Biophysical and pharmacological properties of the voltage-gated potassium current of human pancreatic cells
1 Department of Ion Channels, Merck Research Laboratories, PO Box 2000, Rahway, NJ 07065, USA
2 Farmacología, Departamento de Medicina, Facultad de Medicina, Universidad de Oviedo, Julián Clavería 6, ES-33006, Oviedo, Spain
3 BetaGene Inc., 2600 North Stemmons Freeway, Suite 1, Dallas, TX 75207, USA
Abstract
Voltage-gated potassium (Kv) currents of human pancreatic islet cells were studied by whole-cell patch clamp recording. On average, 75% of the cells tested were identified as cells by single cell, post-recording RT-PCR for insulin mRNA. In most cells, the dominant Kv current was a delayed rectifier. The delayed rectifier activated at potentials above –20 mV and had a V for activation of –5.3 mV. Onset of inactivation was slow for a major component ( = 3.2 s at +20 mV) observed in all cells; a smaller component ( = 0.30 s) with an amplitude of 25% was seen in some cells. Recovery from inactivation had a of 2.5 s at –80 mV and steady-state inactivation had a V of –39 mV. In 12% of cells (21/182) a low-threshold, transient Kv current (A-current) was present. The A-current activated at membrane potentials above –40 mV, inactivated with a time constant of 18.5 ms at –20 mV, and had a V for steady-state inactivation of –52 mV. TEA inhibited total Kv current with an IC50 = 0.54 mM and PAC, a disubstituted cyclohexyl Kv channel inhibitor, inhibited with an IC50 = 0.57 μM. The total Kv current was insensitive to margatoxin (100 nM), agitoxin-2 (50 nM), kaliotoxin (50 nM) and ShK (50 nM). Hanatoxin (100 nM) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose-dependent insulin secretion.
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Introduction
Secretion of insulin by the pancreatic cell in response to glucose or other fuel secretagogues is tightly coupled to the membrane potential of the cell. Glucose triggers closure of the cell KATP channel, leading to membrane depolarization, calcium entry and secretion (Ashcroft & Rorsman, 1989). In the intact islet, glucose triggers synchronized bursts of action potentials that lead to cytosolic calcium oscillations (Santos et al. 1991). This oscillatory bursting behaviour appears to be regulated by the interplay of several types of ion channels (Bertram et al. 1995; Dukes & Philipson, 1996; Roe et al. 1996; Mears et al. 1997; Gopel et al. 1999; Eberhardson et al. 2000; Kanno et al. 2002). Of these, voltage-gated K+ (Kv) channels clearly play an important role in regulating cell excitability (Philipson, 1999).
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Kv channels serve numerous roles in excitable cells. Kv channels are involved in setting the resting potential, repolarizing after an action potential, and determining the interspike interval (Hille, 2001). One class of Kv channels, the delayed rectifier, is thought to be the dominant Kv current of cells and plays a key role in action potential repolarization (Smith et al. 1990). Inhibition of the cell delayed rectifier K+ current would be expected to prolong action potentials and enhance glucose-stimulated insulin secretion. Such a therapeutic strategy would be expected to pose a lower risk for hypoglycaemic events compared to sulphonylurea KATP channel blockers. For this reason, the cell delayed rectifier K+ current has attracted much attention as a potential target for the treatment of type 2 diabetes (Roe et al. 1996; MacDonald & Wheeler, 2003).
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The functional unit of a Kv channel is a tetramer of principal pore-forming subunits. Forty Kv channel genes have been identified in humans and additional complexity can be produced by heteromultimerization, alternative splicing, and additional subunits and interacting proteins (Hille, 2001; Yellen, 2002; Yu & Catterall, 2004). The molecular identities of the Kv channels in cells are unknown. Numerous Kv subunits have been detected in islets and/or cells (MacDonald & Wheeler, 2003; Yan et al. 2004). Of these, the delayed rectifier, Kv2.1, has been detected in many studies (Roe et al. 1996; MacDonald et al. 2001, 2002; MacDonald & Wheeler, 2003; Yan et al. 2004). Adenoviral-mediated expression of a dominant negative C-truncated form of Kv2.1 reduces the delayed rectifier Kv current in rat cells by 60–70% and augments glucose-stimulated insulin secretion (MacDonald et al. 2001). However, the exact molecular composition of Kv channels in pancreatic cells remains unclear.
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The Kv currents of mouse cells have been studied in the most detail. The major Kv current in mouse cells appears to be a delayed rectifier that activates near –20 mV, inactivates slowly ( = 4.5 s at +20 mV) and is blocked by TEA with a KD of 1.4 mM (Rorsman & Trube, 1986; Bokvist et al. 1990; Smith et al. 1990). At least four distinct channels have been observed in single channel recordings. The most frequently observed channel has a conductance of 8–10 pS and is thought to play a key role in action potential repolarization (Smith et al. 1990). In addition to the delayed rectifier, mouse and rat cells appear to possess a small, transient inactivating current (Gopel et al. 2000; MacDonald et al. 2001).
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In contrast to rodent cells, little is known about the properties of the Kv currents of human cells. A delayed rectifier Kv current in human cells has been previously described (Kelly et al. 1991). Given the critical contribution of Kv channels to the control of glucose-dependent insulin secretion, it is important to characterize and determine the molecular composition of this current. In this study, we describe the biophysical and pharmacological properties of Kv currents in identified human cells. We find evidence for the existence of at least two distinct types of Kv currents, a delayed rectifier and a transient A-current.
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Methods
Materials
Cell culture reagents were purchased from Gibco/Invitrogen and chemicals were from Sigma unless otherwise noted. Margatoxin (MgTX), agitoxin-2 (AgTX2) and kaliotoxin (KTX) were provided by M. Garcia (Department of Ion Channels, Merck Research Laboratories) and prepared as previously described (Koch et al. 1997; Koschak et al. 1998). ShK was obtained from Peptides International (Louisville, KY). Hanatoxin was provided courtesy of K. Swartz (NINDS, Bethesda, MD, USA). 4-Phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1- yl]cyclohexanone (PAC) was synthesized by the Medicinal Chemistry Department of Merck Research Laboratories as previously described (Schmalhofer et al. 2002). RNase-free DNase I and RNase inhibitor SUPERaseIn were purchased from Ambion (Austin, TX, USA). The OneStep RT-PCR Kit was purchased from Qiagen (Valencia, CA, USA).
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Donor tissue, islet dissociation and cell culture
Human pancreatic islets were obtained from three sources: BetaGene, Inc. (Dallas, TX, USA), the UCLA/VA Islet Isolation Laboratory (Los Angeles, CA, USA) and the JDRF Human Islet Distribution Program at the University of Alberta (Edmonton, Alberta, Canada).
Islets from one donor were supplied by Betagene, Inc. (donor 1 of Fig. 9). These islets were provided to BetaGene by Dr C. Ricordi's group at the islet isolation facility of the Diabetes Research Institute, University of Miami. Islets had cold ischaemia times > 8 h, and were cultured at ambient temperatures for 1–2 days subsequent to isolation and before shipment to BetaGene. Upon receipt at BetaGene, four assessments of islet quality were made: viability, purity, insulin content and glucose-responsive insulin secretion (Clark et al. 1994, 1997). For the islets used in this study, viability was > 95% and purity was 90%. The insulin content was 1.7 μg/1000 equivalent islet number (EIN), with 99% mature human insulin. In general, islets were not optimally glucose responsive upon receipt, but usually recovered after several days of culture. To facilitate the maintenance of islets and prevent loss with medium changes, the islets were encapsulated in 1.5% alginate bands in medium. The islets were cultured in a custom manufactured medium (JRH Biosciences, Lenexa, KS, USA), optimized for human islets and neuroendocrine cells. The glucose concentration was 7.8 mM. The medium formulation was based on a 1: 1 mixture of M199E and F12 media, supplemented with CuSO4 and an ascorbate to maintain amidating capacity of cells, and an equimolar mixture of ethanolamine–phosphoethanolamine (Clark & Chick, 1990). The medium was supplemented with 2% gamma-irradiated fetal bovine serum (FBS) (JRH Biosciences) and 100 μg ml–1 of streptomycin. Islets were cultured at BetaGene for 2 weeks prior to shipment to Merck. For shipment, islets were removed from the alginate beads by EDTA treatment, pelleted, washed and suspended in 50 ml of medium in a sterile 50 ml tube and shipped at ambient temperature.
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Bar graphs of the properties of Kv current across donors. Bars represent means ± S.E.M. The number of cells was 15, 46, 10, 29, 16, 20, 25 and 17 for donors 1–8, respectively. A, peak current density at +40 mV in pA pF–1. B, percentage of cells that express a detectable low threshold, transient current (A-current). C, voltage of half-activation (activation V) measured from fits of the Boltzmann equation to the tail current amplitude versus command voltage. D, activation time constant () at +40 mV. E, deactivation time constant () measured from single exponential fits to tail currents at –40 mV. F, inactivation time constant () estimated from the decay of current during 10 s steps to +20 mV. In cases where the sum of exponentials were needed to describe the decay, the of the major component was taken.
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Islets from four donors (donors 2–5 in Fig. 9) were obtained from Dr Jonathan Lakey and the JDRF Human Islet Distribution Program at the University of Alberta. The islets from the Edmonton group were isolated as previously described (Shapiro et al. 2000) using procedures approved by the University of Alberta Ethics Committee.
Islets from three donors (donors 6–8 in Fig. 9) were supplied by Dr Yoko Mullen of the UCLA/VA Islet Isolation Laboratory. Collection and isolation of islets by the UCLA laboratory were performed as previously described (Kenmochi et al. 2000) and were approved by the UCLA Institutional Review Boards for the Protection of Human Research Subjects.
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Upon arrival, islets were purified by hand-picking. Some of the islets were used to test for glucose-responsive insulin secretion. In general, about half of the islet preparations showed enhanced insulin secretion in response to elevated glucose. The remainder of the purified islets was dissociated into single cells for electrophysiology. Briefly, islets were placed in Ca2+/Mg2+-free phosphate-buffered saline (PBS) containing trypsin and incubated for 5–10 min, followed by gentle trituration into single cells. The enzyme was quenched by placing the cell suspension in culture medium (RPMI 1640 supplemented with 10% heat-inactivated FBS and penicillin–streptomycin). Following centrifugation, the cells were resuspended in culture medium and plated on poly L-lysine coated glass chips. Cells were used for electrophysiology within 72 h of plating.
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Electrophysiology
Membrane currents were recorded using standard whole-cell voltage clamp techniques (Hamill et al. 1981) with a HEKA EPC9 amplifier (HEKA Elektronik, Lambrecht, Germany) or Dagan 3900A amplifier (Dagan Corporation, Minneapolis, MN, USA). Microelectrodes fabricated from borosilicate glass were coated with Sylgard (Dow Corning, Midland, MI, USA) or beeswax and fire-polished. Electrode resistances were generally 2–4 M when filled with the standard internal saline. The reference electrode was a silver–silver chloride wire within an agar bridge (4% agar in 200 mM KCl). Voltages given in the figures have not been corrected for the small liquid junction potential between the external and internal solutions (2 mV). All experiments were performed at room temperature (22–25°C).
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Generation of analog voltage commands and digitization of membrane currents were controlled with PULSE software (HEKA Elektronik) and an ITC-16 computer interface (Instrutech, Port Washington, NY, USA). Currents were digitized at 5 kHz and digitally filtered at 2 kHz. Digital subtraction of leakage and capacitive currents was performed by the P/n procedure (n = –4 or –5). Voltage steps were applied every 5–20 s from a holding potential of –80 mV.
The standard pipette solution was (mM): 120 KCl, 20 KF, 10 EGTA, 10 Hepes, 2 mM MgATP, pH adjusted to 7.2 with KOH. The standard external solution consisted of (mM): 160 NaCl, 4.5 KCl, 1.8 CaCl2, 1 MgCl2, 10 Hepes, 3 glucose, pH 7.2 with NaOH. Solutions were applied to cells by bath perfusion via gravity. The experimental chamber volume was 0.2 ml and the perfusion rate was 1–2 ml min–1. Flow of solution through the chamber was maintained at all times. Bovine serum albumin (BSA; 0.1% w:v) was added to solutions containing peptides; BSA had no detectable effect on K+ channels. TEA was added directly to the standard extracellular solution from a 1 M stock in water.
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Measurement of current amplitudes was performed with PULSEFIT software (HEKA Elektronik). Fitting of current waveforms or extracted data was performed with PULSEFIT or Igor Pro 4.0 (WaveMetrics, Lake Oswego, OR, USA). The voltage dependence of channel activation and inactivation were fit by the Boltzmann equation:
where Imax is the maximal current, V is the half-activation voltage and k is the voltage dependence of the distribution. In the case of Fig. 3, chord conductance (G) rather than current was analysed. Inhibition of Kv current by pharmacological agents was described by the Hill equation of the form:
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where IC50 is the concentration that produces 50% of the maximal block and n is the apparent Hill coefficient. Experimental values are given as the mean ± S.E.M.
A, membrane current in response to 100 ms voltage steps between –60 mV and +60 mV from a holding potential of –80 mV in 10 mV increments every 5 s. Following the 100 ms command, the membrane potential was stepped to –40 mV for 100 ms. Cell 137 (insulin positive): 7.2 pF. B, plot of chord conductance versus test potential for the cell in A. Conductance was calculated from the equation: G = I/(E – EK), where I is the peak current, E is the test potential and EK is the equilibrium potential for K+ (–90 mV). The continuous line is a fit to the Boltzmann equation. Parameters of the fit are given in the text. C, membrane current in response to voltage steps as described in Fig. 1A. Cell 97: 3.4 pF. D, plot of the membrane currents from C on an expanded y-axis for the –30 mV, –20 mV and –10 mV test potentials. The continuous lines are single exponential fits to the data with time constants of 19 ms (–30 mV), 16 ms (–20 mV), and 13 ms (–10 mV). E, isolation of the transient current by subtraction (details given in the text). Plotted are the currents in response to 100 ms depolarizations to +20 mV following prepulses to –100 mV and –10 mV and the difference currents (–100 mV minus –10 mV). F, plot of the voltage dependence of inactivation measured using the subtraction procedure in E. The continuous line is a fit of the Boltzmann equation to the points. Parameters of the fit are given in the text. Same cell as in C.
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Single-cell RT-PCR
Following recording, the cell contents were collected in the electrode tip by suction and the tip was broken in a PCR tube and stored at –80°C until use. The day of the reaction, the cell contents were thawed by placing reaction solution containing 1 μl RNase-free DNase I (1 U), 1 μl 10x DNase I buffer, and 0.5 μl SUPERaseIn (10 U) and 5 μl distilled H2O in the tube. The total reaction volume was approximately 10 μl. The tube was incubated at 37°C for 30 min to eliminate genomic DNA followed by incubation at 75°C for 5 min to inactivate the DNase I. RT-PCR was performed in the same tube using a Qiagen OneStep RT-PCR Kit as previously described (Yan et al. 2002). Briefly, 40 μl of reaction mix (Sensiscript reverse transcriptase, HotStarTaq polymerase, dNTPs, 5x Qiagen OneStep RT-PCR buffer) and insulin primers (sense primer 5'-CCAGCCGCAGCCTTTGTGA-3', antisense primer 5'-GCTGGTAGAGGGAGCAGAT-3') were added to the tube. The primers were designed from sequences on different exons of the human insulin gene so that PCR products derived from reverse transcribed mRNA and from amplified genomic DNA would be of different size. Only the mRNA-derived product was ever observed, consistent with our previous findings that digestion of genomic DNA is complete with this protocol (Yan et al. 2002). Each reaction (50 μl) contained the DNase I treated RNA of a single cell, 0.6 μM of each of the primers, 400 μM of each of the dNTPs, and 2.0 μl Sensiscript reverse transcriptase–HotStarTaq DNA polymerase enzyme mix. Reverse transcription was allowed to proceed for 30 min at 50°C after which the reaction was heated to 95°C for 15 min to inactivate the reverse transcriptase and activate the HotStarTaq DNA polymerase. The reactions were amplified for 35 cycles followed by a 10 min extension at 72°C. Each cycle consisted of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C. The PCR products were examined on a 2% agarose gel and cells were scored as cells if a 250 bp product corresponding to insulin was present. Positive insulin controls and negative controls, consisting of saline collected from recording bath or ddH2O, were run in parallel with all samples.
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Several attempts were made to perform multiplex PCR for other hormones (e.g. glucagon), Kv subunits or housekeeping genes along with insulin. These experiments generally compromised the robustness and reliability of measuring insulin mRNA. Thus, we routinely performed single-cell PCR for insulin only. A disadvantage of this approach is that it is possible some of the insulin negative cells may in fact be cells but fail to show detectable insulin RNA due to cell collection and/or PCR failure. However, most of the cells for which we performed single-cell PCR for insulin were positive (see Results), suggesting most of the cells we recorded from were cells. Data in the figures from identified cells are denoted in the legends; otherwise the data shown are from unidentified cells.
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Results
Electrophysiological properties of human islet cells
We recorded from a total of 222 human islet cells, collected from eight donors. In response to a 200 ms voltage ramp between –100 mV and +50 mV, most cells displayed large outward currents above 0 mV (Fig. 1A). However, in 12 cells no detectable voltage-gated current was present. These cells are presumably not of endocrine origin and were not studied further. The mean capacitance of all studied cells was 5.7 ± 0.2 pF (n = 210; Fig. 1C).
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A, membrane current in response to a 200 ms voltage ramp between –100 mV and +40 mV is plotted versus voltage. Cell 109 (insulin positive): 8.8 pF. B, plot of membrane capacitance versus current density (pA pF–1) for all cells. Each symbol represents an individual cell (+, unidentified; , insulin positive; , insulin negative). The dashed lines create a window to highlight cells that have current densities between 35 and 400 pA pF–1 and a capacitance greater than 2 pF. C, histogram of cell capacitance for all cells (n = 210). D, histogram of current density for all cells (n = 171). E, histogram of cell capacitance for insulin positive cells only (n = 48). F, histogram of current density for insulin positive cells only (n = 47).
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During the course of these studies we developed and verified a post-recording RT-PCR method to unambiguously identify cells as cells by detection of insulin mRNA. On average, 75% (54 out of 72) of the cells tested were positive for insulin mRNA. Identified cells had a mean capacitance of 7.3 ± 0.4 pF (n = 48; Fig. 1E). Step depolarizations to +40 mV show that the average current density was 188 ± 6 pA pF–1 (n = 171) for all cells (Fig. 1D) and 168 ± 12 pA pF–1 (n = 47) for identified cells (Fig. 1F). Of the 12 cells expressing no measurable voltage-gated current, two were tested by RT-PCR and both tested negative for insulin mRNA supporting the non-endocrine designation of these cells.
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We next sought a way to filter the data so that analysis was primarily restricted to cells. A plot of capacitance versus current density shows identified cells had a current density between 35 and 400 pA pF–1 and a capacitance greater than 2 pF (Fig. 1B, filled circles). The cells that were not positive for insulin by PCR generally fell within this range also, although some clearly did not (Fig. 1B, open circles). However, it is possible that some of the insulin negative cells were indeed cells but failed to show detectable insulin due to technical limitations (see Methods). Thus, we pooled all the data from unidentified cells within this range (Fig. 1B, +) with identified cells for further analysis. Although this approach does not ensure that all of the data comes from cells, our results with single-cell PCR suggest that at least 75% of the cells we recorded from were probably cells. Except for the data in Figs 7 and 8, the major findings of this paper have been confirmed in identified cells.
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A, representative membrane currents in response to 100 ms voltage steps to +20 mV in the absence (control) and the presence of 0.3 μM, 1 μM, 3 μM and 10 μM external PAC. The holding potential was –80 mV and voltage steps were applied every 15 s. Cell 23: 4.5 pF. B, plot of peak current () during the 100 ms step to +20 versus time for the cell in A. Also plotted is the amplitude of the late current () measured at the end of the 100 ms step. The application of PAC is indicated by the bar with the specific concentrations given near the plot. C, plot of the fraction of control peak and late current blocked (Fraction Blocked) versus PAC concentration for the cell in A. The continuous lines are fits of the Hill equation to the data. Parameters of the fit for the peak current are: IC50 = 1.11 μM, n = 0.99 and maximal block = 1.0. Parameters of the fit for the late current are: IC50 = 0.64 μM, n = 1.26 and maximal block = 1.0. D, Plot of fraction blocked versus PAC concentration for all cells along with fits of the Hill equation to the data. The number of determinations are: 8, 11, 9 and 5 for 0.3, 1, 3 and 10 μM PAC, respectively. Parameters of the fit for the peak current are: IC50 = 0.56 μM, n = 1.27 and maximal block = 0.83. Parameters of the fit for the late current are: IC50 = 0. 57 μM, n = 1.67 and maximal block = 0.95.
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A, representative membrane currents in response to 100 ms voltage steps to +20 mV in the absence (control) and the presence of 100 nM hanatoxin. The holding potential was –80 mV. Cell 13: 2.4 pF. B, plot of peak current () during the 100 ms step to +20 versus time for the cell in A. The application of hanatoxin is indicated by the bar. C, plot of the peak current versus voltage in control () and 100 nM hanatoxin () for the cell in A. D, plot of peak current () during the 100 ms step to +20 versus time for a different cell. The application of peptide Kv channel blockers and TEA are indicated by the bars. A second experiment on a different cell was performed and yielded similar results. Cell 35: 3.8 pF.
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An example of typical currents activated in response to step depolarizations is shown in Fig. 2A. Most cells possessed a delayed rectifier current that develops over 50–100 ms and was activated above –20 mV (Fig. 2B). In a subset of cells, in addition to the delayed rectifier, a transient outward current was present at voltages above –30 mV (Fig. 2C and D). An example of currents from a cell expressing more than 400 pA pF–1 is shown in Fig. 2E and F. In this population, 7 out of 15 cells possessed large outward currents that partially inactivated during 100 ms depolarizations above +40 mV. Three cells in this population also displayed fast inward currents greater than 500 pA in amplitude (not shown). Since the hormonal identity of these cells is unclear, these cells were not studied further.
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A, membrane current in response to 100 ms voltage steps between –60 mV and +50 mV from a holding potential of –80 mV in 10 mV increments every 20 s. Following the 100 ms command, the membrane potential was stepped to –40 mV for 20 ms to record tail currents. Cell 105 (insulin positive): 3.5 pF. B, plot of the peak current versus command voltage for the cell in A. C, membrane currents recorded from another cell using the same stimulus protocol as in A. The inset shows the current in response to the step to –30 mV to highlight the small A-current present in this cell. Cell 58: 5.8 pF. D, plot of peak current versus command voltage for the cell in C. E, membrane currents from a cell with current density > 400 pA pF–1 recorded using the same voltage protocol as in A. Cell 79: 3.8 pF. F, plot of the peak current versus command potential for the cell in E.
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Properties of the low threshold, transient outward current
In approximately 12% of cells within the > 2 pF and 35–400 pA pF–1 range (21/182), 100 ms step depolarizations from a holding potential of –80 mV activated a clear low threshold (–40 mV) inactivating outward current. This ‘A-current’ was seen in cells from six out of the eight donors. Except for one donor (donor 1; see below), the A-current was seen in less than 20% of the cells studied from a given donor. In one cell, this current appeared to be dominant (Fig. 3A) and allowed estimation of the voltage dependence of the conductance (Fig. 3B; V = –13.1 mV; k = 14.7 mV, Gmax = 2.52 nS). However, most cells that possessed the low threshold, transient outward current clearly had other currents as well (Fig. 3C). In these cells, the time course of inactivation could be studied only at negative potentials (–30 mV or –20 mV) where the current was present in isolation (Fig. 3D). In these cells, inactivation proceeded with a mean time constant of 19 ms at –20 mV (Table 1).
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To study the voltage dependence of steady-state inactivation, we employed a subtraction procedure to minimize the contribution of other currents. From a holding potential of –80 mV, a 20 ms prepulse to potentials between –110 mV and –10 mV was given followed by a brief (5 ms) step back to the holding potential to allow channel closing. The membrane was then stepped to +20 mV for 100 ms. The currents from the –10 mV prepulse were used to subtract the non-inactivating component of current. As shown in Fig. 3E, this procedure allowed reasonable separation of the transient component of current. The amplitudes of the difference currents are plotted versus the prepulse voltage in Fig. 3F. The voltage dependence of inactivation measured in this way was well described by a Boltzmann distribution with a V of –44 mV and slope factor of 6.3 mV.
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Biophysical properties of the delayed outward current
To study the biophysical properties of the delayed outward current we excluded cells with significant transient current from analysis. The voltage dependence of activation of the delayed rectifier current was measured from the tail current amplitude at –40 mV following 100 ms step depolarizations to various potentials (Fig. 4A). The voltage dependence of the tail current amplitude was well described by a Boltzmann distribution (Fig. 4B). For the cell in Fig. 4, the V was –1.7 mV and the slope factor was 7.3 mV per e-fold change in conductance. The kinetics of activation were measured by fitting an exponential function to the rising phase of the current (Fig. 4C). As expected, the activation proceeded faster with stronger depolarizations, reaching a value of 6.5 ms at +40 mV (Fig. 4D).
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A, membrane current in response to 100 ms voltage steps between –70 mV and +40 mV from a holding potential of –80 mV in 10 mV increments every 5 s. Following the 100 ms step, the membrane potential was stepped to –40 mV for 20 ms. B, plot of tail current amplitude versus test potential for the cell in A. The continuous line is a fit to the Boltzmann equation. Parameters of the fit are given in the text. C, single exponential fits (continuous lines) of currents from A. The time constants are 6.6 ms (+40 mV), 9.6 ms (+20 mV), and 15.1 ms (+10 mV). D, plot of activation time constant versus test potential for the cell in A. E, membrane current in response to a 25 ms voltage step to +40 mV followed by a 30 ms step to between –100 mV and –10 mV in increments of 10 mV from a holding potential of –80 mV. F, plot of tail current amplitude versus repolarization potential for the record in E. Cell 1: 9.0 pF.
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Channel closing upon repolarization, commonly referred to as deactivation, was studied by varying the membrane potential between –10 mV and –100 mV following a 25 ms step depolarization to +40 mV (Fig. 4E). Deactivation was highly voltage dependent. On average, deactivation had a time constant of 7.7 ms at –40 mV. Plotting the tail current amplitude versus repolarization voltage yielded an apparent reversal potential of –82 mV (Fig. 4F). For all cells, the estimated reversal potential was –85 ± 2 mV (n = 13). Such a reversal potential is consistent with a potassium-selective current under these recording conditions (EK = –90 mV; ECl = –9 mV).
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Inactivation of the delayed rectifier K+ current was generally not apparent or very modest with 100 ms step depolarizations (Fig. 5A). To study the onset of inactivation, 10 s depolarizations to +40 mV were given. In general, the current decayed 74 ± 3% (n = 25) over the 10 s step. For the cell shown in Fig. 5, the current decay was best described by the sum of two exponentials with time constants of 0.13 s and 2.58 s (Fig. 5B). In 64% of cells (n = 16), current decay was best described by the sum of two exponentials with average time constants of 0.3 s and 3.3 s. The slower time constant component was 70 ± 3% of the total decay. In the remaining 36% of cells (n = 9), a single exponential with time constant of 2.9 s adequately described inactivation. Recovery from inactivation was studied by first inactivating the current with a 10 s step and varying the time interval at –80 mV before stepping to +40 mV to measure the amount of recovery (Fig. 5C). The time constant of recovery from inactivation was 3.5 s for this cell (Fig. 5D). To study the voltage dependence of steady-state inactivation, 10 s prepulses to potentials between –120 mV and –30 mV were given prior to a step depolarization to +20 mV (Fig. 5E). The voltage dependence of inactivation was well described by a Boltzmann distribution with a V of –46 mV and a slope factor of 7.4 mV (Fig. 5F). Table 1 summarizes the biophysical properties of Kv channels of human cells.
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A, membrane current in response to 100 ms voltage steps between –60 mV and +50 mV from a holding potential of –80 mV in 10 mV increments every 20 s. Following the 100 ms step, the membrane potential was stepped to –40 mV for 20 ms. B, membrane current in response to a 10 s voltage step to +20 mV from a holding potential of –80 mV. The continuous line is a fit of the sum of two exponentials to the current decay with the parameters as shown. C, membrane current in response a voltage protocol designed to measure recovery from inactivation. From a holding potential of –80 mV, the cell was stepped to +20 mV for 10 s (shown at left), followed by a variable interval at –80 mV (ranging from 0.1 s to 31.5 s; not shown) and a 100 ms step to +20 (right) every 90 s. D, plot of relative current amplitude versus recovery duration for the record in C. The continuous line is a single exponential fit to the data with the parameters as shown. E, membrane current in response to a voltage protocol to measure steady-state inactivation. From a holding potential of –80 mV, a 10 s conditioning step to between –120 mV and –30 mV in 10 mV increments was applied (not shown), followed by a short step to –80 mV (10 ms) to deactivate any open channels and a 100 ms step to +20 mV (shown). F, plot of peak current amplitude versus prepulse potential for the record in E. The continuous line is a fit to the Boltzmann equation with parameters as shown. Cell 46: 8.3 pF.
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Pharmacology of the delayed outward current
We next sought to characterize the pharmacology of the delayed rectifier Kv current. As before, we restricted our analysis to cells which had little, if any, transient current. Test compounds were applied while stepping to +40 mV for 100 ms every 15 s. TEA inhibited peak Kv current in a concentration-dependent manner (Fig. 6A and B). For the cell in Fig. 6, TEA inhibited Kv current with an IC50 of 0.74 mM (Fig. 6C). When data from all cells were pooled, the apparent IC50 for TEA block of the delayed rectifier was 0.54 mM (Fig. 6D). Also shown in Fig. 6D are data from PCR-identified cells, which agree well with the pooled data from the entire population.
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A, representative membrane currents in response to 100 ms voltage steps to +20 mV in the absence (control) and the presence of 0.3 mM, 1 mM, 3 mM and 10 mM external TEA. The holding potential was –80 mV and voltage steps were applied every 15 s. Cell 67: 3.2 pF. B, plot of peak current during the 100 ms step to +20 versus time for the cell in A. The application of TEA is indicated by the bar with the specific concentrations given near the plot. C, plot of the fraction of control current blocked (Fraction Blocked) versus TEA concentration for the cell in A. The continuous line is a fit of the Hill equation to the data. Parameters of the fit are: IC50 = 0.74 mM, n = 0.85 and maximal block = 0.99. D, plot of fraction blocked versus TEA concentration for all cells () and PCR-identified cells (). For unidentified cells, the number of determinations are: 3, 13, 44, 7 and 37 for 0.1, 0.3, 1, 3, 10 and 30 mM TEA, respectively. For PCR-identified cells, the number of determinations are 20 and 19 for 1 and 10 mM TEA, respectively. The continuous line is a fit of the Hill equation to the open circles. Parameters of the fit are: IC50 = 0. 54 mM, n = 0.67 and maximal block = 0.95.
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PAC is a disubstituted cyclohexyl analogue which blocks Kv1.3 channels by a mechanism that accelerates channel closing during sustained depolarizations (Schmalhofer et al. 2002). PAC inhibited total current of human cells in a time-dependent manner (Fig. 7A and B). For the cell shown in Fig. 7, the apparent IC50 was 1.11 μM when peak current was measured and 0.64 μM when the current at the end of the 100 ms step was measured (Fig. 7C). When data from all cells are pooled, the apparent IC50 was 0.57 μM (late current; Fig. 7D).
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Hanatoxin is a prototypical gating modifier peptide that was isolated from the venom of the spider Grammostola spatulata based on its ability to inhibit the activity of Kv2.1 channels (Swartz & MacKinnon, 1995). In addition to Kv2.1 channels, hanatoxin inhibits Kv4 channels (IC50 – 100 nM) and, with much lower affinity, other Kv and Ca2+ channels (Swartz & MacKinnon, 1995; Li-Smerin & Swartz, 1998). We tested if the Kv currents of human cells are sensitive to hanatoxin. Application of 100 nM hanatoxin inhibited peak Kv current at +20 mV by 65% (n = 2; Figs 8A and B). Inspection of the current-voltage relation reveals that hanatoxin shifts the voltage dependence of activation of Kv current to more depolarized potentials (Fig. 8C).
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Margatoxin (MgTX), kaliotoxin (KTX), agitoxin-2 (AgTX2) and ShK are inhibitors of Kv1 family channels. Application of 100 nM MgTX, 50 nM KTX, 50 nM AgTX2, or 50 nM ShK had no significant effect on the Kv current of human cells (Fig. 8D). These data are consistent with the lack of detection of Kv1 subunits in human islets by RT-PCR (MacDonald & Wheeler, 2003; Yan et al. 2004). Since ShK also potently blocks Kv3.2 channels, the lack of effect on the Kv current of human cells suggests Kv3.2 does not contribute significantly to the delayed rectifier in these cells, as reported previously (Yan et al. 2005).
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Donor analysis
We next compared the basic electrophysiological properties described above across the different donors. Current density, deactivation time constant and inactivation time constant showed no or very modest differences between donors (Figs 9A, E and F). Interestingly, the V for activation was more positive and the activation time constant slower for donor 2 and donor 3 compared to the others (Fig. 9C and D). One striking difference between donors was the variable presence of the low threshold, transient current (Fig. 9B). In one donor (donor 1) 46% of cells had this current while in others the current was completely absent (donor 3 and donor 5) or in less than 20% of cells (donors 2, 4 and 6–8).
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Discussion
Kv channels of pancreatic cells have received much attention as potential therapeutic targets for type 2 diabetes (Roe et al. 1996; MacDonald & Wheeler, 2003). A clear understanding of the properties of the Kv current of human cells will be needed to realize this aim. In this study, we define the properties of the Kv current of human cells. Prior to this work, human cell Kv current was described in a single report where the delayed rectifier Kv current was described (Kelly et al. 1991). Here we provide a detailed biophysical and pharmacological characterization of the delayed rectifier potassium current of human cells. We also find that a subset of human cells possess a low threshold, transient A-current.
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Properties of delayed rectifier current
The delayed rectifier of human cells has the following basic properties: activation near –20 mV, deactivation of about 8 ms at –40 mV, slow onset and recovery from inactivation (3 s at +20 mV and –80 mV, respectively). These biophysical properties are similar to those of the delayed rectifier found in mouse cells (Rorsman & Trube, 1986; Bokvist et al. 1990; Smith et al. 1990). The current was fully blocked by TEA with an IC50 of 0.54 mM and by PAC with an IC50 of 0.57 μM, and partially blocked by 100 nM hanatoxin.
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Identity of the delayed rectifier current
Several studies have suggested that in rodent cells, Kv2.1 is expressed and contributes to forming the delayed rectifier current (Roe et al. 1996; MacDonald et al. 2001; MacDonald et al. 2002). Indeed, the most frequent Kv channel seen in cell attached patches from mouse cells has a conductance of 8–10 pS (Smith et al. 1990), which is consistent with the conductance of heterologously expressed Kv2.1 channels. In on-cell recordings from human cells, we have also observed a 10 pS channel (O. B. McManus, unpublished observations). Like the rodent cell, many other of the biophysical properties (e.g. activation and inactivation time constants) of the human cell delayed rectifier current described here are consistent with Kv2.1. Kv2.1 has been detected in human islets and cells by RT-PCR and immunocytochemistry (MacDonald & Wheeler, 2003; Yan et al. 2004). However, given that Kv channel kinetics can be regulated by interaction with auxillary subunits, small calcium-binding proteins and phospholipids (Hanlon & Wallace, 2002; Pourrier et al. 2003; Oliver et al. 2004), it is impossible to use biophysical properties alone to identify the molecular correlate of a given Kv current.
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Pharmacological tools provide important probes for identification of channels in native tissue. In this study, we find the delayed rectifier of human cells is partially inhibited by the gating modifier peptide hanatoxin. Hanatoxin inhibits Kv2.1 channels, but is known to inhibit other Kv channels as well (Swartz & MacKinnon, 1995). Although hanatoxin blocks Kv4 channels, the slow activation and inactivation of the cell delayed rectifier current make it unlikely that the hanatoxin-sensitve current is due to Kv4 channels. Hanatoxin inhibits Kv2.1 by altering the energetics of gating, causing a shift in the current–voltage relation to more positive potentials (Swartz & MacKinnon, 1997). Hanatoxin shifted the voltage dependence of activation of the human cell current in a manner similar to, but distinct from, Kv2.1 (see below). Given that hanatoxin can act on other channels in addition to Kv2.1, the sensitivity of the human cell current to this peptide must be interpreted with caution.
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PAC, a disubstituted cyclohexanone, blocked the delayed rectifier in a time-dependent manner with the degree of block increasing over a 100 ms depolarizing pulse and an IC50 of 0.6 μM for block at the end of the pulse. PAC causes state-dependent block of Kv1.3 channels in human T-lymphocytes with at IC50 of 0.3 μM for block of peak current after a single 1 s depolarizing pulse, and blocks other Kv1 channels in 86Rb efflux assays at 0.2–0.4 μM (Schmalhofer et al. 2002). Block of Kv2.1 and Kv3.2 occurred at 20- to 30-fold higher concentrations. Thus, PAC blocked the human cell delayed rectifier at higher concentrations than has been reported for block of Kv1 channels, in agreement with the lack of effect of Kv1-blocking peptides on the delayed rectifier, and blocked the delayed rectifier at lower concentrations than reported for block of Kv2.1 or Kv3.2.
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The TEA sensitivity of the delayed rectifier current of human cells is particularly revealing. We found that the IC50 for TEA block of human cell current was 0.54 mM. Kv2.1 is relatively insensitive to TEA, with an IC50 of 4–10 mM (Coetzee et al. 1999) (J. Herrington & O. B. McManus, unpublished observations). Thus, it is unlikely that the major component of delayed rectifier current in the human cell is a homotetramer of Kv.2.1. Interestingly, other Kv channel subunits are known to assemble with Kv2.1. These so-called ‘electrically silent’ subunits (Kv5–11 families) do not form functional channels when expressed by themselves, but can combine with Kv2.1 to form channels with distinct properties (Hugnot et al. 1996; Salinas et al. 1997; Kramer et al. 1998; Stocker et al. 1999; Ottschytsch et al. 2002). Of these, Kv6.2, Kv9.3, Kv10.1 and Kv11.1 are expressed in human islets, with Kv6.2 and Kv9.3 known to be in cells (Yan et al. 2004). Thus, it is quite possible that the human cell delayed rectifier K+ channel is formed from the combination of more than one K+ channel gene, possibly including Kv2.1.
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Our data also suggest that the human cell delayed rectifier current may arise from more than one distinct type of Kv channel. For example, we found that, in most cells, inactivation of the delayed rectifier proceeded with two kinetically distinct phases while, in some cells, the inactivation was well described by a single exponential. This kinetic behaviour may arise from two distinct channel types. Alternatively, the complex kinetic behaviour of a single population of channels could also account for this result. The shallow slope (n = 0.67) of the TEA inhibition curve for the delayed rectifier is also consistent with multiple channel or subunit types combining to form the delayed rectifier. Another piece of evidence that hints at multiple underlying channels is the action of hanatoxin. Careful inspection of the current–voltage relation in hanatoxin (see Fig. 8C) shows that inhibition was less prominent at –10 mV compared to +10 mV. Hanatoxin does not produce this effect on Kv2.1, even at non-saturating doses (Swartz & MacKinnon, 1997). The simplest explanation for this result is that human cells express both a hanatoxin-sensitive and a hanatoxin-insensitive delayed rectifier channel.
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Data from previous studies also suggest the expression of multiple delayed rectifier K+ channels in cells (MacDonald & Wheeler, 2003; Yan et al. 2004). For example, at least four distinct Kv channels have been observed in single channel recordings from mouse cells (Smith et al. 1990). One intriguing candidate is Kv3.2. Kv3.2 is present in human cells (Yan et al. 2004) and gives rise to TEA-sensitive (IC50 – 0.3 mM), delayed rectifier currents when expressed in heterologous systems (Coetzee et al. 1999; Yan et al. 2005). However, ShK, a potent blocker of Kv3.2 as well as Kv1 channels, has no effect on the delayed rectifier current of human cells (Yan et al. 2005; Fig. 8), thus ruling out a major contribution of Kv3.2. Although the composition of the underlying channels remains unclear, it appears likely that multiple distinct Kv channels comprise the delayed rectifier current of the human cell.
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Transient potassium current
In addition to the delayed rectifier current, some human cells expressed a low threshold, transient inactivating current. A qualitatively similar current has been described in mouse and rat cells (Gopel et al. 2000; MacDonald et al. 2001). The human cell current has the following basic properties: activation at membrane potentials above –40 mV, inactivation time constant of about 20 ms at –20 mV, and a V for steady-state inactivation of –52 mV.
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Like the delayed rectifier current, the molecular composition of the channel underlying the low threshold, transient current is unclear. Several Kv -subunits have been detected in rodent islets and/or cells that generate inactivating currents in heterologous expression systems. These include members of the Kv1, Kv3 and Kv4 families (reviewed in MacDonald & Wheeler, 2003). In human islets and cells, no Kv -subunits have been detected that are obvious candidates for the rapid inactivating current we describe here (MacDonald & Wheeler, 2003; Yan et al. 2004). Our results with various Kv1 peptide blockers (Fig. 8) suggest that this family does not contribute significantly to the total Kv current of human cells. The pharmacological properties of the transient current were not specifically examined due to its small size, variable expression and the difficulties in studying it in isolation. As an initial approach, we examined the time course of remaining currents after applying blockers in order to estimate pharmacological differences between the transient and the delayed rectifier currents. Seventeen percent of the peak current remaining at maximal PAC concentrations displayed a transient time course (Fig. 7), but this may result from time-dependent block of a non-inactivating current by PAC, as has been seen for PAC block of other currents (Schmalhofer et al. 2002). TEA can block potassium channels without significantly affecting activation kinetics and was applied to 57 cells, of which seven expressed transient currents. In most cells, the kinetics of the currents did not significantly change as TEA concentration increased and nearly all current was blocked at the highest (10–30 mM) concentrations. In two of the seven cells, a small transient component could be resolved as higher concentrations of TEA were applied which may reflect higher transient current expression in these cells or heterogeneity. In sum, TEA blocked the transient current at similar concentrations to those needed to block the delayed rectifier.
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We find that there is considerable variation in the presence of the transient current across different donors. For example, in one donor (donor 1) 46% of cells had a low threshold, transient current whereas in two donors it was completely absent. Interestingly, the set of islets with a high prevalence of transient current were cultured for 2 weeks before shipment, unlike the islets from the other sources which were shipped to us within 3 days post mortem and used immediately. Importantly, the prevalence of the transient current in acutely isolated islets is unknown. A more complete analysis of the Kv currents across populations, metabolic state and islet isolation and culture procedures will be needed before a complete understanding of the role of specific Kv currents in human cell physiology is possible.
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Summary
In summary, we have shown that human cells possess at least two distinct voltage-gated potassium currents. Considerable attention in recent years has focused on developing blockers of cell Kv current as glucose-dependent insulin secretagogues for the treatment of type 2 diabetes. These efforts are largely based on early studies showing potentiation of glucose-stimulated insulin secretion from rodent islets by non-specific Kv blockers (e.g. TEA) (Henquin, 1977; Henquin et al. 1979). More recent studies have suggested Kv2.1 as the leading candidate for the delayed rectifier current in rodent cells. Our results show that the human cell delayed rectifier is unlikely to be a homotetramer of Kv2.1. Also, we find that these cells possess a second, distinct Kv current. Determining the molecular identities of the channels underlying these currents will be important for understanding their role in cell physiology and capitalizing on their potential as drug targets for type 2 diabetes.
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Footnotes
J. Herrington and M. Sanchez contributed equally to this work.
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2 Farmacología, Departamento de Medicina, Facultad de Medicina, Universidad de Oviedo, Julián Clavería 6, ES-33006, Oviedo, Spain
3 BetaGene Inc., 2600 North Stemmons Freeway, Suite 1, Dallas, TX 75207, USA
Abstract
Voltage-gated potassium (Kv) currents of human pancreatic islet cells were studied by whole-cell patch clamp recording. On average, 75% of the cells tested were identified as cells by single cell, post-recording RT-PCR for insulin mRNA. In most cells, the dominant Kv current was a delayed rectifier. The delayed rectifier activated at potentials above –20 mV and had a V for activation of –5.3 mV. Onset of inactivation was slow for a major component ( = 3.2 s at +20 mV) observed in all cells; a smaller component ( = 0.30 s) with an amplitude of 25% was seen in some cells. Recovery from inactivation had a of 2.5 s at –80 mV and steady-state inactivation had a V of –39 mV. In 12% of cells (21/182) a low-threshold, transient Kv current (A-current) was present. The A-current activated at membrane potentials above –40 mV, inactivated with a time constant of 18.5 ms at –20 mV, and had a V for steady-state inactivation of –52 mV. TEA inhibited total Kv current with an IC50 = 0.54 mM and PAC, a disubstituted cyclohexyl Kv channel inhibitor, inhibited with an IC50 = 0.57 μM. The total Kv current was insensitive to margatoxin (100 nM), agitoxin-2 (50 nM), kaliotoxin (50 nM) and ShK (50 nM). Hanatoxin (100 nM) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose-dependent insulin secretion.
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Introduction
Secretion of insulin by the pancreatic cell in response to glucose or other fuel secretagogues is tightly coupled to the membrane potential of the cell. Glucose triggers closure of the cell KATP channel, leading to membrane depolarization, calcium entry and secretion (Ashcroft & Rorsman, 1989). In the intact islet, glucose triggers synchronized bursts of action potentials that lead to cytosolic calcium oscillations (Santos et al. 1991). This oscillatory bursting behaviour appears to be regulated by the interplay of several types of ion channels (Bertram et al. 1995; Dukes & Philipson, 1996; Roe et al. 1996; Mears et al. 1997; Gopel et al. 1999; Eberhardson et al. 2000; Kanno et al. 2002). Of these, voltage-gated K+ (Kv) channels clearly play an important role in regulating cell excitability (Philipson, 1999).
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Kv channels serve numerous roles in excitable cells. Kv channels are involved in setting the resting potential, repolarizing after an action potential, and determining the interspike interval (Hille, 2001). One class of Kv channels, the delayed rectifier, is thought to be the dominant Kv current of cells and plays a key role in action potential repolarization (Smith et al. 1990). Inhibition of the cell delayed rectifier K+ current would be expected to prolong action potentials and enhance glucose-stimulated insulin secretion. Such a therapeutic strategy would be expected to pose a lower risk for hypoglycaemic events compared to sulphonylurea KATP channel blockers. For this reason, the cell delayed rectifier K+ current has attracted much attention as a potential target for the treatment of type 2 diabetes (Roe et al. 1996; MacDonald & Wheeler, 2003).
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The functional unit of a Kv channel is a tetramer of principal pore-forming subunits. Forty Kv channel genes have been identified in humans and additional complexity can be produced by heteromultimerization, alternative splicing, and additional subunits and interacting proteins (Hille, 2001; Yellen, 2002; Yu & Catterall, 2004). The molecular identities of the Kv channels in cells are unknown. Numerous Kv subunits have been detected in islets and/or cells (MacDonald & Wheeler, 2003; Yan et al. 2004). Of these, the delayed rectifier, Kv2.1, has been detected in many studies (Roe et al. 1996; MacDonald et al. 2001, 2002; MacDonald & Wheeler, 2003; Yan et al. 2004). Adenoviral-mediated expression of a dominant negative C-truncated form of Kv2.1 reduces the delayed rectifier Kv current in rat cells by 60–70% and augments glucose-stimulated insulin secretion (MacDonald et al. 2001). However, the exact molecular composition of Kv channels in pancreatic cells remains unclear.
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The Kv currents of mouse cells have been studied in the most detail. The major Kv current in mouse cells appears to be a delayed rectifier that activates near –20 mV, inactivates slowly ( = 4.5 s at +20 mV) and is blocked by TEA with a KD of 1.4 mM (Rorsman & Trube, 1986; Bokvist et al. 1990; Smith et al. 1990). At least four distinct channels have been observed in single channel recordings. The most frequently observed channel has a conductance of 8–10 pS and is thought to play a key role in action potential repolarization (Smith et al. 1990). In addition to the delayed rectifier, mouse and rat cells appear to possess a small, transient inactivating current (Gopel et al. 2000; MacDonald et al. 2001).
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In contrast to rodent cells, little is known about the properties of the Kv currents of human cells. A delayed rectifier Kv current in human cells has been previously described (Kelly et al. 1991). Given the critical contribution of Kv channels to the control of glucose-dependent insulin secretion, it is important to characterize and determine the molecular composition of this current. In this study, we describe the biophysical and pharmacological properties of Kv currents in identified human cells. We find evidence for the existence of at least two distinct types of Kv currents, a delayed rectifier and a transient A-current.
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Methods
Materials
Cell culture reagents were purchased from Gibco/Invitrogen and chemicals were from Sigma unless otherwise noted. Margatoxin (MgTX), agitoxin-2 (AgTX2) and kaliotoxin (KTX) were provided by M. Garcia (Department of Ion Channels, Merck Research Laboratories) and prepared as previously described (Koch et al. 1997; Koschak et al. 1998). ShK was obtained from Peptides International (Louisville, KY). Hanatoxin was provided courtesy of K. Swartz (NINDS, Bethesda, MD, USA). 4-Phenyl-4-[3-(2-methoxyphenyl)-3-oxo-2-azaprop-1- yl]cyclohexanone (PAC) was synthesized by the Medicinal Chemistry Department of Merck Research Laboratories as previously described (Schmalhofer et al. 2002). RNase-free DNase I and RNase inhibitor SUPERaseIn were purchased from Ambion (Austin, TX, USA). The OneStep RT-PCR Kit was purchased from Qiagen (Valencia, CA, USA).
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Donor tissue, islet dissociation and cell culture
Human pancreatic islets were obtained from three sources: BetaGene, Inc. (Dallas, TX, USA), the UCLA/VA Islet Isolation Laboratory (Los Angeles, CA, USA) and the JDRF Human Islet Distribution Program at the University of Alberta (Edmonton, Alberta, Canada).
Islets from one donor were supplied by Betagene, Inc. (donor 1 of Fig. 9). These islets were provided to BetaGene by Dr C. Ricordi's group at the islet isolation facility of the Diabetes Research Institute, University of Miami. Islets had cold ischaemia times > 8 h, and were cultured at ambient temperatures for 1–2 days subsequent to isolation and before shipment to BetaGene. Upon receipt at BetaGene, four assessments of islet quality were made: viability, purity, insulin content and glucose-responsive insulin secretion (Clark et al. 1994, 1997). For the islets used in this study, viability was > 95% and purity was 90%. The insulin content was 1.7 μg/1000 equivalent islet number (EIN), with 99% mature human insulin. In general, islets were not optimally glucose responsive upon receipt, but usually recovered after several days of culture. To facilitate the maintenance of islets and prevent loss with medium changes, the islets were encapsulated in 1.5% alginate bands in medium. The islets were cultured in a custom manufactured medium (JRH Biosciences, Lenexa, KS, USA), optimized for human islets and neuroendocrine cells. The glucose concentration was 7.8 mM. The medium formulation was based on a 1: 1 mixture of M199E and F12 media, supplemented with CuSO4 and an ascorbate to maintain amidating capacity of cells, and an equimolar mixture of ethanolamine–phosphoethanolamine (Clark & Chick, 1990). The medium was supplemented with 2% gamma-irradiated fetal bovine serum (FBS) (JRH Biosciences) and 100 μg ml–1 of streptomycin. Islets were cultured at BetaGene for 2 weeks prior to shipment to Merck. For shipment, islets were removed from the alginate beads by EDTA treatment, pelleted, washed and suspended in 50 ml of medium in a sterile 50 ml tube and shipped at ambient temperature.
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Bar graphs of the properties of Kv current across donors. Bars represent means ± S.E.M. The number of cells was 15, 46, 10, 29, 16, 20, 25 and 17 for donors 1–8, respectively. A, peak current density at +40 mV in pA pF–1. B, percentage of cells that express a detectable low threshold, transient current (A-current). C, voltage of half-activation (activation V) measured from fits of the Boltzmann equation to the tail current amplitude versus command voltage. D, activation time constant () at +40 mV. E, deactivation time constant () measured from single exponential fits to tail currents at –40 mV. F, inactivation time constant () estimated from the decay of current during 10 s steps to +20 mV. In cases where the sum of exponentials were needed to describe the decay, the of the major component was taken.
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Islets from four donors (donors 2–5 in Fig. 9) were obtained from Dr Jonathan Lakey and the JDRF Human Islet Distribution Program at the University of Alberta. The islets from the Edmonton group were isolated as previously described (Shapiro et al. 2000) using procedures approved by the University of Alberta Ethics Committee.
Islets from three donors (donors 6–8 in Fig. 9) were supplied by Dr Yoko Mullen of the UCLA/VA Islet Isolation Laboratory. Collection and isolation of islets by the UCLA laboratory were performed as previously described (Kenmochi et al. 2000) and were approved by the UCLA Institutional Review Boards for the Protection of Human Research Subjects.
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Upon arrival, islets were purified by hand-picking. Some of the islets were used to test for glucose-responsive insulin secretion. In general, about half of the islet preparations showed enhanced insulin secretion in response to elevated glucose. The remainder of the purified islets was dissociated into single cells for electrophysiology. Briefly, islets were placed in Ca2+/Mg2+-free phosphate-buffered saline (PBS) containing trypsin and incubated for 5–10 min, followed by gentle trituration into single cells. The enzyme was quenched by placing the cell suspension in culture medium (RPMI 1640 supplemented with 10% heat-inactivated FBS and penicillin–streptomycin). Following centrifugation, the cells were resuspended in culture medium and plated on poly L-lysine coated glass chips. Cells were used for electrophysiology within 72 h of plating.
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Electrophysiology
Membrane currents were recorded using standard whole-cell voltage clamp techniques (Hamill et al. 1981) with a HEKA EPC9 amplifier (HEKA Elektronik, Lambrecht, Germany) or Dagan 3900A amplifier (Dagan Corporation, Minneapolis, MN, USA). Microelectrodes fabricated from borosilicate glass were coated with Sylgard (Dow Corning, Midland, MI, USA) or beeswax and fire-polished. Electrode resistances were generally 2–4 M when filled with the standard internal saline. The reference electrode was a silver–silver chloride wire within an agar bridge (4% agar in 200 mM KCl). Voltages given in the figures have not been corrected for the small liquid junction potential between the external and internal solutions (2 mV). All experiments were performed at room temperature (22–25°C).
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Generation of analog voltage commands and digitization of membrane currents were controlled with PULSE software (HEKA Elektronik) and an ITC-16 computer interface (Instrutech, Port Washington, NY, USA). Currents were digitized at 5 kHz and digitally filtered at 2 kHz. Digital subtraction of leakage and capacitive currents was performed by the P/n procedure (n = –4 or –5). Voltage steps were applied every 5–20 s from a holding potential of –80 mV.
The standard pipette solution was (mM): 120 KCl, 20 KF, 10 EGTA, 10 Hepes, 2 mM MgATP, pH adjusted to 7.2 with KOH. The standard external solution consisted of (mM): 160 NaCl, 4.5 KCl, 1.8 CaCl2, 1 MgCl2, 10 Hepes, 3 glucose, pH 7.2 with NaOH. Solutions were applied to cells by bath perfusion via gravity. The experimental chamber volume was 0.2 ml and the perfusion rate was 1–2 ml min–1. Flow of solution through the chamber was maintained at all times. Bovine serum albumin (BSA; 0.1% w:v) was added to solutions containing peptides; BSA had no detectable effect on K+ channels. TEA was added directly to the standard extracellular solution from a 1 M stock in water.
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Measurement of current amplitudes was performed with PULSEFIT software (HEKA Elektronik). Fitting of current waveforms or extracted data was performed with PULSEFIT or Igor Pro 4.0 (WaveMetrics, Lake Oswego, OR, USA). The voltage dependence of channel activation and inactivation were fit by the Boltzmann equation:
where Imax is the maximal current, V is the half-activation voltage and k is the voltage dependence of the distribution. In the case of Fig. 3, chord conductance (G) rather than current was analysed. Inhibition of Kv current by pharmacological agents was described by the Hill equation of the form:
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where IC50 is the concentration that produces 50% of the maximal block and n is the apparent Hill coefficient. Experimental values are given as the mean ± S.E.M.
A, membrane current in response to 100 ms voltage steps between –60 mV and +60 mV from a holding potential of –80 mV in 10 mV increments every 5 s. Following the 100 ms command, the membrane potential was stepped to –40 mV for 100 ms. Cell 137 (insulin positive): 7.2 pF. B, plot of chord conductance versus test potential for the cell in A. Conductance was calculated from the equation: G = I/(E – EK), where I is the peak current, E is the test potential and EK is the equilibrium potential for K+ (–90 mV). The continuous line is a fit to the Boltzmann equation. Parameters of the fit are given in the text. C, membrane current in response to voltage steps as described in Fig. 1A. Cell 97: 3.4 pF. D, plot of the membrane currents from C on an expanded y-axis for the –30 mV, –20 mV and –10 mV test potentials. The continuous lines are single exponential fits to the data with time constants of 19 ms (–30 mV), 16 ms (–20 mV), and 13 ms (–10 mV). E, isolation of the transient current by subtraction (details given in the text). Plotted are the currents in response to 100 ms depolarizations to +20 mV following prepulses to –100 mV and –10 mV and the difference currents (–100 mV minus –10 mV). F, plot of the voltage dependence of inactivation measured using the subtraction procedure in E. The continuous line is a fit of the Boltzmann equation to the points. Parameters of the fit are given in the text. Same cell as in C.
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Single-cell RT-PCR
Following recording, the cell contents were collected in the electrode tip by suction and the tip was broken in a PCR tube and stored at –80°C until use. The day of the reaction, the cell contents were thawed by placing reaction solution containing 1 μl RNase-free DNase I (1 U), 1 μl 10x DNase I buffer, and 0.5 μl SUPERaseIn (10 U) and 5 μl distilled H2O in the tube. The total reaction volume was approximately 10 μl. The tube was incubated at 37°C for 30 min to eliminate genomic DNA followed by incubation at 75°C for 5 min to inactivate the DNase I. RT-PCR was performed in the same tube using a Qiagen OneStep RT-PCR Kit as previously described (Yan et al. 2002). Briefly, 40 μl of reaction mix (Sensiscript reverse transcriptase, HotStarTaq polymerase, dNTPs, 5x Qiagen OneStep RT-PCR buffer) and insulin primers (sense primer 5'-CCAGCCGCAGCCTTTGTGA-3', antisense primer 5'-GCTGGTAGAGGGAGCAGAT-3') were added to the tube. The primers were designed from sequences on different exons of the human insulin gene so that PCR products derived from reverse transcribed mRNA and from amplified genomic DNA would be of different size. Only the mRNA-derived product was ever observed, consistent with our previous findings that digestion of genomic DNA is complete with this protocol (Yan et al. 2002). Each reaction (50 μl) contained the DNase I treated RNA of a single cell, 0.6 μM of each of the primers, 400 μM of each of the dNTPs, and 2.0 μl Sensiscript reverse transcriptase–HotStarTaq DNA polymerase enzyme mix. Reverse transcription was allowed to proceed for 30 min at 50°C after which the reaction was heated to 95°C for 15 min to inactivate the reverse transcriptase and activate the HotStarTaq DNA polymerase. The reactions were amplified for 35 cycles followed by a 10 min extension at 72°C. Each cycle consisted of 30 s at 94°C, 30 s at 56°C, and 1 min at 72°C. The PCR products were examined on a 2% agarose gel and cells were scored as cells if a 250 bp product corresponding to insulin was present. Positive insulin controls and negative controls, consisting of saline collected from recording bath or ddH2O, were run in parallel with all samples.
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Several attempts were made to perform multiplex PCR for other hormones (e.g. glucagon), Kv subunits or housekeeping genes along with insulin. These experiments generally compromised the robustness and reliability of measuring insulin mRNA. Thus, we routinely performed single-cell PCR for insulin only. A disadvantage of this approach is that it is possible some of the insulin negative cells may in fact be cells but fail to show detectable insulin RNA due to cell collection and/or PCR failure. However, most of the cells for which we performed single-cell PCR for insulin were positive (see Results), suggesting most of the cells we recorded from were cells. Data in the figures from identified cells are denoted in the legends; otherwise the data shown are from unidentified cells.
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Results
Electrophysiological properties of human islet cells
We recorded from a total of 222 human islet cells, collected from eight donors. In response to a 200 ms voltage ramp between –100 mV and +50 mV, most cells displayed large outward currents above 0 mV (Fig. 1A). However, in 12 cells no detectable voltage-gated current was present. These cells are presumably not of endocrine origin and were not studied further. The mean capacitance of all studied cells was 5.7 ± 0.2 pF (n = 210; Fig. 1C).
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A, membrane current in response to a 200 ms voltage ramp between –100 mV and +40 mV is plotted versus voltage. Cell 109 (insulin positive): 8.8 pF. B, plot of membrane capacitance versus current density (pA pF–1) for all cells. Each symbol represents an individual cell (+, unidentified; , insulin positive; , insulin negative). The dashed lines create a window to highlight cells that have current densities between 35 and 400 pA pF–1 and a capacitance greater than 2 pF. C, histogram of cell capacitance for all cells (n = 210). D, histogram of current density for all cells (n = 171). E, histogram of cell capacitance for insulin positive cells only (n = 48). F, histogram of current density for insulin positive cells only (n = 47).
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During the course of these studies we developed and verified a post-recording RT-PCR method to unambiguously identify cells as cells by detection of insulin mRNA. On average, 75% (54 out of 72) of the cells tested were positive for insulin mRNA. Identified cells had a mean capacitance of 7.3 ± 0.4 pF (n = 48; Fig. 1E). Step depolarizations to +40 mV show that the average current density was 188 ± 6 pA pF–1 (n = 171) for all cells (Fig. 1D) and 168 ± 12 pA pF–1 (n = 47) for identified cells (Fig. 1F). Of the 12 cells expressing no measurable voltage-gated current, two were tested by RT-PCR and both tested negative for insulin mRNA supporting the non-endocrine designation of these cells.
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We next sought a way to filter the data so that analysis was primarily restricted to cells. A plot of capacitance versus current density shows identified cells had a current density between 35 and 400 pA pF–1 and a capacitance greater than 2 pF (Fig. 1B, filled circles). The cells that were not positive for insulin by PCR generally fell within this range also, although some clearly did not (Fig. 1B, open circles). However, it is possible that some of the insulin negative cells were indeed cells but failed to show detectable insulin due to technical limitations (see Methods). Thus, we pooled all the data from unidentified cells within this range (Fig. 1B, +) with identified cells for further analysis. Although this approach does not ensure that all of the data comes from cells, our results with single-cell PCR suggest that at least 75% of the cells we recorded from were probably cells. Except for the data in Figs 7 and 8, the major findings of this paper have been confirmed in identified cells.
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A, representative membrane currents in response to 100 ms voltage steps to +20 mV in the absence (control) and the presence of 0.3 μM, 1 μM, 3 μM and 10 μM external PAC. The holding potential was –80 mV and voltage steps were applied every 15 s. Cell 23: 4.5 pF. B, plot of peak current () during the 100 ms step to +20 versus time for the cell in A. Also plotted is the amplitude of the late current () measured at the end of the 100 ms step. The application of PAC is indicated by the bar with the specific concentrations given near the plot. C, plot of the fraction of control peak and late current blocked (Fraction Blocked) versus PAC concentration for the cell in A. The continuous lines are fits of the Hill equation to the data. Parameters of the fit for the peak current are: IC50 = 1.11 μM, n = 0.99 and maximal block = 1.0. Parameters of the fit for the late current are: IC50 = 0.64 μM, n = 1.26 and maximal block = 1.0. D, Plot of fraction blocked versus PAC concentration for all cells along with fits of the Hill equation to the data. The number of determinations are: 8, 11, 9 and 5 for 0.3, 1, 3 and 10 μM PAC, respectively. Parameters of the fit for the peak current are: IC50 = 0.56 μM, n = 1.27 and maximal block = 0.83. Parameters of the fit for the late current are: IC50 = 0. 57 μM, n = 1.67 and maximal block = 0.95.
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A, representative membrane currents in response to 100 ms voltage steps to +20 mV in the absence (control) and the presence of 100 nM hanatoxin. The holding potential was –80 mV. Cell 13: 2.4 pF. B, plot of peak current () during the 100 ms step to +20 versus time for the cell in A. The application of hanatoxin is indicated by the bar. C, plot of the peak current versus voltage in control () and 100 nM hanatoxin () for the cell in A. D, plot of peak current () during the 100 ms step to +20 versus time for a different cell. The application of peptide Kv channel blockers and TEA are indicated by the bars. A second experiment on a different cell was performed and yielded similar results. Cell 35: 3.8 pF.
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An example of typical currents activated in response to step depolarizations is shown in Fig. 2A. Most cells possessed a delayed rectifier current that develops over 50–100 ms and was activated above –20 mV (Fig. 2B). In a subset of cells, in addition to the delayed rectifier, a transient outward current was present at voltages above –30 mV (Fig. 2C and D). An example of currents from a cell expressing more than 400 pA pF–1 is shown in Fig. 2E and F. In this population, 7 out of 15 cells possessed large outward currents that partially inactivated during 100 ms depolarizations above +40 mV. Three cells in this population also displayed fast inward currents greater than 500 pA in amplitude (not shown). Since the hormonal identity of these cells is unclear, these cells were not studied further.
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A, membrane current in response to 100 ms voltage steps between –60 mV and +50 mV from a holding potential of –80 mV in 10 mV increments every 20 s. Following the 100 ms command, the membrane potential was stepped to –40 mV for 20 ms to record tail currents. Cell 105 (insulin positive): 3.5 pF. B, plot of the peak current versus command voltage for the cell in A. C, membrane currents recorded from another cell using the same stimulus protocol as in A. The inset shows the current in response to the step to –30 mV to highlight the small A-current present in this cell. Cell 58: 5.8 pF. D, plot of peak current versus command voltage for the cell in C. E, membrane currents from a cell with current density > 400 pA pF–1 recorded using the same voltage protocol as in A. Cell 79: 3.8 pF. F, plot of the peak current versus command potential for the cell in E.
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Properties of the low threshold, transient outward current
In approximately 12% of cells within the > 2 pF and 35–400 pA pF–1 range (21/182), 100 ms step depolarizations from a holding potential of –80 mV activated a clear low threshold (–40 mV) inactivating outward current. This ‘A-current’ was seen in cells from six out of the eight donors. Except for one donor (donor 1; see below), the A-current was seen in less than 20% of the cells studied from a given donor. In one cell, this current appeared to be dominant (Fig. 3A) and allowed estimation of the voltage dependence of the conductance (Fig. 3B; V = –13.1 mV; k = 14.7 mV, Gmax = 2.52 nS). However, most cells that possessed the low threshold, transient outward current clearly had other currents as well (Fig. 3C). In these cells, the time course of inactivation could be studied only at negative potentials (–30 mV or –20 mV) where the current was present in isolation (Fig. 3D). In these cells, inactivation proceeded with a mean time constant of 19 ms at –20 mV (Table 1).
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To study the voltage dependence of steady-state inactivation, we employed a subtraction procedure to minimize the contribution of other currents. From a holding potential of –80 mV, a 20 ms prepulse to potentials between –110 mV and –10 mV was given followed by a brief (5 ms) step back to the holding potential to allow channel closing. The membrane was then stepped to +20 mV for 100 ms. The currents from the –10 mV prepulse were used to subtract the non-inactivating component of current. As shown in Fig. 3E, this procedure allowed reasonable separation of the transient component of current. The amplitudes of the difference currents are plotted versus the prepulse voltage in Fig. 3F. The voltage dependence of inactivation measured in this way was well described by a Boltzmann distribution with a V of –44 mV and slope factor of 6.3 mV.
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Biophysical properties of the delayed outward current
To study the biophysical properties of the delayed outward current we excluded cells with significant transient current from analysis. The voltage dependence of activation of the delayed rectifier current was measured from the tail current amplitude at –40 mV following 100 ms step depolarizations to various potentials (Fig. 4A). The voltage dependence of the tail current amplitude was well described by a Boltzmann distribution (Fig. 4B). For the cell in Fig. 4, the V was –1.7 mV and the slope factor was 7.3 mV per e-fold change in conductance. The kinetics of activation were measured by fitting an exponential function to the rising phase of the current (Fig. 4C). As expected, the activation proceeded faster with stronger depolarizations, reaching a value of 6.5 ms at +40 mV (Fig. 4D).
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A, membrane current in response to 100 ms voltage steps between –70 mV and +40 mV from a holding potential of –80 mV in 10 mV increments every 5 s. Following the 100 ms step, the membrane potential was stepped to –40 mV for 20 ms. B, plot of tail current amplitude versus test potential for the cell in A. The continuous line is a fit to the Boltzmann equation. Parameters of the fit are given in the text. C, single exponential fits (continuous lines) of currents from A. The time constants are 6.6 ms (+40 mV), 9.6 ms (+20 mV), and 15.1 ms (+10 mV). D, plot of activation time constant versus test potential for the cell in A. E, membrane current in response to a 25 ms voltage step to +40 mV followed by a 30 ms step to between –100 mV and –10 mV in increments of 10 mV from a holding potential of –80 mV. F, plot of tail current amplitude versus repolarization potential for the record in E. Cell 1: 9.0 pF.
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Channel closing upon repolarization, commonly referred to as deactivation, was studied by varying the membrane potential between –10 mV and –100 mV following a 25 ms step depolarization to +40 mV (Fig. 4E). Deactivation was highly voltage dependent. On average, deactivation had a time constant of 7.7 ms at –40 mV. Plotting the tail current amplitude versus repolarization voltage yielded an apparent reversal potential of –82 mV (Fig. 4F). For all cells, the estimated reversal potential was –85 ± 2 mV (n = 13). Such a reversal potential is consistent with a potassium-selective current under these recording conditions (EK = –90 mV; ECl = –9 mV).
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Inactivation of the delayed rectifier K+ current was generally not apparent or very modest with 100 ms step depolarizations (Fig. 5A). To study the onset of inactivation, 10 s depolarizations to +40 mV were given. In general, the current decayed 74 ± 3% (n = 25) over the 10 s step. For the cell shown in Fig. 5, the current decay was best described by the sum of two exponentials with time constants of 0.13 s and 2.58 s (Fig. 5B). In 64% of cells (n = 16), current decay was best described by the sum of two exponentials with average time constants of 0.3 s and 3.3 s. The slower time constant component was 70 ± 3% of the total decay. In the remaining 36% of cells (n = 9), a single exponential with time constant of 2.9 s adequately described inactivation. Recovery from inactivation was studied by first inactivating the current with a 10 s step and varying the time interval at –80 mV before stepping to +40 mV to measure the amount of recovery (Fig. 5C). The time constant of recovery from inactivation was 3.5 s for this cell (Fig. 5D). To study the voltage dependence of steady-state inactivation, 10 s prepulses to potentials between –120 mV and –30 mV were given prior to a step depolarization to +20 mV (Fig. 5E). The voltage dependence of inactivation was well described by a Boltzmann distribution with a V of –46 mV and a slope factor of 7.4 mV (Fig. 5F). Table 1 summarizes the biophysical properties of Kv channels of human cells.
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A, membrane current in response to 100 ms voltage steps between –60 mV and +50 mV from a holding potential of –80 mV in 10 mV increments every 20 s. Following the 100 ms step, the membrane potential was stepped to –40 mV for 20 ms. B, membrane current in response to a 10 s voltage step to +20 mV from a holding potential of –80 mV. The continuous line is a fit of the sum of two exponentials to the current decay with the parameters as shown. C, membrane current in response a voltage protocol designed to measure recovery from inactivation. From a holding potential of –80 mV, the cell was stepped to +20 mV for 10 s (shown at left), followed by a variable interval at –80 mV (ranging from 0.1 s to 31.5 s; not shown) and a 100 ms step to +20 (right) every 90 s. D, plot of relative current amplitude versus recovery duration for the record in C. The continuous line is a single exponential fit to the data with the parameters as shown. E, membrane current in response to a voltage protocol to measure steady-state inactivation. From a holding potential of –80 mV, a 10 s conditioning step to between –120 mV and –30 mV in 10 mV increments was applied (not shown), followed by a short step to –80 mV (10 ms) to deactivate any open channels and a 100 ms step to +20 mV (shown). F, plot of peak current amplitude versus prepulse potential for the record in E. The continuous line is a fit to the Boltzmann equation with parameters as shown. Cell 46: 8.3 pF.
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Pharmacology of the delayed outward current
We next sought to characterize the pharmacology of the delayed rectifier Kv current. As before, we restricted our analysis to cells which had little, if any, transient current. Test compounds were applied while stepping to +40 mV for 100 ms every 15 s. TEA inhibited peak Kv current in a concentration-dependent manner (Fig. 6A and B). For the cell in Fig. 6, TEA inhibited Kv current with an IC50 of 0.74 mM (Fig. 6C). When data from all cells were pooled, the apparent IC50 for TEA block of the delayed rectifier was 0.54 mM (Fig. 6D). Also shown in Fig. 6D are data from PCR-identified cells, which agree well with the pooled data from the entire population.
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A, representative membrane currents in response to 100 ms voltage steps to +20 mV in the absence (control) and the presence of 0.3 mM, 1 mM, 3 mM and 10 mM external TEA. The holding potential was –80 mV and voltage steps were applied every 15 s. Cell 67: 3.2 pF. B, plot of peak current during the 100 ms step to +20 versus time for the cell in A. The application of TEA is indicated by the bar with the specific concentrations given near the plot. C, plot of the fraction of control current blocked (Fraction Blocked) versus TEA concentration for the cell in A. The continuous line is a fit of the Hill equation to the data. Parameters of the fit are: IC50 = 0.74 mM, n = 0.85 and maximal block = 0.99. D, plot of fraction blocked versus TEA concentration for all cells () and PCR-identified cells (). For unidentified cells, the number of determinations are: 3, 13, 44, 7 and 37 for 0.1, 0.3, 1, 3, 10 and 30 mM TEA, respectively. For PCR-identified cells, the number of determinations are 20 and 19 for 1 and 10 mM TEA, respectively. The continuous line is a fit of the Hill equation to the open circles. Parameters of the fit are: IC50 = 0. 54 mM, n = 0.67 and maximal block = 0.95.
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PAC is a disubstituted cyclohexyl analogue which blocks Kv1.3 channels by a mechanism that accelerates channel closing during sustained depolarizations (Schmalhofer et al. 2002). PAC inhibited total current of human cells in a time-dependent manner (Fig. 7A and B). For the cell shown in Fig. 7, the apparent IC50 was 1.11 μM when peak current was measured and 0.64 μM when the current at the end of the 100 ms step was measured (Fig. 7C). When data from all cells are pooled, the apparent IC50 was 0.57 μM (late current; Fig. 7D).
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Hanatoxin is a prototypical gating modifier peptide that was isolated from the venom of the spider Grammostola spatulata based on its ability to inhibit the activity of Kv2.1 channels (Swartz & MacKinnon, 1995). In addition to Kv2.1 channels, hanatoxin inhibits Kv4 channels (IC50 – 100 nM) and, with much lower affinity, other Kv and Ca2+ channels (Swartz & MacKinnon, 1995; Li-Smerin & Swartz, 1998). We tested if the Kv currents of human cells are sensitive to hanatoxin. Application of 100 nM hanatoxin inhibited peak Kv current at +20 mV by 65% (n = 2; Figs 8A and B). Inspection of the current-voltage relation reveals that hanatoxin shifts the voltage dependence of activation of Kv current to more depolarized potentials (Fig. 8C).
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Margatoxin (MgTX), kaliotoxin (KTX), agitoxin-2 (AgTX2) and ShK are inhibitors of Kv1 family channels. Application of 100 nM MgTX, 50 nM KTX, 50 nM AgTX2, or 50 nM ShK had no significant effect on the Kv current of human cells (Fig. 8D). These data are consistent with the lack of detection of Kv1 subunits in human islets by RT-PCR (MacDonald & Wheeler, 2003; Yan et al. 2004). Since ShK also potently blocks Kv3.2 channels, the lack of effect on the Kv current of human cells suggests Kv3.2 does not contribute significantly to the delayed rectifier in these cells, as reported previously (Yan et al. 2005).
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Donor analysis
We next compared the basic electrophysiological properties described above across the different donors. Current density, deactivation time constant and inactivation time constant showed no or very modest differences between donors (Figs 9A, E and F). Interestingly, the V for activation was more positive and the activation time constant slower for donor 2 and donor 3 compared to the others (Fig. 9C and D). One striking difference between donors was the variable presence of the low threshold, transient current (Fig. 9B). In one donor (donor 1) 46% of cells had this current while in others the current was completely absent (donor 3 and donor 5) or in less than 20% of cells (donors 2, 4 and 6–8).
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Discussion
Kv channels of pancreatic cells have received much attention as potential therapeutic targets for type 2 diabetes (Roe et al. 1996; MacDonald & Wheeler, 2003). A clear understanding of the properties of the Kv current of human cells will be needed to realize this aim. In this study, we define the properties of the Kv current of human cells. Prior to this work, human cell Kv current was described in a single report where the delayed rectifier Kv current was described (Kelly et al. 1991). Here we provide a detailed biophysical and pharmacological characterization of the delayed rectifier potassium current of human cells. We also find that a subset of human cells possess a low threshold, transient A-current.
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Properties of delayed rectifier current
The delayed rectifier of human cells has the following basic properties: activation near –20 mV, deactivation of about 8 ms at –40 mV, slow onset and recovery from inactivation (3 s at +20 mV and –80 mV, respectively). These biophysical properties are similar to those of the delayed rectifier found in mouse cells (Rorsman & Trube, 1986; Bokvist et al. 1990; Smith et al. 1990). The current was fully blocked by TEA with an IC50 of 0.54 mM and by PAC with an IC50 of 0.57 μM, and partially blocked by 100 nM hanatoxin.
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Identity of the delayed rectifier current
Several studies have suggested that in rodent cells, Kv2.1 is expressed and contributes to forming the delayed rectifier current (Roe et al. 1996; MacDonald et al. 2001; MacDonald et al. 2002). Indeed, the most frequent Kv channel seen in cell attached patches from mouse cells has a conductance of 8–10 pS (Smith et al. 1990), which is consistent with the conductance of heterologously expressed Kv2.1 channels. In on-cell recordings from human cells, we have also observed a 10 pS channel (O. B. McManus, unpublished observations). Like the rodent cell, many other of the biophysical properties (e.g. activation and inactivation time constants) of the human cell delayed rectifier current described here are consistent with Kv2.1. Kv2.1 has been detected in human islets and cells by RT-PCR and immunocytochemistry (MacDonald & Wheeler, 2003; Yan et al. 2004). However, given that Kv channel kinetics can be regulated by interaction with auxillary subunits, small calcium-binding proteins and phospholipids (Hanlon & Wallace, 2002; Pourrier et al. 2003; Oliver et al. 2004), it is impossible to use biophysical properties alone to identify the molecular correlate of a given Kv current.
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Pharmacological tools provide important probes for identification of channels in native tissue. In this study, we find the delayed rectifier of human cells is partially inhibited by the gating modifier peptide hanatoxin. Hanatoxin inhibits Kv2.1 channels, but is known to inhibit other Kv channels as well (Swartz & MacKinnon, 1995). Although hanatoxin blocks Kv4 channels, the slow activation and inactivation of the cell delayed rectifier current make it unlikely that the hanatoxin-sensitve current is due to Kv4 channels. Hanatoxin inhibits Kv2.1 by altering the energetics of gating, causing a shift in the current–voltage relation to more positive potentials (Swartz & MacKinnon, 1997). Hanatoxin shifted the voltage dependence of activation of the human cell current in a manner similar to, but distinct from, Kv2.1 (see below). Given that hanatoxin can act on other channels in addition to Kv2.1, the sensitivity of the human cell current to this peptide must be interpreted with caution.
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PAC, a disubstituted cyclohexanone, blocked the delayed rectifier in a time-dependent manner with the degree of block increasing over a 100 ms depolarizing pulse and an IC50 of 0.6 μM for block at the end of the pulse. PAC causes state-dependent block of Kv1.3 channels in human T-lymphocytes with at IC50 of 0.3 μM for block of peak current after a single 1 s depolarizing pulse, and blocks other Kv1 channels in 86Rb efflux assays at 0.2–0.4 μM (Schmalhofer et al. 2002). Block of Kv2.1 and Kv3.2 occurred at 20- to 30-fold higher concentrations. Thus, PAC blocked the human cell delayed rectifier at higher concentrations than has been reported for block of Kv1 channels, in agreement with the lack of effect of Kv1-blocking peptides on the delayed rectifier, and blocked the delayed rectifier at lower concentrations than reported for block of Kv2.1 or Kv3.2.
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The TEA sensitivity of the delayed rectifier current of human cells is particularly revealing. We found that the IC50 for TEA block of human cell current was 0.54 mM. Kv2.1 is relatively insensitive to TEA, with an IC50 of 4–10 mM (Coetzee et al. 1999) (J. Herrington & O. B. McManus, unpublished observations). Thus, it is unlikely that the major component of delayed rectifier current in the human cell is a homotetramer of Kv.2.1. Interestingly, other Kv channel subunits are known to assemble with Kv2.1. These so-called ‘electrically silent’ subunits (Kv5–11 families) do not form functional channels when expressed by themselves, but can combine with Kv2.1 to form channels with distinct properties (Hugnot et al. 1996; Salinas et al. 1997; Kramer et al. 1998; Stocker et al. 1999; Ottschytsch et al. 2002). Of these, Kv6.2, Kv9.3, Kv10.1 and Kv11.1 are expressed in human islets, with Kv6.2 and Kv9.3 known to be in cells (Yan et al. 2004). Thus, it is quite possible that the human cell delayed rectifier K+ channel is formed from the combination of more than one K+ channel gene, possibly including Kv2.1.
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Our data also suggest that the human cell delayed rectifier current may arise from more than one distinct type of Kv channel. For example, we found that, in most cells, inactivation of the delayed rectifier proceeded with two kinetically distinct phases while, in some cells, the inactivation was well described by a single exponential. This kinetic behaviour may arise from two distinct channel types. Alternatively, the complex kinetic behaviour of a single population of channels could also account for this result. The shallow slope (n = 0.67) of the TEA inhibition curve for the delayed rectifier is also consistent with multiple channel or subunit types combining to form the delayed rectifier. Another piece of evidence that hints at multiple underlying channels is the action of hanatoxin. Careful inspection of the current–voltage relation in hanatoxin (see Fig. 8C) shows that inhibition was less prominent at –10 mV compared to +10 mV. Hanatoxin does not produce this effect on Kv2.1, even at non-saturating doses (Swartz & MacKinnon, 1997). The simplest explanation for this result is that human cells express both a hanatoxin-sensitive and a hanatoxin-insensitive delayed rectifier channel.
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Data from previous studies also suggest the expression of multiple delayed rectifier K+ channels in cells (MacDonald & Wheeler, 2003; Yan et al. 2004). For example, at least four distinct Kv channels have been observed in single channel recordings from mouse cells (Smith et al. 1990). One intriguing candidate is Kv3.2. Kv3.2 is present in human cells (Yan et al. 2004) and gives rise to TEA-sensitive (IC50 – 0.3 mM), delayed rectifier currents when expressed in heterologous systems (Coetzee et al. 1999; Yan et al. 2005). However, ShK, a potent blocker of Kv3.2 as well as Kv1 channels, has no effect on the delayed rectifier current of human cells (Yan et al. 2005; Fig. 8), thus ruling out a major contribution of Kv3.2. Although the composition of the underlying channels remains unclear, it appears likely that multiple distinct Kv channels comprise the delayed rectifier current of the human cell.
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Transient potassium current
In addition to the delayed rectifier current, some human cells expressed a low threshold, transient inactivating current. A qualitatively similar current has been described in mouse and rat cells (Gopel et al. 2000; MacDonald et al. 2001). The human cell current has the following basic properties: activation at membrane potentials above –40 mV, inactivation time constant of about 20 ms at –20 mV, and a V for steady-state inactivation of –52 mV.
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Like the delayed rectifier current, the molecular composition of the channel underlying the low threshold, transient current is unclear. Several Kv -subunits have been detected in rodent islets and/or cells that generate inactivating currents in heterologous expression systems. These include members of the Kv1, Kv3 and Kv4 families (reviewed in MacDonald & Wheeler, 2003). In human islets and cells, no Kv -subunits have been detected that are obvious candidates for the rapid inactivating current we describe here (MacDonald & Wheeler, 2003; Yan et al. 2004). Our results with various Kv1 peptide blockers (Fig. 8) suggest that this family does not contribute significantly to the total Kv current of human cells. The pharmacological properties of the transient current were not specifically examined due to its small size, variable expression and the difficulties in studying it in isolation. As an initial approach, we examined the time course of remaining currents after applying blockers in order to estimate pharmacological differences between the transient and the delayed rectifier currents. Seventeen percent of the peak current remaining at maximal PAC concentrations displayed a transient time course (Fig. 7), but this may result from time-dependent block of a non-inactivating current by PAC, as has been seen for PAC block of other currents (Schmalhofer et al. 2002). TEA can block potassium channels without significantly affecting activation kinetics and was applied to 57 cells, of which seven expressed transient currents. In most cells, the kinetics of the currents did not significantly change as TEA concentration increased and nearly all current was blocked at the highest (10–30 mM) concentrations. In two of the seven cells, a small transient component could be resolved as higher concentrations of TEA were applied which may reflect higher transient current expression in these cells or heterogeneity. In sum, TEA blocked the transient current at similar concentrations to those needed to block the delayed rectifier.
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We find that there is considerable variation in the presence of the transient current across different donors. For example, in one donor (donor 1) 46% of cells had a low threshold, transient current whereas in two donors it was completely absent. Interestingly, the set of islets with a high prevalence of transient current were cultured for 2 weeks before shipment, unlike the islets from the other sources which were shipped to us within 3 days post mortem and used immediately. Importantly, the prevalence of the transient current in acutely isolated islets is unknown. A more complete analysis of the Kv currents across populations, metabolic state and islet isolation and culture procedures will be needed before a complete understanding of the role of specific Kv currents in human cell physiology is possible.
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Summary
In summary, we have shown that human cells possess at least two distinct voltage-gated potassium currents. Considerable attention in recent years has focused on developing blockers of cell Kv current as glucose-dependent insulin secretagogues for the treatment of type 2 diabetes. These efforts are largely based on early studies showing potentiation of glucose-stimulated insulin secretion from rodent islets by non-specific Kv blockers (e.g. TEA) (Henquin, 1977; Henquin et al. 1979). More recent studies have suggested Kv2.1 as the leading candidate for the delayed rectifier current in rodent cells. Our results show that the human cell delayed rectifier is unlikely to be a homotetramer of Kv2.1. Also, we find that these cells possess a second, distinct Kv current. Determining the molecular identities of the channels underlying these currents will be important for understanding their role in cell physiology and capitalizing on their potential as drug targets for type 2 diabetes.
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Footnotes
J. Herrington and M. Sanchez contributed equally to this work.
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