ramat-Gan 52900, Israel
Abstract
Airway ciliated cells express an ATP-gated P2X receptor channel of unknown subunit composition (P2Xcilia) which is modulated by Na+ and by long exposures to ATP. P2Xcilia was investigated by recording currents from freshly dissociated rabbit airway ciliated cells with the patch-clamp technique in the whole-cell configuration. During the initial continuous exposure to extracellular ATP, P2Xcilia currents gradually increase in magnitude (priming), yet the permeability to N-methyl-D-glucamine (NMDG) does not change, indicating that priming does not arise from a progressive change in pore diameter. Na+, which readily permeates P2Xcilia receptor channels, was found to inhibit the channel extracellular to the electric field. The rank order of permeability to various monovalent cations is: Li+, Na+, K+, Rb+, Cs+, NMDG+ and TEA+, with a relative permeability of 1.35, 1.0, 0.99, 0.91, 0.79, 0.19 and 0.10, respectively. The rank order for the alkali cations follows an Eisenman series XI for a high-strength field site. Ca2+ has been estimated to be 7-fold more permeant than Na+. The rise in [Ca2+]i in ciliated cells, induced by the activation of P2Xcilia, is largely inhibited by either Brilliant Blue G or KN-62, indicating that P2X7 may be a part of P2Xcilia. P2Xcilia is augmented by Zn2+ and by ivermectin, and P2X4 receptor protein is detected by immunolabelling at the basal half of the cilia, strongly suggesting that P2X4 is a component of P2Xcilia receptor channels. Taken together, these results suggest that P2Xcilia is either assembled from P2X4 and P2X7 subunits, or formed from modified P2X4 subunits.
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Introduction
Cation-selective ion channels activated by extracellular ATP (P2X receptor channels) are widely distributed in electrically excitable cells such as neurones, and in non-excitable cells such as immune cells and epithelial cells (North, 2002). Seven subunits of the mammalian P2X receptor family (P2X1 to P2X7) have been cloned, and all of them can form functional homomeric receptor channels in heterologous expression systems (North, 2002; Jones et al. 2004). Six functional heteromeric receptor channels (P2X1/P2X2, P2X1/P2X4, P2X1/P2X5, P2X2/P2X3, P2X2/P2X6 and P2X4/P2X6) have also been characterized (Torres et al. 1998; Le et al. 1998, 1999; King et al. 2000; Brown et al. 2002; Nicke et al. 2005). Co-immunoprecipitation assays performed on HEK293 cells co-expressing different P2X subunits suggest that additional hetero-oligomeric assemblies are possible among the different P2X receptor subunits (Torres et al. 1999). The pharmacological and biophysical properties of native P2X receptor channels often deviate from those of heterologously expressed P2X receptor channels. This suggests that native P2X receptor channels might be processed differently or that they are heteromeric channels of yet uncharacterized combinations of P2X subunits.
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Airway ciliated cells express a P2X receptor channel (P2Xcilia) of unknown composition. This channel is interesting both from a physiological and from a biophysical standpoint because of its modulation by both ATP and Na+. P2Xcilia receptor currents gradually increase over tens of seconds during the first exposure to ATP whereas subsequent applications of ATP activate the current significantly faster (Korngreen et al. 1998). This priming phenomenon can be observed for several minutes after the initial application of ATP, and thus ATP can be considered both an agonist and a long-term modulator of the P2Xcilia receptor channel. P2Xcilia receptor channels are also modulated by Na+ (Ma et al. 1999). The effect of Na+ can be overcome by raising the concentration of ATP, suggesting an allosteric mechanism of regulation. While the physiological role of P2Xcilia receptor channels has not yet been resolved, comparing the effects of Na+ on cilia activation induced by different concentrations of ATP suggests that P2Xcilia receptor channels contribute to the mechanisms sustaining the cilia at a high level of activation during continued exposure to ATP (Silberberg et al. 2001).
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The unusual properties of P2Xcilia receptor channels, and their potential physiological significance, prompted us to investigate the relative permeability of the channel to various cations and the pharmacological properties of the channel in order to address four specific questions. First, does the gradual increase in membrane current observed during long exposures to ATP result from a change in ion selectivity, as has been proposed for P2X2, P2X4 and P2X7 receptor channels Second, if Na+ can permeate P2Xcilia receptor channels, is the binding site for inhibition by Na+ located on the extracellular or intracellular side of the channel The answer to this question could help guide the development of strategies to exploit P2Xcilia receptor channels to enhance mucociliary clearance. Third, are P2Xcilia receptor channels highly permeable to Ca2+, as might be expected if the channel contributes to cilia activation by ATP (Korngreen & Priel, 1994, 1996) Fourth, is P2Xcilia composed of P2X7 receptor subunits, as suggested by some of its properties Here we show that the channels do not undergo pore dilatation, that the binding site for Na+ is extracellular, and that the channels are highly permeable to Ca2+. In addition, the results suggest that the pore of P2Xcilia receptor channels contains a high-strength field site, and put constrains on the diameter of the narrow part of the ion conduction pore. Pharmacological and immunohistochemical experiments suggest that P2Xcilia receptor channels may contain both P2X4 and P2X7 subunits.
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Methods
Isolation of single ciliated cells
Male New Zealand white rabbits (2–3 months old) were killed as previously described (Korngreen et al. 1998), according to the guidelines laid down by the animal welfare committee of Ben-Gurion University, by gradual exposure to carbon dioxide followed by exsanguination. Care was taken to slowly increase the gas flow over several minutes to prevent any visual signs of distress. The trachea was removed and the epithelium surgically separated from the cartilage and cut into pieces of approximately 0.3 cm x 0.3 cm. The tissue was maintained in physiological extracellular solution (DPBS) containing (mM): NaCl 137, KCl 2.7, CaCl2 0.9, MgCl2 0.5, Na2HPO4 8, KH2PO4 1.47 and D-glucose 5; pH 7.4. The epithelium was incubated for 25 min, at 37°C, in DPBS supplemented with 13 U ml–1 papain (Sigma), 1 mg ml–1 bovine serum albumin (BSA) (Sigma, Fraction V, essentially fatty acid free) and 1 mg ml–1 1,4-dithiothreitol (Merck, Darmstadt, Germany). Cells were then dispersed by repeated aspiration with a fire-polished Pasteur pipette, concentrated by centrifugation 800 g for 10 min, and re-suspended in DPBS. The isolated cells were transferred to 35-mm tissue culture dishes and used immediately. Single ciliated cells remained active for several hours after isolation and increased cilia beating in response to ATP. Part of the tissue was maintained overnight in DPBS at 4°C and used on the next day. No obvious differences in the experimental results, in cell viability or in sensitivity to ATP, were observed when cells were used on the next day (Ma et al. 2002).
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Solutions and drugs
Solutions were made with highly purified water (NANOpure, Barnstead, Debuque, IA, USA), using chemicals of analytical grade. The standard extracellular solution contained (mM): CsCl 151, CaCl2 1.58, Tes 5, D-glucose 10 and 3,4-diaminopyridine (DAP) 1; pH adjusted to 7.4 with CsOH (295–300 mosmol l–1). The standard pipette solution contained (mM): CsCl 155, Tes 5, EGTA 0.1, Mg-ATP 1, Na2GTP 0.1 and CaCl2 0.096 (0.1 μM Ca2+, estimated by Max Chelator 6.0, Chris Patton, Stanford University, CA, USA); pH adjusted to 7.2 with CsOH (285–290 mosmol l–1). Deviations from these solutions are noted in the text. K2ATP, Mg-ATP, 2'-3'-O-(4-benzoylbenzoyl) ATP triethylammonium salt (BzATP), Brilliant Blue G (BBG), 1-[N,O-Bis(5-isoquinoline sulphonyl)benzyl]-2-(4-phenylpiperazine)ethyl]-5-isoquinoline sulphonamide (KN-62) and ivermectin (IVM) were from Sigma. Na2GTP was from Boehringer Mannheim. DAP was from Fluka. TEA was from Fluka and from Sigma. Solutions containing ATP or other nucleotides were freshly prepared each day and the pH of the solution was readjusted.
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Whole-cell current recording
Membrane currents were recorded using the standard whole-cell configuration of the patch-clamp technique (Hamill et al. 1981), as previously described (Korngreen et al. 1998). Briefly, patch pipettes (2–3 M, when filled with intracellular solution) were fabricated from borosilicate glass (GC150F-7.5, Clark Electromedical Instruments, Reading, UK), coated with Sylgard 184 (Dow Corning, Midland, MI) and fire-polished shortly before use. Capacitive currents were electronically compensated. Series resistance was not compensated in most experiments, as the currents activated by ATP were small and slow. Once the whole-cell configuration was established, the cell was continuously superfused with the desired extracellular solutions via a gravity-fed perfusion system. The bathing solution was changed using a Teflon rotary valve, which selected among one of six reservoirs. The continuous perfusion of the cell prevented fluctuations in the concentration of ATP due to degradation of ATP by ecto-nucleotidases, and prevented the possible build-up of degradation products such as ADP. Liquid junction potentials were determined by adjusting the bath potential to zero with a patch pipette containing internal solution placed in internal solution, and then measuring the bath potential in each of the external solutions (Neher & Sakmann, 1992). Liquid junction potentials were at most 1.5 mV and hence were not corrected. All measurements were at room temperature (18–22°C).
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Data acquisition and analysis
Experiments were initiated at least 2 min after establishing the whole-cell configuration to allow equilibration of the cytosol with the pipette solution. Membrane currents were recorded under voltage clamp using an Axopatch 200A patch-clamp amplifier (Axon Instruments, Union City, CA, USA). Stable recordings were typically maintained for 20–30 min. Membrane currents were low-pass filtered using an 8-pole Bessel filter (Frequency Devices, Haverhill, MA, USA) in series with the integral 4-pole Bessel filter of the patch-clamp amplifier and stored on video tape for subsequent analysis (VR-10B, Instrutech). Membrane currents were also digitized on-line using a Digidata 1200 interface board and pClamp 6.03 software (Axon Instruments). The sampling frequency was set to at least two times the corner frequency of the low-pass filter. SigmaPlot scientific software was used for non-linear curve fitting. Experimental results were consistently observed in cells from two or more animals; hence, all the results for a particular experiment were pooled and displayed as means ±S.E.M.; groups were compared with Student's t test.
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The permeability ratio, PX/PCs, was quantified by fitting the reversal potentials (Vrev) measured experimentally with the Goldman–Hodgkin–Katz (GHK) equation:
(1)
where R is the universal gas constant, T is absolute temperature, F is Faraday's constant, PX and PCs are the permeability (P) to any monovalent cation X and Cs+, respectively, X and Cs are the activity coefficients () of X+ and Cs+, respectively, and [X]0 and [Cs]i are the concentrations of X+ and Cs+ in the extracellular and intracellular solutions, respectively.
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When the extracellular solution contained Cs+ and Na+, the permeability ratio, PNa/PCs, was estimated using the equation:
(2)
Calculating the permeability of Ca2+ over Cs+
As P2Xcilia receptor channels are modulated by divalent cations (Korngreen et al. 1998), the relative permeability to Ca2+ was estimated by measuring the effects of 0.1–2 mM extracellular Ca2+ on the reversal potential of the ATP-activated currents. The relationship between Vrev and the relative ion permeability predicted by the GHK equation is described by the equation (Korngreen et al. 1998):
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(3)
The estimated permeability ratio PCa/PCs depends on the choice of activity coefficients for these ions (Ca and Cs). In mixed electrolyte solutions, each salt influences the activity coefficient of the other electrolytes in the solution. However, in mixed solutions of total ionic strength up to 0.2 M, it can be assumed that the activity of an individual ion is determined solely by the ionic strength of the solution, and not by the chemical nature of the other ions in the solution (Shatkay, 1968). Hence, the activity coefficients for CsCl and CaCl2 (Robinson & Stokes, 1959) and the activity coefficient for TEA-Cl (Lindenbaum & Boyd, 1964) were adjusted for total ionic strength, and used to estimate the activity coefficient of Cs+, Ca2+ and TEA+, respectively. For example, we used activity coefficients of 0.515 and 0.48 for 0.1 M and 0.2 M CaCl2, respectively. Assuming a linear relationship between ionic strength and activity coefficient in this range of concentrations, the activity coefficient of CaCl2 for a solution with an ionic strength of 0.15 M would be 0.4975. Various assumptions have been made in order to estimate the activity coefficient of a divalent cation (++) from the mean activity coefficient of the salt (±) in mixed solutions (Butler, 1968). In the present study, two conceptually different assumptions were used, ++=± (Shatkay, 1968) and ++= (±)2 (Butler, 1968). No difference in the estimate of PCa/PCs was observed.
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Measurement of intracellular calcium
Experiments were carried out on explants of epithelium from rabbit trachea using the procedure previously described (Korngreen & Priel, 1996). Briefly, the ciliary epithelium was peeled off the cartilage and cut into small pieces. Two or three pieces of epithelium were placed on a glass coverslip and incubated in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 20 units ml–1 penicillin, 2.5 units ml–1 nystatin and 20 μg ml–1 streptomycin at 37°C with 5% CO2. The medium was replaced every 3 days. Measurements were performed after 6–19 days in culture. Measurements of intracellular calcium were performed as previously described (Korngreen & Priel, 1994). Briefly, the cells were preloaded with fura-2 by incubating the tissue in serum-free growth medium, containing 5 μM of the acetoxymethyl ester (AM) form of fura-2 (fura-2AM) and 500 μM probenecid, for 60 min at 32°C in a rotating water bath, followed by washing for 30 min in Ringer solution containing (mM): NaCl 150, KCl 2.5, CaCl2 1.5, MgCl2 1.5, D-glucose 5 and Hepes 5. Probenecid, an inhibitor of membrane organic anion transporters, is widely used to prevent extrusion of loaded fura-2 from the cells. The dye-loaded cells were epi-illuminated with light from a 75-W xenon lamp (Ushio Inc., Japan) filtered through 340 and 380 nm interference filters (Oriel Corp., Stamford, CT, USA) mounted on a four-position, rotating, filter wheel. The fluorescence, emitted at 510 nm, was detected by a photon-counting photomultiplier (H3460-53, Hamamatsu, Japan). The 340/380 nm fluorescence ratio, averaged over a period of 1 s, was stored in a computer. A calibration curve of the calcium concentration was created by titrating an external calibration solution with a solution of the same composition containing 10 mM CaCl2 (Grynkiewicz et al. 1985; Korngreen & Priel, 1994). To account for the difference between the fura-2 fluorescence signal in the intracellular medium and in the calibration solution, the maximal and minimal values of the 340/380 nm fluorescence ratio were measured from the cells. The maximal value was obtained by adding 5 μM ionomycin to the cells, which flooded the cells with Ca2+. The minimal value was obtained by adding ionomycin to the cells in a Ca2+-free medium. The calibration curve was corrected according to the obtained results. The non-specific signal was estimated by adding ionomycin to the cells in the presence of 1 mM Mn2+, which leads to quenching of fura-2 fluorescence. The calcium concentration was calculated directly from the corrected calibration curve by interpolation using an algorithm table. Prior to any treatment, the Ringer solution over the explants was changed twice. The basal [Ca2+]i from a single cell was measured for 1–5 min in the appropriate solution, and the results of this measurement taken as the reference value. Then, the solution was changed to the test solution and [Ca2+]i monitored from the same cell for an additional 10 min.
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Immunohistochemistry
Immunohistochemistry was carried out on paraffin embedded sections as previously described (Humason, 1979). The trachea was cleaned and washed with cold phosphate-buffered solution (PBS) containing (mM): Na2HPO4 77.5, NaH2PO4 22.5 and NaCl 150; pH 7.4. Slices (width, 5 mm) were obtained from each cleaned trachea, fixed in 4% formaldehyde in PBS overnight at 4°C, and embedded in paraffin blocks. The blocks were sliced (10 μm thick), mounted on SuperFrost Plus slides (Menzel-Glaser, Braunschweig, Germany) and stored at –20°C. To remove paraffin, sections were washed twice for 20 min each in xylene followed by gradual re-hydration with ethanol (100%, 95%, 80% and 70%, 5 min in each) and finally in PBS. The slides were transferred to 50 mM sodium citrate solution (pH 6.0) and submitted to antigen retrieval by boiling in a microwave for 10 min. Non-specific binding sites were blocked by 30-min incubation at room temperature with 5% BSA and 5% normal goat serum (NGS) in PBS. The slides were incubated overnight at 4°C in PBS containing 1% NGS, 0.1% Triton X-100 and either 1: 400 primary antibody or primary antibody pre-absorbed overnight with a 10-fold excess of the homologous peptide antigen. Finally, the slides were exposed in PBS to the secondary goat anti-rabbit antibody 1: 500 Alexa flour 594 in the dark for 1 h. The slides were washed and covered by coverslips using Fluramount-G (Southern Biothechnology Associates, Inc. Birmingham, AL, USA). Immunoreactivity was visualized using a Zeiss LSM510 confocal microscope.
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Results
Long applications of ATP gradually activate a membrane conductance in ciliated cells
P2Xcilia receptor channels exhibit a time-dependent increase in current during the initial long exposure to ATP (Korngreen et al. 1998). This phenomenon was called ‘priming’ because the response to a subsequent application of ATP applied up to several minutes later activates more rapidly (Korngreen et al. 1998). Figure 1A is an example of such priming, showing a whole-cell current trace recorded in the standard intracellular and extracellular CsCl solutions, at a holding potential of –40 mV, from a ciliated cell freshly dissociated from rabbit airway epithelium. Exposing the cell to 200 μM ATP activated an inward current, which slowly increased in the presence of ATP to a maximal level of –65 pA. The ATP-activated current reached half of the maximal level after 87 s and deactivated to half the maximal current level within 5 s upon removal of the ATP. During a second application of ATP, the current activated significantly faster, reaching half maximal activation within 3 s. This gradual increase in the magnitude of the current during the initial sustained application of ATP and the much faster response to subsequent exposures to ATP was seen in all ciliated cells tested (n > 150).
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A, continuous whole-cell current trace recorded at a holding potential of –40 mV, from a ciliated cell not previously exposed to ATP. ATP (200 μM) was applied twice (indicated by the bars). The first application gradually activated an inward current, which opened significantly faster upon the second application of ATP. Standard intracellular and extracellular solutions. B and C, net ATP-activated current responses to voltage ramps measured during priming. The extracellular solution contained (mM): NMDG-Cl 151, Tes 5, D-glucose 10, DAP 1 and either CaCl2 1.58 (B) or BaCl2 0.1 (C). The pipette contained the standard CsCl intracellular solution. In both cases, the reversal potential did not change during priming, indicating that P2XCilia receptor channels do not exhibit a time-dependent change in ion selectivity during long exposures to ATP.
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The gradual increase in membrane conductance does not result from a time-dependent change in ion selectivity of the P2Xcilia receptor channel
An enhancement in membrane conductance can result from an increase in single-channel conductance, a rise in channel open probability (by increasing ATP affinity or efficacy) and/or from recruitment of silent channels. If the priming phenomenon described above results from changes in ion channel conductance, the selectivity of the channel would be likely to change with time. It is interesting that cells heterologously expressing rat P2X2, P2X4, P2X7 and heteromeric P2X2/P2X3 receptor channels, bullfrog P2X5 or Schistosoma mansoni P2X receptor channels exhibit time-dependent changes in ion selectivity with continuous application of agonist, which has led to the suggestion that some of the P2X receptor channels undergo pore dilatation during activation (Surprenant et al. 1996; Khakh et al. 1999a; Virginio et al. 1999; Jensik et al. 2001; Raouf et al. 2005; for review see Khakh & Lester, 1999,). We therefore investigated whether the gradual increase in membrane current observed in the ciliated cells during long applications of ATP (Fig. 1A) is also accompanied by a change in ion selectivity. In these experiments, whole-cell currents were recorded with the standard intracellular Cs+ solution and with Cs+ replaced by NMDG+ in the extracellular solution. Current–voltage relationships were constructed by applying voltage ramps throughout the priming process, and the net current activated by ATP was assessed by subtracting the current recorded under control conditions from the current recorded in the presence of ATP. Figure 1B shows current–voltage relationship measured at different times throughout the priming by ATP, as indicated by each current trace. Despite the progressive increase in current with time, the reversal potential of the current remained at –37 mV, indicating that the permeability to NMDG+ relative to Cs+ did not change during priming. A lack of change in the reversal potential during priming was observed in 7 out of 7 experiments. When the extracellular solution contained a low concentration of divalent cations, the reversal potential also remained constant during priming (Fig. 1C). The difference between the reversal potentials shown in Fig. 1B and C arises from permeability to Ca2+, as discussed later. These results show that during the priming process P2Xcilia receptor channels do not undergo pore dilatation, as suggested for some P2X receptors (Khakh & Lester, 1999,). As the permeability of P2Xcilia receptor channels does not change during priming, for the remainder of the experiments, measurements were conducted on fully primed channels.
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Na+ both inhibits and permeates P2Xcilia receptor channels
We have previously reported that extracellular Na+ directly inhibits P2Xcilia receptor channels, and that the degree of inhibition by Na+ depends on the interplay between the effective concentrations of ATP and Na+ (Ma et al. 1999). To establish whether P2Xcilia receptor channels are permeable to Na+, net whole-cell currents activated by ATP (200 μM) in response to voltage ramps were recorded in extracellular solutions containing different concentrations of NaCl. The ionic strength of the extracellular solution was maintained constant by replacing CsCl with equimolar amounts of NaCl. Figure 2A shows examples of membrane currents recorded in the different extracellular solutions as indicated by each current trace. The rightward shifts in the zero-current potential induced by increasing the concentration of Na+ relative to Cs+ indicate that Na+ is more permeable than Cs+. Figure 2B shows a graph of the mean (±S.E.M.) reversal potential of the current activated by ATP in extracellular solutions containing different concentrations of NaCl (, averaged from nine experiments of the type shown in Fig. 2A). The permeability ratio, PNa/PCs, was quantified by fitting Vrev measured experimentally using the GHK equation (eqn (2)), and found to be 1.27: 1 (continuous line). Permeability ratios of 1.4: 1 and 1.2: 1 are also shown for comparison (dashed lines). Clearly, in addition to inhibiting P2Xcilia receptor channels, Na+ permeates the channels.
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A, mean net whole-cell currents activated by ATP (200 μM) in response to voltage ramps in extracellular solutions containing either 15, 30, 75 or 120 mM NaCl (the total alkali cation concentration was adjusted to 151 mM with CsCl). The extracellular solution also contained (mM): Tes 5, D-glucose 10, and BaCl2 0.1. Standard intracellular solution. Five to 10 consecutive current traces were averaged for each experimental condition. B, mean (±S.E.M.) reversal potentials of the current activated by ATP in extracellular solutions containing different concentrations of NaCl (, averaged from nine experiments of the type shown in A). The relative permeability of Na+ and Cs+ (PNa/PCs) was estimated to be 1.27: 1 using eqn (2) (continuous line). Permeability ratios of 1.4: 1 and 1.2: 1 are also shown (dashed lines).
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The binding site for Na+ is extracellular
The permeability of P2Xcilia receptor channels to Na+ (Fig. 2) raises the possibility that Na+ enters the cell during channel activation and inhibits the channel from the inside. To explore this possibility, the effects of intracellular Na+ on the current activated by ATP were investigated by replacing CsCl in the pipette solution with an equimolar amount of NaCl (30 mM). Although it is possible to change the composition of the solution in the pipette during whole-cell recording, this is a rather slow process and it is difficult to determine when complete solution exchange has taken place. We therefore measured the currents activated by ATP under a single experimental condition in each cell, and made comparisons between cells by estimating the current density (pA/pF). The cells were exposed to ATP at least 2 min after establishing the whole-cell configuration to allow equilibration of the cytosol with the pipette solution. We chose a concentration of 30 mM Na+ because application of 30 mM Na+ to the extracellular solution reversibly inhibited the ATP-activated current by 90% (Fig. 3A and B). Thus, if Na+ inhibits the channel from outside the cell, significantly less inhibition would be expected when the Na+ is included in the pipette solution. This was indeed the case. Examples of current density traces recorded under control conditions and in the presence of 30 mM Na+ in the pipette solution are shown in Fig. 3C. The highly significant (P < 0.0001) greater inhibition by extracellular Na+ (Fig. 3B) suggests that the binding site for inhibition by Na+ is more readily accessible to extracellular Na+.
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A, continuous whole-cell trace recorded at a holding potential of –40 mV, from a cell exposed to ATP (300 μM) for the duration indicated by the top bar. At the time indicated by the lower bar, 30 mM of the extracellular CsCl was replaced by 30 mM NaCl. Standard intracellular solution. The cell was primed with ATP before the start of the experiment. B, mean (±S.E.M.) current in the presence of either extracellular (n= 17) or intracellular (n= 17) NaCl (30 mM), normalized to the current in the absence of NaCl (n= 24). C, continuous whole-cell current traces recorded at a holding potential of –40 mV, from cells exposed to ATP (300 μM) for the duration indicated by the bars. Standard extracellular solution. The intracellular solution contained either 155 mM CsCl (left-hand trace) or 30 mM NaCl and 125 mM CsCl (right-hand trace). The intracellular solution also contained (mM): Tes 5, EGTA 0.1, Mg-ATP 1, Na2GTP 0.1 and CaCl2 0.096.
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In order to determine whether inhibition by Na+ is voltage dependent, the effect of 15 mM extracellular NaCl on the current activated by ATP (200 μM) was measured at different holding potentials. On average, the current activated by ATP (200 μM) at –80, –60, –40 or +40 mV was attenuated by 15 mM NaCl by 68.4 ± 2.3% (n= 11), 68.5 ± 2.9% (n= 17), 69.4 ± 3.8% (n= 10) and 59.6 ± 4.4% (n= 9), respectively. The absence of significant voltage dependence of Na+ inhibition, suggests that the binding site for Na+ is not within the electric field.
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P2Xcilia receptor channels exhibit little discrimination between the alkali cations
The relative permeability of P2Xcilia receptor channels to the other alkali cations was estimated under bi-ionic conditions using the same experimental approach described above. The reversal potential of the net current activated by ATP was measured using the standard intracellular solution and an extracellular solution containing (mM): XCl 151, BaCl2 0.1, Tes 5, D-glucose 10, DAP 1; pH 7.4, where X represents a monovalent cation. Figure 4A shows an example of net ATP-activated current responses to voltage ramps measured from a cell in extracellular solutions containing Cs+, K+ or Li+, as indicated. As the reversal potentials of K+ and Li+ are positive, it can be concluded that these cations are more permeable than Cs+. An illustration of the relative permeability of the P2Xcilia receptor channel to the various cations is presented in Fig. 4B, which shows representative net ATP-activated current responses to voltage ramps assembled from different cells. The reversal potential in each extracellular solution served to calculate the relative permeability of different monovalent cations using eqn (1). The permeability (relative to Cs+) of Li+, K+, Rb+, NMDG+ and TEA+ were estimated to be 1.71, 1.26, 1.16, 0.24 and 0.13, respectively (Table 1). It is interesting that, despite the relatively weak discrimination between the alkali cations, the permeability of P2Xcilia receptor channels is greatest for Li+ and decreases with atomic number (Li+ > Na+ > K+ > Rb+ > Cs+), consistent with a site of high electrostatic field strength in the permeation pathway (Eisenman & Horn, 1983). The TEA+ permeability ratio reported here (PTEA/PNa, 0.1) is similar (within a factor of two) to that of the P2X receptor channel of rat parasympathetic ganglia (PTEA/PNa, < 0.1; Liu & Adams, 2001), and to that of rat P2X2 receptor channel (PTEA/PNa, 0.04; Evans et al. 1996; Ding & Sachs, 1999). The measurable permeability to TEA+ indicates that the diameter of the narrowest part of the pore of P2Xcilia receptor channels is somewhat greater than 0.66 nm (i.e. the mean diameter of TEA+). A similar estimate for the minimum pore diameter (< 0.7 nm) was obtained for the native P2X receptor channel in neurones from rat parasympathetic ganglia (Liu & Adams, 2001).
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A, net ATP-activated current responses to voltage ramps measured in extracellular solutions containing 151 mM of the indicated cation. Five to 10 consecutive current traces were averaged in each experimental condition. B, representative net ATP-activated current responses to voltage ramps measured in extracellular solutions containing the indicated monovalent cation. The currents were assembled from different cells. Calibration bar: 2 pA for NMDG+, Cs+ and TEA+, 1 pA for K+ and Li+, and 4 pA for Rb+.
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P2Xcilia receptor channels are 9-fold more permeable to Ca2+ than to Cs+
P2Xcilia receptor channel currents are strongly attenuated by extracellular divalent cations (Korngreen et al. 1998). This is demonstrated in Fig. 5A, which shows a continuous whole-cell current trace recorded at a holding potential of –40 mV, in the standard CsCl solutions. The inward current activated by ATP (200 μM) was completely inhibited by replacing the extracellular 151 mM CsCl solution by a solution containing 136 mM CsCl and 10 mM CaCl2. Switching back to the 151 mM CsCl solution then restored the current. The permeability of the channel to Ca2+ was therefore estimated by measuring the effects of 0.1–2 mM extracellular Ca2+ on the reversal potential of the ATP-activated current. To this end, TEA+ was used as the major extracellular cation, because the permeability of P2Xcilia receptor channels to TEA+ is 10-fold lower than to Cs+ (Table 1). The ATP-activated (difference) currents for one such experiment are shown in Fig. 5B. The reversal potential in 0.1, 0.3, 1.0 and 2.0 mM extracellular Ca2+ was –53, –49, –40 and –33 mV, respectively. When the extracellular solution contained a highly permeable cation (Cs+) in place of TEA+ (Fig. 5B, inset), Ca2+ (0.1–2 mM) did not shift the reversal potential, indicating that the observed Ca2+-dependent shifts in reversal potential are not due to surface charge effects. Figure 5C summarizes the dependence of the reversal potential on the concentration of extracellular Ca2+, determined in experiments of the type shown in Fig. 5B. A permeability ratio for PCa/PCs of 9: 1 was estimated by fitting the reversal potential data with eqn (3) (continuous line in Fig. 5C). Predicted reversal potentials assuming permeability ratios for PCa/PCs of 15: 1 and 6: 1 are also shown for comparison (dashed lines). The results shown in Fig. 5C clearly indicate that the conductance activated by ATP is more permeable to Ca2+ than to Cs+, and suggest that Ca2+ permeates the ATP-activated conductance even better than Ba2+, given that PBa/PCs was reported to be 4: 1 (Korngreen et al. 1998). To confirm this point, the relative permeability of Ca2+ and Ba2+ was compared in the same cell (Fig. 5D). Replacing Ba2+ (1 mM) by Ca2+ (1 mM) shifted the reversal potential from –44 to –36 mV. On average, the reversal potential in 1 mM Ca2+ and in 1 mM Ba2+ was –35.8 ± 1.4 and –42.5 ± 1.2 mV, respectively (n= 5), confirming that P2Xcilia receptor channels are more permeable to Ca2+ than to Ba2+.
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A, continuous whole-cell current trace recorded at a holding potential of –40 mV, from a cell exposed to ATP (200 μM) for the duration indicated by the top bar. The extracellular solutions contained either 1.58 mM CaCl2 and 151 mM CsCl or 10 mM CaCl2 and 136 mM CsCl (lower bar). The extracellular solutions also contained (mM): Tes 5, D-glucose 10 and DAP 1. The cell was primed with ATP before the start of the experiment. B, mean whole-cell currents activated by ATP (200 μM) in response to voltage ramps in extracellular solutions containing 0.1, 0.3, 1 or 2 mM CaCl2, as indicated by each current trace. The extracellular solution also contained (mM): Tes 5, D-glucose 10, DAP 1 and TEA-Cl (or CsCl in the inset) at either 151, 151, 149, or 147, respectively. Standard CsCl intracellular solution. Five to 10 consecutive current traces were averaged in each experimental condition. C, mean (±S.E.M.) reversal potentials of the current activated by ATP in extracellular solutions containing different concentrations of CaCl2 (). Each point is the average of 20–28 experiments of the type shown in A. The relative permeability of Ca2+ and Cs+ (PCa/PCs) was estimated from eqn (3) to be 9: 1 (continuous line). Permeability ratios of 15: 1 and 6: 1 are also shown (dashed lines). D, net ATP-activated current responses to voltage ramps measured in extracellular TEA-Cl solution containing either 1 mM CaCl2 or 1 mM BaCl2. Both extracellular solutions contained in addition (mM): TEA-Cl 149, Tes 5, D-glucose 10 and DAP 1; pH was adjusted to 7.4 with TEA-OH.
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P2Xcilia receptor channels share properties in common with P2X7 receptor channels
P2Xcilia receptor channels display properties that are commonly attributed to P2X7 receptor channels including: low sensitivity to ATP, significant attenuation by physiological concentrations of extracellular divalent cations, and modulation by extracellular Na+ (Korngreen et al. 1998; Ma et al. 1999). As with P2X7 receptor channels, P2Xcilia receptor channels are also more sensitive to BzATP than to ATP (the EC50 for BzATP and for ATP in a CsCl extracellular solution containing 1 mM CaCl2 was 100 μM and 530 μM, respectively). However, a greater sensitivity to BzATP is not a property unique to P2X7 receptor channels (Bianchi et al. 1999). Could P2Xcilia receptor channels be composed exclusively of P2X7 receptor subunits To further examine this possibility we measured the effects of known P2X7 receptor channel inhibitors on the changes in [Ca2+]i induced by BzATP in explants of rabbit airway epithelium.
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Average time course of changes in [Ca2+]i in ciliated cells induced by BzATP (100 μM) in Ringer solution (), in low Na+-containing Ringer solution (), and in low Na+-containing Ringer solution plus 10 μM BBG (&;). Reducing the Na+ concentration in the Ringer solution from 150 to 10 mM (replaced by NMDG+) did not influenced the basal [Ca2+]i or the initial fast rise in [Ca2+]i, while the sustained rise in [Ca2+]i was dramatically increased. The strong elevation in [Ca2+]i in low Na+-containing Ringer solution induced by BzATP was largely abolished by 10-min preincubation of the explants with 10 μM BBG. Each data point is the average of eight to nine experiments from four rabbits. The data points are at 10-s intervals, and every fifth S.E.M. is shown. Each experiment was performed on a single ciliated cell.
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P2Xcilia receptor channels also share properties in common with P2X4 receptor channels
P2X7 receptor channels are inhibited by 1 μM Zn2+ (Virginio et al. 1997). In contrast, P2Xcilia receptor channels were found to be augmented by extracellular Zn2+ (1 μM), as demonstrated in Fig. 7A. On average, Zn2+ augmented the P2Xcilia receptor channel currents 1.5-fold (Fig. 7B). This finding prompted us to investigate the possibility that P2Xcilia receptor channels share properties in common with the P2X4 receptor channel, which is also augmented by Zn2+ (Soto et al. 1996; North, 2002). Therefore, airway ciliated cells were exposed to ivermectin (IVM), a semisynthetic derivative of the natural fermentation products of Streptomyces avermitilis, which augments the current of P2X4 but not of P2X7 receptor channels (Khakh et al. 1999b). Figure 7C shows current traces recorded from a ciliated cell prior to (left-hand trace) and after exposing the cell to 10 μM IVM for 5 min (right-hand trace). We found, that IVM increased the current of P2Xcilia receptor channels in all tested cells (Fig. 7D), raising the possibility that P2Xcilia receptor channels contain both P2X4 and P2X7 subunits.
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A, continuous whole-cell current trace recorded at a holding potential of –40 mV, from a ciliated cell exposed to ATP (200 μM) for the duration indicated by the top bar. The addition of 1 μM Zn2+ to the extracellular solution (indicated by lower bar) reversibly increased the current. B, mean (±S.E.M.) ATP-activated current under control conditions (left) and in the presence of 1 μM Zn2+ (right) averaged from five cells. C, whole-cell current trace recorded at a holding potential of –40 mV, from a ciliated cell exposed to ATP (200 μM) before (left) and after (right) 5-min exposure to 10 μM IVM. D, mean (±S.E.M.) ATP-activated current under control conditions (left) and after 5 min exposure to 10 μM IVM (right) averaged from six cells. Statistical significance in B and D was determined with a paired Students t test with P < 0.05. Standard intracellular and extracellular solutions.
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Airway ciliated cells display P2X4 immunoreactivity
Immunohistochemical techniques were used to test the hypothesis that P2Xcilia receptor channels contain both P2X4 and P2X7 subunits. Robust immunohistochemical labelling localized primarily to the basal portion of the cilia was detected with the anti-P2X4 receptor antibody in rabbit airway epithelium (Fig. 8A). The specificity of the immunoreaction was verified by absorbing the primary antibody with an excess of the homologous peptide antigen, followed by the second antibody (Fig. 8B). In contrast to the pronounced labelling with anti-P2X4 antibodies, no conclusive specific labelling was detected with either of two anti-P2X7 receptor antibodies.
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A, immunolocalization of P2X4 receptors in rabbit airway epithelium. P2X4 immunostaining is evident in the proximal half of the cilia. Calibration bar, 10 μm. B, P2X4 immunostaining of the cilia is absent when the primary antibody is blocked by the antigen. GC, goblet cell; BM, basal membrane; Ci, cilia.
Discussion
The aim of the present study was to further characterize the P2X receptor channel expressed in airway ciliated cells. We have shown that in contrast to homomeric mammalian P2X2, P2X4 and P2X7 receptor channels, the gradual increase in membrane conductance observed with P2Xcilia receptor channels during long exposures to ATP is not associated with a time-dependent change in ion selectivity (Fig. 1). This made it possible to conduct reliable selectivity measurements, which indicate that Na+ both inhibits and permeates P2Xcilia receptor channels (Figs 2 and 3), and that the rank order of relative permeability for the alkali cations follows an Eisenman series XI for a site of high field strength (Fig. 4). In addition, we have shown that P2Xcilia receptor channels are highly permeable to Ca2+ (Fig. 5), consistent with their proposed role in regulating cilia activity (Ma et al. 1999; Silberberg et al. 2001). Finally, results have been presented which suggest that the P2Xcilia receptor channel is either an unusual variant of P2X4 or a heteromeric channel containing both P2X4 and P2X7 subunits.
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Priming
An intriguing property of P2Xcilia receptor channels is the time-dependent increase in current (priming) during the first long exposure to ATP (Korngreen et al. 1998; Fig. 1). The mechanism by which ATP primes the P2Xcilia receptor channels is not known. Nevertheless, priming is unlikely to be mediated by the P2Y pathway because priming persists when the G proteins are inhibited by GDP-S (Korngreen et al. 1998). Also, ambient release of ATP is not likely to play a significant role in priming because the total concentration of nucleotides released from airway epithelial cells is less than 200 nM, of which ATP is a small fraction (Lazarowski et al. 2004), whereas in dissociated airway ciliated cells, P2Xcilia receptor channels are relatively insensitive to ATP (EC50 is >200 μM in an extracellular solution containing 1.5 mM CaCl2; Korngreen et al. 1998). Moreover, all the experiments were performed with continuous perfusion of ATP to circumvent the possible effects of ambient ATP and/or ectoATPases. Priming appears to be distinct from the mechanism underlying current growth reported for P2X7 receptor channels. Repeated or prolonged activation of P2X7 receptor channels, in low extracellular divalent cation solutions (but not in the presence of physiological concentrations of divalent cations), leads to an increase in membrane permeability to large cations such as NMDG+ (Surprenant et al. 1996; Jiang et al. 2005). In contrast, priming of P2Xcilia receptor channels is observed at physiological levels of extracellular divalent cations, and the permeability of P2Xcilia receptor channels to NMDG+ does not change throughout the priming process (Fig. 1B and C). Fujiwara & Kubo (2004) have reported that changes in ion selectivity of P2X2 receptor channels are seen only when channel density is high. It is not known whether a higher density of P2Xcilia receptor channels would lead to changes in ion selectivity during long exposures to ATP. Nonetheless, it is clear that the native density of channels is sufficient to induce current growth which is not accompanied by increased permeability to large cations (Fig. 1). It was recently shown that the ATP-induced increase in permeability to NMDG+ and to the fluorescent dye YO-PRO-1 in P2X7-expressing cells arises from two separate permeation pathways, and they proposed that dilatation of the P2X7 receptor channel gives rise to the increase in permeability to NMDG+ (Jiang et al. 2005). A similar mechanism cannot account for the priming of P2Xcilia receptor channels because the relative permeability of NMDG+ and Cs+ is the same before and after priming. Thus, priming could result either from the recruitment of silent P2Xcilia receptor channels, from a progressive increase in sensitivity to ATP, a mechanism that may contribute to current growth of P2X7 receptor channels (Hibell et al. 2000), or from a mode shift of the channels to a conformation that supports high open probabilities. In either case, priming provides an additional dimension to the regulation of P2Xcilia receptor channel activity, and thus may have an important physiological function.
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Modulation by Na+
Extracellular Na+ inhibits P2Xcilia receptor channel activity (Ma et al. 1999; Silberberg et al. 2001). In the present study it was found that Na+ also permeates the channel (Fig. 2), which led us to investigate whether Na+ inhibits the channel from the extracellular or intracellular side of the membrane. Inhibition by 30 mM Na+ was significantly less when Na+ was applied from the inside, suggesting that the binding site for Na+ is more readily accessible from the extracellular side (Fig. 3). Furthermore, inhibition by Na+ is insensitive to voltage indicating that the Na+-binding site is not within the electric field. In N-type calcium channels, extracellular Na+ inhibits the channels by competing with Ca2+ for a binding site within the permeation pathway, but outside the electric field (Polo-Parada & Korn, 1997). It is unlikely that a similar mechanism of block of the ion permeation pathway also accounts for the effects of Na+ on the P2Xcilia receptor channel because Na+ inhibition can be overcome by raising the concentration of ATP (Ma et al. 1999). It is more likely that Na+ either directly reduces the affinity of the P2Xcilia receptor channel to ATP or acts as an allosteric modulator of channel gating. It is therefore tempting to predict that analogues of ATP with significantly greater efficacy could help improve mucociliary clearance in chronic obstruction airway disorders.
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Permeation properties of the pore
P2Xcilia receptor channels discriminate poorly between the alkali cations and are permeable to large cations such as TEA+ and NMDG+ (Fig. 4 and Table 1). These findings suggest that the selectivity filter of P2Xcilia receptor channels may not be as well defined in comparison to other more selective channels such as the voltage-dependent Na+, K+ and Ca2+ channels. It is surprising that, despite the relatively poor discrimination between the alkali cations, the permeability of P2Xcilia receptor channels is greatest for Li+ and decreases with atomic number (Li+ > Na+ > K+ > Rb+ > Cs+), consistent with a site of high field strength in the permeation pathway (Eisenman & Horn, 1983). The slightly greater relative permeability to NMDG+ in comparison to TEA+ is inconsistent with their respective mean diameters: 0.73 nm (Artigas & Gadsby, 2004) and 0.66 nm (Liu & Adams, 2001). This is probably due to the elongated shape of NMDG+. The dimensions of NMDG+ are 0.50 x 0.64 x 1.20 nm (Burnashev et al. 1996), and hence NMDG+ might fit through the pore better than TEA+. Judging from the relative permeability to TEA+, the diameter of the narrow part of the pore of the P2Xcilia receptor channel is slightly greater than 0.66 nm. Similarly, the minimum pore diameter of rat P2X1, P2X2 and undilated P2X7 receptor channels has been estimated to be less than 0.8 nm (Evans et al. 1996; Surprenant et al. 1996). It is noteworthy that human P2X5 receptor channels exhibit a time-independent high permeability to NMDG+ (PNMDG/PNa, 0.4), suggesting that the pore of these channels might be larger than that of other P2X receptor channels (Bo et al. 2003a).
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Reversal potential measurements were used to estimate PCa/PNa in rat P2X1, P2X2, P2X3 and P2X4 receptor channels and found to be 4 (Evans et al. 1996), 2.5 (Virginio et al. 1998), 1.2 (Virginio et al. 1998), and 4.2 (Buell et al. 1996; Soto et al. 1996), respectively. Although it is difficult to make comparisons between studies due to different experimental conditions and assumptions, it is interesting to note that direct measurements of fractional Ca2+ currents show that in P2X1, P2X2, P2X3 and P2X4 receptor channels approximately 12%, 6%, 3% and 11%, respectively, of the current is carried by Ca2+ (Egan & Khakh, 2004). Thus, in P2X receptor channels there appears to be a correlation between relative permeability to Ca2+ and the fractional Ca2+ current. From this it can be inferred that more than 10% of the current through P2Xcilia receptor channels is carried by Ca2+ under physiological conditions, consistent with their proposed role in regulating cilia activity (Ma et al. 1999; Silberberg et al. 2001).
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Possible subunit composition of P2Xcilia receptor channels
Relating the diverse physiological responses mediated by native P2X receptor channels to the cloned P2X receptor subtypes is a major challenge. ATP-activated currents with characteristics that do not coincide with any of the cloned channels could arise from splice variations, post translational modification, regulation by auxiliary proteins, or the expression of a mixed population of channels. The ATP-activated currents in airway ciliated cells have properties that are unique to P2X4 receptor channels and properties that are unique to P2X7 receptor channels (see below). Nevertheless, it is unlikely that P2Xcilia receptor channel currents arise from a mixed population of P2X4 and P2X7 homomeric receptor channels because these channels have very different sensitivities to ATP in the presence of physiological concentrations of divalent cations (North, 2002), yet the dose–response relationship for activation of P2Xcilia receptor currents by ATP is well described by a simple Hill equation (Korngreen et al. 1998). Furthermore, P2Xcilia receptor currents can be fully inhibited by extracellular Na+, whereas P2X4 receptor channels are largely insensitive to variations in the extracellular concentration of Na+. It might be that during the priming process the population of P2X4 receptor channels are desensitized by the relatively high concentrations of ATP (200 μM) and that these channels can be rescued from the desensitized state by either Zn2+ or IVM. However, this is unlikely because 1 μM Zn2+ is unable to activate desensitized rat P2X4 receptor channels (S.D. Silberberg, unpublished observations). Based on experiments in cell lines derived from airway epithelium it was recently proposed that the P2Xcilia receptor channel is a hetero-multimer of P2X4 and P2X6 subunits (P2X4+6; Liang et al. 2005). However, this is unlikely to be the case because P2X4+6 receptor channels appear to be relatively insensitive to extracellular Na+ (Le et al. 1998), and because P2X4+6 receptor channels have a sensitivity to ATP similar to that of homomeric P2X4 receptor channels (EC5010 μM; Le et al. 1998), whereas P2Xcilia receptor channels have a low sensitivity to ATP (Korngreen et al. 1998).
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P2Xcilia receptor channels and homomeric P2X7 receptor channels share a low sensitivity to ATP (EC50, 200 μM) at physiological concentrations of divalent cations (North, 2002). In addition, calcium influx induced by ATP in human lymphocytes is inhibited by extracellular Na+ (Pizzo et al. 1991; Wiley et al. 1992), extracellular Na+ reduces the potency of BzATP in HEK293 cells expressing the human P2X7 receptor channel (Michel et al. 1999), and Na+ prevents the changes in NMDG+ permeability induced by BzATP in HEK293 cells expressing the rat P2X7 receptor channel (Jiang et al. 2005). All these results suggest that P2X7 receptor channels are also directly modulated by extracellular Na+. Furthermore, P2Xcilia and P2X7 receptor channels are both significantly attenuated by physiological concentrations of extracellular divalent cations (Surprenant et al. 1996; Korngreen et al. 1998; Fig. 5) and are inhibited by BBG and by KN-62 (Humphreys et al. 1998; Jiang et al. 2000; Fig. 6). These properties are not shared by P2X4 receptor channels. On the other hand, P2X7 receptor channels are insensitive to IVM and are inhibited by 1 μM Zn2+ (Virginio et al. 1997), whereas both IVM and Zn2+ augment P2Xcilia receptor channel currents (Fig. 7). Of the non-desensitizing cloned P2X receptors, both P2X2 and P2X4 are augmented by Zn2+; however, only P2X4 is sensitive to IVM, indicating a special relationship between P2Xcilia and P2X4 receptor channels. Could P2Xcilia receptor channels be a hetero-oligomeric assembly containing P2X4 and P2X7 receptor subunits
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Co-immunoprecipitation assays performed on HEK293 cells co-expressing different P2X subunits suggest that P2X7 does not form channels with other P2X receptor channel subunits (Torres et al. 1999). However, it is conceivable that such interactions do occur in native tissue. P2X7 receptor channel protein was strongly detected in mouse lung (Sim et al. 2004), whereas P2X4 protein was identified by immunoreactivity in rat bronchus epithelium (Bo et al. 2003b). Furthermore, both P2X4 and P2X7 mRNA were detected by RT-PCR in epithelial cells freshly isolated from rat trachea (Marino et al. 1999), and P2X4 and P2X7 protein was detected by Western blot in pig airway epithelium (E. Ben-Tal Cohen, S. Weil and S.D. Silberberg, unpublished observations). None of these studies, however, specifically targeted ciliated cells nor did they demonstrate colocalization of these subunits. Thus, we used specific antibodies in combination with fluorescence microscopy to try to determine whether P2Xcilia receptor channels are composed of P2X4 and P2X7 subunits. The results for P2X4 are compelling. In intact trachea, specific binding of the anti-P2X4 antibody was detected in the proximal half of the cilia (Fig. 8). In contrast, we were unable to reliably detect P2X7 immunoreactivity using two different antibodies (see Methods). This finding does not exclude the possibility that P2X7 is expressed by these cells, as P2X7 receptor channels interact with several other proteins which could mask the binding sites for the antibodies (Kim et al. 2001). Taken together, these results suggest that P2Xcilia receptor channels contain at least one P2X4 subunit. Whether the inhibition of P2Xcilia receptor channels by extracellular Na+, extracellular divalent cations, KN-62 and BBG, as well as their low sensitivity to ATP arise from the contribution of P2X7 subunits or from modification/modulation of P2X4 subunits in a homomeric structure remains to be determined.
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The targeting of P2Xcilia receptor channels to the proximal half of the cilia suggests that they are poised to modulate cilia function. The restricted volume of the cilium combined with a relatively high permeability to Ca2+ for P2Xcilia receptor channels suggest that even at low open probabilities (i.e. high extracellular Na+ concentration) these channels could influence cilia activity. In view of the location of the channels, it is tempting to speculate that in addition to providing a pathway for ions, these proteins may have other functions in regulating cilia activity, analogous to the role of calcium channels in excitation–contraction coupling in skeletal muscle.
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