Activation of the Endothelial Store-Operated ISOC Ca2+ Channel Requires Interaction of Protein 4.1 With TRPC4
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循环研究杂志 2005年第11期
the Center for Lung Biology (D.L.C., S.W., M.A., T.S.), Department of Pharmacology (D.L.C., S.W., T.S.)
Department of Cell Biology and Neuroscience (M.A.), the University of South Alabama College of Medicine, Mobile
Department of Molecular and Cell Biology (S.R.G.), the University of Texas at Dallas
Department of Neuroscience and Center for Molecular Neurobiology (M.X.Z.), the Ohio State University, Columbus.
Abstract
Store-operated calcium (SOC) entry represents the principal Ca2+ entry pathway into nonexcitable cells. Despite intensive investigation, mechanisms underlying activation of SOC entry have remained elusive. The endothelial ISOC channel is a Ca2+-selective SOC entry channel to which the transient receptor potential (TRP) proteins TRPC1 and TRPC4 contribute subunits. Activation of ISOC is specifically regulated by the spectrineCactin membrane skeleton; however, the nature of coupling between the ISOC channel and membrane skeleton is unknown. Here we demonstrate that protein 4.1 is an essential component of the ISOC channel gating mechanism. Protein 4.1 interacts with TRPC4 and the membrane skeleton. Deletion of the protein 4.1 binding domain on TRPC4 or peptide competition to the protein 4.1 binding domain prevents ISOC activation. These findings reveal that interaction of protein 4.1 with TRPC4 is required for activation of the endothelial ISOC channel.
Key Words: cytoskeleton ISOC spectrin store-operated Ca2+ entry TRPC
Introduction
Elevation in cytosolic calcium, [Ca2+]i, is an essential step in numerous physiological and pathophysiological signal transduction events. Ion channels allowing for the entry of extracellular Ca2+ into the cytosol are the principal source of increased [Ca2+]i. In nonexcitable cells, store-operated calcium (SOC), and to a lesser extent, receptor-operated calcium (ROC), entry channels represent the main source of calcium entry.1,2 Although it is generally recognized that SOC entry occurs through Ca2+-selective and nonselective channels, the molecular makeup, regulation, and downstream targets of specific channels remain to be determined.
In endothelial cells, activation of SOC entry contributes to the formation of intercellular gaps, leading to endothelial barrier disruption,3eC5 a common factor in the development and progression of systemic and pulmonary diseases, including hypertension, atherosclerosis, and acute respiratory distress syndrome. It is therefore therapeutically desirable to identify specific SOC entry channel(s) involved in endothelial barrier disruption. We recently reported that activation of the Ca2+-selective SOC entry current ISOC contributes to the increase in [Ca2+]i necessary to disrupt the endothelial barrier.6 However, the molecular makeup and regulation of the channel that provides ISOC (ie, the ISOC channel) are incompletely understood.
Transient receptor potential (TRP) proteins encode SOC and ROC entry channels. The TRPC (canonical) subfamily consists of seven known members.7 It is not currently clear which TRPC homologues contribute to SOC versus ROC entry channels. Some studies have shown that TRPCs 1, 3, 4, and 5 can be activated by depletion of endoplasmic reticulum Ca2+, suggesting they contribute to SOC entry channels, whereas other studies have proposed contribution of these homologues to ROC entry channels.8 For the purpose of this study, we focused specifically on endogenous TRPC proteins that contribute to SOC entry in endothelial cells. Given this criterion, TRPC1 and TRPC4 contribute to the endothelial Ca2+-selective ISOC channel. Previously, our group used antisense technology to assess the contribution of endogenous TRPC1 to ISOC in pulmonary artery endothelial cells (PAECs).9 Inhibition of TRPC1 reduced but did not eliminate ISOC. On the other hand, endothelial cells obtained from TRPC4-deficient mice completely lacked ISOC.10 These studies suggest that although TRPC1 and TRPC4 contribute subunits to the ISOC channel molecular makeup, it is TRPC4 that is required for channel activation.
Activation of Drosophila melanogaster TRP and mammalian TRPC1 channels requires an intact C terminus.11eC13 However, to date, mechanisms regulating SOC entry channel activation are still unclear. Early studies in endothelial cells and platelets provided evidence supporting cytoskeletal regulation of SOC entry channel activation.5,14,15 Actin and spectrin principally comprise cellular membrane and cytoskeletons, whereas protein 4.1 and ankyrin function to link transmembrane proteins to the underlying membrane skeleton.16 Our group recently demonstrated that the specific interaction of protein 4.1 with spectrin was required for ISOC activation.17 Thus, we proposed that protein 4.1 functionally links the endothelial ISOC channel to the membrane skeleton.
Although the spectrineCprotein 4.1 interaction is necessary for ISOC activation, the mode of coupling between the membrane skeleton and ISOC channel is unknown. Because TRPC4 appears to be the subunit important for ISOC channel activation, we sought to determine whether protein 4.1 interacts with TRPC4. We report that protein 4.1 binds to TRPC4, likely within a conserved protein 4.1 binding domain located on the C terminus of TRPC4. Deletion of the protein 4.1 binding domain on TRPC4, as well as peptide transfection targeted to disrupt endogenous TRPC4eCprotein 4.1 interaction, similarly prevents ISOC activation. Collectively, these data support a regulatory role for protein 4.1 in ISOC channel activation.
Materials and Methods
Reagents
All reagents were obtained from Sigma-Aldrich unless specified otherwise. Antibodies to TRPC4 were provided by M.Z. or purchased from Sigma-Aldrich; protein 4.1 antibody was received from S.R.G. Secondary antibodies and chemiluminescence reagents were purchased from Santa Cruz Biotechnology or Molecular Probes, Inc.
Isolation and Culture of Pulmonary Endothelial Cells
PAECs were isolated and cultured using a method described by Creighton et al.18
Immunocytochemistry
PAEC monolayers were methanol fixed and permeabilized. Primary antibody to TRPC4 (Sigma-Aldrich) was added at 1:200 dilution for 1 hour followed by secondary antibody (Alexa Fluor 488 goat anti-rabbit; Molecular Probes, Inc.) at 1:300 dilution for 40 minutes. Fluorescent and Normarski differential interference contrast images were obtained using a Leica TCS SP2 confocal laser scanning microscope fitted with a 63x oil immersion objective.
Preparation and Detergent Extraction of Membrane/Cytoskeleton Fractions19
For detailed protocol, refer to the online supplement, available at http://circres.ahajournals.org.
Thapsigargin Treatment
Cells were exposed to 1 eol/L thapsigargin (Calbiochem) for 2.5 to 3 minutes at room temperature before beginning lysing procedure. In these experiments, the total time from addition of thapsigargin to end of lysing procedure (ie, samples at eC80°C) was 12 to 14 minutes.
Immunoprecipitation Reactions and Western Blot Analysis
Immunoprecipitation Reactions
To 150 to 250 e蘈 of supernatant was added TRPC4 antibody (1:100 dilution) or protein 4.1 antibody (1:130 dilution), and 2% protease inhibitor cocktail (10x). The sample was rocked at 4°C (typically overnight). This was followed by addition of 40 to 50 e蘈 EZ View Red Protein A affinity gel (Sigma-Aldrich) and 2% protease inhibitor cocktail (10x). The sample was rocked for an additional 4 to 5 hours, pelleted, washed 3x750 e蘈 ice-cold PBS/0.1% protease inhibitor cocktail (10x), and resuspended in 4x lithium dodecylsulfate (Invitrogen).
Western Blot Analysis
For SDS-PAGE, 7%, 8%, or 4% to 12% gradient (Invitrogen) gels were typically used. Samples were transferred to nitro-cellulose or polyvinylidene difluoride membranes at 30 V. Membranes were rocked with primary antibody for 2.5 hours at room temperature or overnight at 4°C and with secondary antibody for 1 hour. Proteins were visualized via enhanced chemiluminescence detection (Luminol Reagent; Santa Cruz Biotechnology).
Adenoviral Constructs
Recombinant adenoviruses were generated according to established protocols20 using commercially available plasmids (AdMax; Microbix). For protocol description, refer to the online supplement.
Adenovirus Infection
Endothelial cells were seeded on 100- or 35-mm culture dishes±25-mm circle microscope glass coverslips (Fisher Scientific) and grown to &60% to 90% confluence. Cells were infected with adenovirus constructs at multiplicities of infection equal to 50:1, 25:1, or 10:1. Ca2+ and ISOC measurements were performed at 36 to 50 hours after infection.
Peptide Transfection
Two peptides were synthesized by Alpha Diagnostic International, Inc. The prr/4.1 peptide corresponds to the region on TRPC4 encompassing the protein 4.1 binding domain and adjacent proline-rich region. A peptide with scrambled sequence was also prepared. Peptides were transfected into PAECs using Chariot reagent (Active Motif) generally following the suggested protocol of the manufacturer. For protocol description, refer to the online supplement.
Ca2+ Measurements
Endothelial cells were seeded onto 25-mm glass coverslips and grown to confluence. [Ca2+]i was estimated with the fluorophore fura 2-acetoxymethylester (fura-2 AM; Molecular Probes) according to methods described previously.21
Patch-Clamp Electrophysiology
Conventional whole-cell voltage clamp configuration was performed to measure transmembrane currents in single rat PAECs using the protocol described by Wu et al.17
Results
TRPC4 Localizes to Sites of CelleCCell Adhesion
TRPC4 is a subunit of the endothelial ISOC channel that, when activated, promotes endothelial barrier disruption through the formation of interendothelial cell gaps. Immunocytochemistry was performed to determine cellular localization of endogenous TRPC4. Positive TRPC4 staining was identified in the plasma membrane of PAECs. Staining was particularly strong in areas of the membrane involved in celleCcell adhesion (Figure 1). Additional punctate staining was evident, likely because of the presence of TRPC4 in cholesterol-rich (eg, caveolae) areas of the membrane.22 The localization of TRPC4 at sites of celleCcell adhesion supports a role for the endothelial ISOC channel in intercellular gap formation.6
TRPC4 Binds to the PAEC Membrane Skeleton
Interaction between spectrin and protein 4.1 is required for activation of the ISOC channel.17 However, the nature of interaction between the membrane skeleton and proteins encoding the ISOC channel is unknown. Because TRPC4 appears to be required for channel activation, we sought to determine whether TRPC4 physically interacts with the membrane skeleton. Membrane/cytoskeleton fractions of PAECs were detergent extracted using octyl--D-glucopyranoside to yield pellet and supernatant fractions. SDS-PAGE and immunoblotting of these fractions found TRPC4 exclusively in the pellet (Figure 2a). Actin, a membrane skeleton marker, was also found in the pellet, whereas endothelial myosin light chain kinase 1 resided in the supernatant. Protein 4.1 was detected in pellet and supernatant fractions. Thus, we found that TRPC4 physically interacts with the membrane skeleton of PAECs because it was found in the pellet fraction extracted from membrane/cytoskeleton preparations. It is noteworthy that this physical association appears to be constitutive (ie, not requiring channel activation).
TRPC4 Interacts With the Membrane Skeleton in a Salt-Sensitive Manner
Having demonstrated that TRPC4 physically associates with the membrane skeleton, we next questioned the nature of this interaction. Salts are ionic compounds that weaken ionic and strengthen hydrophobic proteineCprotein interactions.23 When PAEC membrane/cytoskeleton fractions were detergent extracted in the presence of 1 mol/L potassium iodide (KI), TRPC4 was found in the supernatant fraction instead of the pellet (data not shown). This suggested that TRPC4 associates with the membrane skeleton through an ionic interaction, which is sensitive to KI disruption. To more selectively probe the TRPC4eCmembrane skeleton interaction, we repeated the experiments using lower salt concentrations. Even at 150 mmol/L KI, TRPC4 resided in the supernatant (Figure 2b). We also probed for protein 4.1. Similar to TRPC4, the interaction of protein 4.1 with the membrane skeleton was sensitive to 150 mmol/L KI because the majority of protein 4.1 was now found in the supernatant. These data indicate that TRPC4 and protein 4.1 associate with the membrane skeleton through an ionic interaction sensitive to KI disruption.
To determine the minimal KI concentration necessary to dissociate TRPC4 from the membrane skeleton, we performed detergent/KI extractions over a low KI concentration range (0 to 100 mmol/L KI). Immunoblot analysis revealed that the interaction of TRPC4 with the membrane skeleton is exquisitely sensitive to this concentration range because it exhibited a dose-dependent shift from the pellet fraction to the supernatant (Figure 2c). Protein 4.1 was also sensitive to this KI concentration range (ie, at 0 mmol/L KI, the majority of protein 4.1 resided in the pellet fraction, whereas at 100 mmol/L KI, protein 4.1 was found predominantly in the supernatant). Actin, on the other hand, was found mainly in the pellet fraction irrespective of solution ionic strength. Collectively, these observations demonstrate that TRPC4 and protein 4.1 associate with the membrane skeleton through similar ionic interactions. These findings suggested to us that the KI-mediated dissociation of TRPC4 and protein 4.1 from the membrane skeleton occurred in the form of a TRPC4eCprotein 4.1 complex.
TRPC4 Interacts With Protein 4.1
If TRPC4 interacts with protein 4.1 and a TPRC4eCprotein 4.1 complex dissociates from the membrane skeleton in the presence of KI, then these proteins should coimmunoprecipitate from the salt-dissociated supernatant (Figure 3a). This is predicated on the idea that TRPC4eCprotein 4.1 binding is neither ionic in nature nor sensitive to disruption by KI. TRPC4 was immunoprecipitated from 25 and 100 mmol/L KI salt-dissociated supernatants and the coimmunoprecipitation of protein 4.1 assessed by immunoblot. Protein 4.1 coimmunoprecipitated with TRPC4 at both KI concentrations; these data indicate that indeed protein 4.1 binds to TRPC4 (Figure 3b). We next performed the reciprocal immunoprecipitation experiment and observed that TRPC4 coimmunoprecipitated with protein 4.1 (Figure 3c). Interestingly, in this reverse experiment, we observed that the TRPC4 coimmunoprecipitation was dependent on the salt concentration (ie, at 25 mmol/L KI, the coimmunoprecipitated TRPC4 appeared as a weak band, whereas at 100 mmol/L KI, the TRPC4 band was much stronger. Together, these data reveal that protein 4.1 binds to TRPC4, and further, increased KI concentrations promote interaction of TRPC4 with protein 4.1. Because salt strengthens hydrophobic interactions, we conclude that the interaction of TRPC4 with protein 4.1 is hydrophobic in nature.
TRPC4 Contains a Protein 4.1 Binding Domain
We screened TRPC4 for potential protein 4.1 binding domains and identified a sequence on the C terminus between amino acids 675 and 685 that resembled a consensus protein 4.1 binding sequence [(S/T)XXX(K/R)4] found in membrane-associated guanylate kinase proteins.24 Interestingly, the putative protein 4.1 binding sequence resides only 50 amino acids downstream from where the sixth transmembrane region exits the membrane, placing it in direct proximity to the pore region of the channel (Figure 4a and 4b). A proline-rich region (amino acids 654 to 663) lies immediately upstream of the protein 4.1 binding domain. Proline-rich regions are well recognized in signal transduction events, mediating dynamic proteineCprotein interactions.25,26 Typically, proline-rich regions act through hydrophobic attractive forces,27 which, in some systems, can be modulated by phosphorylation.28,29 The proline-rich region adjacent to the protein 4.1 binding domain contains threonine and serine as potential phosphorylation targets. Indeed, activation of SOC entry decreased gel mobility of TRPC4 isolated from the salt-dissociated supernatant, which was reversed by treatment with alkaline phosphatase (data not shown), suggesting SOC entry activation induced TRPC4 phosphorylation. Thus, we propose that protein 4.1 binds to the protein 4.1 binding domain on TRPC4 to form an interaction dynamically mediated by the adjacent proline-rich region and that functions to regulate ISOC channel activation.
ISOC Activation Requires the Protein 4.1 Binding Domain/Proline-Rich Region on TRPC4
If the protein 4.1 binding domain and adjacent proline-rich region on the C terminus of TRPC4 are critical for the binding of protein 4.1 and regulation of ISOC channel function, deletion of this region should disrupt thapsigargin-induced SOC entry. To test this hypothesis, adenoviral constructs were prepared to express either wild-type TRPC4 (TRPC4wt) or a mutant TRPC4 lacking the protein 4.1 binding domain and adjacent proline-rich region (TRPC4; Figure 5a). PAECs were infected with adenoviruseCTRPC4 or adenoviruseCTRPC4wt at multiplicities of infection 50:1, 25:1, and 10:1. The global [Ca2+]i response to thapsigargin was decreased in a dose-dependent manner in PAECs expressing TRPC4 compared with TRPC4wt (data not shown). At a multiplicity of infection of 25:1, the thapsigargin response was decreased 38% at the plateau phase (Figure 5b). Because the global [Ca2+]i response reflects all SOC entry channels activated by thapsigargin, we conclude that the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 is critical for activation of a subset of SOC entry channels. To determine the importance of the protein 4.1 binding domain and adjacent proline-rich region to ISOC channel activation, whole-cell patch-clamp analysis was used. In PAECs expressing TRPC4wt, a typical thapsigargin-induced ISOC current was observed (Figure 5c). Specifically, this current was small (50 pA at eC100 mV), inwardly rectifying, and exhibited a reversal potential near +30 mV.9,17 In PAECs expressing TRPC4, the thapsigargin-induced ISOC current was abolished (Figure 5c). The inward Ca2+ current was reduced to instrument noise level and the reversal potential left-shifted to 0 mV. Together, these data demonstrate a functional requirement for the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 for ISOC activation.
ISOC Activation Is Disrupted by Peptide Competition With the Protein 4.1 Binding Domain/Proline-Rich Region on TRPC4
Heterologous expression of TRPC4 in PAECs, lacking the protein 4.1 binding domain and adjacent proline-rich region, reduced global SOC entry and blocked ISOC activation. These studies clearly demonstrate functional significance of the protein 4.1 binding domain and adjacent proline-rich region on TRPC4. However, we could not determine from these data whether this effect was specifically attributable to the inability of protein 4.1 to bind to the TRPC4 subunit or whether the TRPC4 protein itself caused mistargeting of the ISOC channel such that it was not able to insert properly into the plasma membrane. We therefore chose an alternate approach to determine whether the protein 4.1 binding domain and adjacent proline-rich region is critical for ISOC channel function. Here, peptide competition experiments were performed. A 41-aa residue peptide corresponding to the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 (prr/4.1) and a scrambled peptide were synthesized. We reasoned that if the protein 4.1 binding domain (+/eCadjacent proline-rich region) on TRPC4 is the site of direct binding between protein 4.1 and TRPC4 needed for ISOC activation, then transfection of the prr/4.1 peptide into PAECs would disrupt endogenous TRPC4eCprotein 4.1 interaction and prevent ISOC activation. Peptides were transfected into PAECs using the Chariot reagent. In cells transfected with prr/4.1 peptide, the global [Ca2+]i response to thapsigargin was decreased 37% at the plateau phase compared with scrambled peptide (Figure 6a). Experiments performed in low Ca2+ buffer indicated no difference in Ca2+ release (Figure 6b). Patch-clamp experiments showed that although thapsigargin-induced ISOC was normal in PAECs transfected with scrambled peptide, it was completely blocked by the prr/4.1 peptide in eight of eleven experiments (Figure 6c). Similar to our observations in PAECs expressing TRPC4, the inward Ca2+ current was reduced to instrument noise level, and the reversal potential left-shifted to 0 mV. These results demonstrate a functional requirement for the protein 4.1 binding domain and adjacent proline-rich region located on the C terminus of TRPC4. We conclude that this region is essential for endothelial ISOC activation, likely through direct interaction between protein 4.1 and TRPC4. Collectively, these data support a regulatory role for protein 4.1 in ISOC channel activation.
Discussion
SOC entry was first described by Putney nearly two decades ago.30 Since then, the structural and functional components underlying activation of SOC entry have been aggressively studied, hotly debated, and, to date, still remain elusive. Although most would agree that the TRPCs 1, 3, 4, and 5 proteins can form SOC entry channels, no one has yet described the oligomeric state of even one endogenous SOC entry channel. Equally puzzling is the regulatory mechanism(s) linking store depletion to activation of SOC entry channels. Although these questions remain to be answered, the physiological implications of SOC entry are beginning to be understood. In the pulmonary circulation, for example, activation of the endothelial ISOC channel promotes endothelial barrier disruption,6 and as such, the ISOC channel may represent an attractive therapeutic target.
In this study, we identified protein 4.1 as a binding partner of TRPC4. This interaction likely occurs through a protein 4.1 binding domain +/eCadjacent proline-rich region found on the C terminus of TRPC4. The TRPC4eCprotein 4.1 interaction appears to be a high affinity, constitutive interaction, such that an intact TRPC4eCprotein 4.1 complex can be dissociated from the endothelial membrane skeleton. It is important that this interaction is constitutive and does not require activation of SOC entry. Thus, physical association between the ISOC channel and membrane skeleton is already established before Ca2+ store depletion. This observation supports our earlier evidence that disruption of the spectrineCprotein 4.1 interaction abolishes ISOC.17
A number of binding domains have been identified on the C terminus of TRPC4 as well as other TRPC homologues.31 Although all the domains have not yet been studied, the majority of data to date show that discrete proteineCprotein interactions serve specific functions, including modulation of SOC entry channel activation states and proper assembly within the signaling scaffold. Rosado and Sage32 demonstrated a requirement for interaction between the inositol-1,4,5-trisphosphate receptor (II) and TRPC1 for SOC entry in platelets; and Singh et al33 showed that a TRPC1eCcalmodulin interaction was important for channel inactivation in human submandibular gland cells. The proline-rich region adjacent to the protein 4.1 binding domain represents an additional site of proteineCprotein interaction. Parts of this region have already been implicated in immunophilin FKBP52 binding to TRPCs 1, 4, and 534 and Homer binding to TRPC1.35 The PSD-95/discs large protein/zonula occludens 1 (PDZ) binding domain located at the distal C terminus of TRPC4 and TRPC5 binds to the Na+/H+ exchange regulatory factor (NHERF) scaffolding protein36 (Figure 7). In addition to coupling TRPC4 to other members of the signaling cascade, such as G-proteineCcoupled receptors37 and phospholipase C,36,38 NHERF may function to indirectly link TRPC4 to the membrane skeleton. NHERF binds to members of the ezrin-radixin-moesin protein family,39 which can interact with transmembrane proteins and also bind directly to actin.40 The TRPC4eCNHERF interaction appears to be required for signalplex localization to organize signaling events into spatially41 and temporally efficient cascades.
In this study, we demonstrated a functional requirement of the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 for ISOC channel activation. ISOC was abolished by deletion of the protein 4.1 binding domain and adjacent proline-rich region. Transfection of a competitive peptide to this region also prevented ISOC activation. Indeed, this is the first evidence for an ISOC regulatory mechanism. Collectively, our data suggest that a protein 4.1 bridge between TRPC4 and the membrane skeleton is an absolute requirement for the regulation of channel activation and ion permeation.
Acknowledgments
This work was supported by National Institutes of Health grants HL66299 and HL60024 to T.S. and NS42183 to M.X.Z. D.L.C. received American Heart Association predoctoral fellowship 0215100B. The authors thank Linn Ayers, Anna Penton, and Tray Hamil for assistance with cell culture and Ingrid Durcova for performing immunocytochemistry.
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Vennekens R, Voets T, Bindels RJ, Droogmans G, Nilius B. Current understanding of mammalian TRP homologues. Cell Calcium. 2002; 31: 253eC264.
Clapham DE, Runnels LW, Streing C. The TRP ion channel family. Nat Rev Neurosci. 2001; 2: 387eC396.(Donna L. Cioffi, Songwei )
Department of Cell Biology and Neuroscience (M.A.), the University of South Alabama College of Medicine, Mobile
Department of Molecular and Cell Biology (S.R.G.), the University of Texas at Dallas
Department of Neuroscience and Center for Molecular Neurobiology (M.X.Z.), the Ohio State University, Columbus.
Abstract
Store-operated calcium (SOC) entry represents the principal Ca2+ entry pathway into nonexcitable cells. Despite intensive investigation, mechanisms underlying activation of SOC entry have remained elusive. The endothelial ISOC channel is a Ca2+-selective SOC entry channel to which the transient receptor potential (TRP) proteins TRPC1 and TRPC4 contribute subunits. Activation of ISOC is specifically regulated by the spectrineCactin membrane skeleton; however, the nature of coupling between the ISOC channel and membrane skeleton is unknown. Here we demonstrate that protein 4.1 is an essential component of the ISOC channel gating mechanism. Protein 4.1 interacts with TRPC4 and the membrane skeleton. Deletion of the protein 4.1 binding domain on TRPC4 or peptide competition to the protein 4.1 binding domain prevents ISOC activation. These findings reveal that interaction of protein 4.1 with TRPC4 is required for activation of the endothelial ISOC channel.
Key Words: cytoskeleton ISOC spectrin store-operated Ca2+ entry TRPC
Introduction
Elevation in cytosolic calcium, [Ca2+]i, is an essential step in numerous physiological and pathophysiological signal transduction events. Ion channels allowing for the entry of extracellular Ca2+ into the cytosol are the principal source of increased [Ca2+]i. In nonexcitable cells, store-operated calcium (SOC), and to a lesser extent, receptor-operated calcium (ROC), entry channels represent the main source of calcium entry.1,2 Although it is generally recognized that SOC entry occurs through Ca2+-selective and nonselective channels, the molecular makeup, regulation, and downstream targets of specific channels remain to be determined.
In endothelial cells, activation of SOC entry contributes to the formation of intercellular gaps, leading to endothelial barrier disruption,3eC5 a common factor in the development and progression of systemic and pulmonary diseases, including hypertension, atherosclerosis, and acute respiratory distress syndrome. It is therefore therapeutically desirable to identify specific SOC entry channel(s) involved in endothelial barrier disruption. We recently reported that activation of the Ca2+-selective SOC entry current ISOC contributes to the increase in [Ca2+]i necessary to disrupt the endothelial barrier.6 However, the molecular makeup and regulation of the channel that provides ISOC (ie, the ISOC channel) are incompletely understood.
Transient receptor potential (TRP) proteins encode SOC and ROC entry channels. The TRPC (canonical) subfamily consists of seven known members.7 It is not currently clear which TRPC homologues contribute to SOC versus ROC entry channels. Some studies have shown that TRPCs 1, 3, 4, and 5 can be activated by depletion of endoplasmic reticulum Ca2+, suggesting they contribute to SOC entry channels, whereas other studies have proposed contribution of these homologues to ROC entry channels.8 For the purpose of this study, we focused specifically on endogenous TRPC proteins that contribute to SOC entry in endothelial cells. Given this criterion, TRPC1 and TRPC4 contribute to the endothelial Ca2+-selective ISOC channel. Previously, our group used antisense technology to assess the contribution of endogenous TRPC1 to ISOC in pulmonary artery endothelial cells (PAECs).9 Inhibition of TRPC1 reduced but did not eliminate ISOC. On the other hand, endothelial cells obtained from TRPC4-deficient mice completely lacked ISOC.10 These studies suggest that although TRPC1 and TRPC4 contribute subunits to the ISOC channel molecular makeup, it is TRPC4 that is required for channel activation.
Activation of Drosophila melanogaster TRP and mammalian TRPC1 channels requires an intact C terminus.11eC13 However, to date, mechanisms regulating SOC entry channel activation are still unclear. Early studies in endothelial cells and platelets provided evidence supporting cytoskeletal regulation of SOC entry channel activation.5,14,15 Actin and spectrin principally comprise cellular membrane and cytoskeletons, whereas protein 4.1 and ankyrin function to link transmembrane proteins to the underlying membrane skeleton.16 Our group recently demonstrated that the specific interaction of protein 4.1 with spectrin was required for ISOC activation.17 Thus, we proposed that protein 4.1 functionally links the endothelial ISOC channel to the membrane skeleton.
Although the spectrineCprotein 4.1 interaction is necessary for ISOC activation, the mode of coupling between the membrane skeleton and ISOC channel is unknown. Because TRPC4 appears to be the subunit important for ISOC channel activation, we sought to determine whether protein 4.1 interacts with TRPC4. We report that protein 4.1 binds to TRPC4, likely within a conserved protein 4.1 binding domain located on the C terminus of TRPC4. Deletion of the protein 4.1 binding domain on TRPC4, as well as peptide transfection targeted to disrupt endogenous TRPC4eCprotein 4.1 interaction, similarly prevents ISOC activation. Collectively, these data support a regulatory role for protein 4.1 in ISOC channel activation.
Materials and Methods
Reagents
All reagents were obtained from Sigma-Aldrich unless specified otherwise. Antibodies to TRPC4 were provided by M.Z. or purchased from Sigma-Aldrich; protein 4.1 antibody was received from S.R.G. Secondary antibodies and chemiluminescence reagents were purchased from Santa Cruz Biotechnology or Molecular Probes, Inc.
Isolation and Culture of Pulmonary Endothelial Cells
PAECs were isolated and cultured using a method described by Creighton et al.18
Immunocytochemistry
PAEC monolayers were methanol fixed and permeabilized. Primary antibody to TRPC4 (Sigma-Aldrich) was added at 1:200 dilution for 1 hour followed by secondary antibody (Alexa Fluor 488 goat anti-rabbit; Molecular Probes, Inc.) at 1:300 dilution for 40 minutes. Fluorescent and Normarski differential interference contrast images were obtained using a Leica TCS SP2 confocal laser scanning microscope fitted with a 63x oil immersion objective.
Preparation and Detergent Extraction of Membrane/Cytoskeleton Fractions19
For detailed protocol, refer to the online supplement, available at http://circres.ahajournals.org.
Thapsigargin Treatment
Cells were exposed to 1 eol/L thapsigargin (Calbiochem) for 2.5 to 3 minutes at room temperature before beginning lysing procedure. In these experiments, the total time from addition of thapsigargin to end of lysing procedure (ie, samples at eC80°C) was 12 to 14 minutes.
Immunoprecipitation Reactions and Western Blot Analysis
Immunoprecipitation Reactions
To 150 to 250 e蘈 of supernatant was added TRPC4 antibody (1:100 dilution) or protein 4.1 antibody (1:130 dilution), and 2% protease inhibitor cocktail (10x). The sample was rocked at 4°C (typically overnight). This was followed by addition of 40 to 50 e蘈 EZ View Red Protein A affinity gel (Sigma-Aldrich) and 2% protease inhibitor cocktail (10x). The sample was rocked for an additional 4 to 5 hours, pelleted, washed 3x750 e蘈 ice-cold PBS/0.1% protease inhibitor cocktail (10x), and resuspended in 4x lithium dodecylsulfate (Invitrogen).
Western Blot Analysis
For SDS-PAGE, 7%, 8%, or 4% to 12% gradient (Invitrogen) gels were typically used. Samples were transferred to nitro-cellulose or polyvinylidene difluoride membranes at 30 V. Membranes were rocked with primary antibody for 2.5 hours at room temperature or overnight at 4°C and with secondary antibody for 1 hour. Proteins were visualized via enhanced chemiluminescence detection (Luminol Reagent; Santa Cruz Biotechnology).
Adenoviral Constructs
Recombinant adenoviruses were generated according to established protocols20 using commercially available plasmids (AdMax; Microbix). For protocol description, refer to the online supplement.
Adenovirus Infection
Endothelial cells were seeded on 100- or 35-mm culture dishes±25-mm circle microscope glass coverslips (Fisher Scientific) and grown to &60% to 90% confluence. Cells were infected with adenovirus constructs at multiplicities of infection equal to 50:1, 25:1, or 10:1. Ca2+ and ISOC measurements were performed at 36 to 50 hours after infection.
Peptide Transfection
Two peptides were synthesized by Alpha Diagnostic International, Inc. The prr/4.1 peptide corresponds to the region on TRPC4 encompassing the protein 4.1 binding domain and adjacent proline-rich region. A peptide with scrambled sequence was also prepared. Peptides were transfected into PAECs using Chariot reagent (Active Motif) generally following the suggested protocol of the manufacturer. For protocol description, refer to the online supplement.
Ca2+ Measurements
Endothelial cells were seeded onto 25-mm glass coverslips and grown to confluence. [Ca2+]i was estimated with the fluorophore fura 2-acetoxymethylester (fura-2 AM; Molecular Probes) according to methods described previously.21
Patch-Clamp Electrophysiology
Conventional whole-cell voltage clamp configuration was performed to measure transmembrane currents in single rat PAECs using the protocol described by Wu et al.17
Results
TRPC4 Localizes to Sites of CelleCCell Adhesion
TRPC4 is a subunit of the endothelial ISOC channel that, when activated, promotes endothelial barrier disruption through the formation of interendothelial cell gaps. Immunocytochemistry was performed to determine cellular localization of endogenous TRPC4. Positive TRPC4 staining was identified in the plasma membrane of PAECs. Staining was particularly strong in areas of the membrane involved in celleCcell adhesion (Figure 1). Additional punctate staining was evident, likely because of the presence of TRPC4 in cholesterol-rich (eg, caveolae) areas of the membrane.22 The localization of TRPC4 at sites of celleCcell adhesion supports a role for the endothelial ISOC channel in intercellular gap formation.6
TRPC4 Binds to the PAEC Membrane Skeleton
Interaction between spectrin and protein 4.1 is required for activation of the ISOC channel.17 However, the nature of interaction between the membrane skeleton and proteins encoding the ISOC channel is unknown. Because TRPC4 appears to be required for channel activation, we sought to determine whether TRPC4 physically interacts with the membrane skeleton. Membrane/cytoskeleton fractions of PAECs were detergent extracted using octyl--D-glucopyranoside to yield pellet and supernatant fractions. SDS-PAGE and immunoblotting of these fractions found TRPC4 exclusively in the pellet (Figure 2a). Actin, a membrane skeleton marker, was also found in the pellet, whereas endothelial myosin light chain kinase 1 resided in the supernatant. Protein 4.1 was detected in pellet and supernatant fractions. Thus, we found that TRPC4 physically interacts with the membrane skeleton of PAECs because it was found in the pellet fraction extracted from membrane/cytoskeleton preparations. It is noteworthy that this physical association appears to be constitutive (ie, not requiring channel activation).
TRPC4 Interacts With the Membrane Skeleton in a Salt-Sensitive Manner
Having demonstrated that TRPC4 physically associates with the membrane skeleton, we next questioned the nature of this interaction. Salts are ionic compounds that weaken ionic and strengthen hydrophobic proteineCprotein interactions.23 When PAEC membrane/cytoskeleton fractions were detergent extracted in the presence of 1 mol/L potassium iodide (KI), TRPC4 was found in the supernatant fraction instead of the pellet (data not shown). This suggested that TRPC4 associates with the membrane skeleton through an ionic interaction, which is sensitive to KI disruption. To more selectively probe the TRPC4eCmembrane skeleton interaction, we repeated the experiments using lower salt concentrations. Even at 150 mmol/L KI, TRPC4 resided in the supernatant (Figure 2b). We also probed for protein 4.1. Similar to TRPC4, the interaction of protein 4.1 with the membrane skeleton was sensitive to 150 mmol/L KI because the majority of protein 4.1 was now found in the supernatant. These data indicate that TRPC4 and protein 4.1 associate with the membrane skeleton through an ionic interaction sensitive to KI disruption.
To determine the minimal KI concentration necessary to dissociate TRPC4 from the membrane skeleton, we performed detergent/KI extractions over a low KI concentration range (0 to 100 mmol/L KI). Immunoblot analysis revealed that the interaction of TRPC4 with the membrane skeleton is exquisitely sensitive to this concentration range because it exhibited a dose-dependent shift from the pellet fraction to the supernatant (Figure 2c). Protein 4.1 was also sensitive to this KI concentration range (ie, at 0 mmol/L KI, the majority of protein 4.1 resided in the pellet fraction, whereas at 100 mmol/L KI, protein 4.1 was found predominantly in the supernatant). Actin, on the other hand, was found mainly in the pellet fraction irrespective of solution ionic strength. Collectively, these observations demonstrate that TRPC4 and protein 4.1 associate with the membrane skeleton through similar ionic interactions. These findings suggested to us that the KI-mediated dissociation of TRPC4 and protein 4.1 from the membrane skeleton occurred in the form of a TRPC4eCprotein 4.1 complex.
TRPC4 Interacts With Protein 4.1
If TRPC4 interacts with protein 4.1 and a TPRC4eCprotein 4.1 complex dissociates from the membrane skeleton in the presence of KI, then these proteins should coimmunoprecipitate from the salt-dissociated supernatant (Figure 3a). This is predicated on the idea that TRPC4eCprotein 4.1 binding is neither ionic in nature nor sensitive to disruption by KI. TRPC4 was immunoprecipitated from 25 and 100 mmol/L KI salt-dissociated supernatants and the coimmunoprecipitation of protein 4.1 assessed by immunoblot. Protein 4.1 coimmunoprecipitated with TRPC4 at both KI concentrations; these data indicate that indeed protein 4.1 binds to TRPC4 (Figure 3b). We next performed the reciprocal immunoprecipitation experiment and observed that TRPC4 coimmunoprecipitated with protein 4.1 (Figure 3c). Interestingly, in this reverse experiment, we observed that the TRPC4 coimmunoprecipitation was dependent on the salt concentration (ie, at 25 mmol/L KI, the coimmunoprecipitated TRPC4 appeared as a weak band, whereas at 100 mmol/L KI, the TRPC4 band was much stronger. Together, these data reveal that protein 4.1 binds to TRPC4, and further, increased KI concentrations promote interaction of TRPC4 with protein 4.1. Because salt strengthens hydrophobic interactions, we conclude that the interaction of TRPC4 with protein 4.1 is hydrophobic in nature.
TRPC4 Contains a Protein 4.1 Binding Domain
We screened TRPC4 for potential protein 4.1 binding domains and identified a sequence on the C terminus between amino acids 675 and 685 that resembled a consensus protein 4.1 binding sequence [(S/T)XXX(K/R)4] found in membrane-associated guanylate kinase proteins.24 Interestingly, the putative protein 4.1 binding sequence resides only 50 amino acids downstream from where the sixth transmembrane region exits the membrane, placing it in direct proximity to the pore region of the channel (Figure 4a and 4b). A proline-rich region (amino acids 654 to 663) lies immediately upstream of the protein 4.1 binding domain. Proline-rich regions are well recognized in signal transduction events, mediating dynamic proteineCprotein interactions.25,26 Typically, proline-rich regions act through hydrophobic attractive forces,27 which, in some systems, can be modulated by phosphorylation.28,29 The proline-rich region adjacent to the protein 4.1 binding domain contains threonine and serine as potential phosphorylation targets. Indeed, activation of SOC entry decreased gel mobility of TRPC4 isolated from the salt-dissociated supernatant, which was reversed by treatment with alkaline phosphatase (data not shown), suggesting SOC entry activation induced TRPC4 phosphorylation. Thus, we propose that protein 4.1 binds to the protein 4.1 binding domain on TRPC4 to form an interaction dynamically mediated by the adjacent proline-rich region and that functions to regulate ISOC channel activation.
ISOC Activation Requires the Protein 4.1 Binding Domain/Proline-Rich Region on TRPC4
If the protein 4.1 binding domain and adjacent proline-rich region on the C terminus of TRPC4 are critical for the binding of protein 4.1 and regulation of ISOC channel function, deletion of this region should disrupt thapsigargin-induced SOC entry. To test this hypothesis, adenoviral constructs were prepared to express either wild-type TRPC4 (TRPC4wt) or a mutant TRPC4 lacking the protein 4.1 binding domain and adjacent proline-rich region (TRPC4; Figure 5a). PAECs were infected with adenoviruseCTRPC4 or adenoviruseCTRPC4wt at multiplicities of infection 50:1, 25:1, and 10:1. The global [Ca2+]i response to thapsigargin was decreased in a dose-dependent manner in PAECs expressing TRPC4 compared with TRPC4wt (data not shown). At a multiplicity of infection of 25:1, the thapsigargin response was decreased 38% at the plateau phase (Figure 5b). Because the global [Ca2+]i response reflects all SOC entry channels activated by thapsigargin, we conclude that the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 is critical for activation of a subset of SOC entry channels. To determine the importance of the protein 4.1 binding domain and adjacent proline-rich region to ISOC channel activation, whole-cell patch-clamp analysis was used. In PAECs expressing TRPC4wt, a typical thapsigargin-induced ISOC current was observed (Figure 5c). Specifically, this current was small (50 pA at eC100 mV), inwardly rectifying, and exhibited a reversal potential near +30 mV.9,17 In PAECs expressing TRPC4, the thapsigargin-induced ISOC current was abolished (Figure 5c). The inward Ca2+ current was reduced to instrument noise level and the reversal potential left-shifted to 0 mV. Together, these data demonstrate a functional requirement for the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 for ISOC activation.
ISOC Activation Is Disrupted by Peptide Competition With the Protein 4.1 Binding Domain/Proline-Rich Region on TRPC4
Heterologous expression of TRPC4 in PAECs, lacking the protein 4.1 binding domain and adjacent proline-rich region, reduced global SOC entry and blocked ISOC activation. These studies clearly demonstrate functional significance of the protein 4.1 binding domain and adjacent proline-rich region on TRPC4. However, we could not determine from these data whether this effect was specifically attributable to the inability of protein 4.1 to bind to the TRPC4 subunit or whether the TRPC4 protein itself caused mistargeting of the ISOC channel such that it was not able to insert properly into the plasma membrane. We therefore chose an alternate approach to determine whether the protein 4.1 binding domain and adjacent proline-rich region is critical for ISOC channel function. Here, peptide competition experiments were performed. A 41-aa residue peptide corresponding to the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 (prr/4.1) and a scrambled peptide were synthesized. We reasoned that if the protein 4.1 binding domain (+/eCadjacent proline-rich region) on TRPC4 is the site of direct binding between protein 4.1 and TRPC4 needed for ISOC activation, then transfection of the prr/4.1 peptide into PAECs would disrupt endogenous TRPC4eCprotein 4.1 interaction and prevent ISOC activation. Peptides were transfected into PAECs using the Chariot reagent. In cells transfected with prr/4.1 peptide, the global [Ca2+]i response to thapsigargin was decreased 37% at the plateau phase compared with scrambled peptide (Figure 6a). Experiments performed in low Ca2+ buffer indicated no difference in Ca2+ release (Figure 6b). Patch-clamp experiments showed that although thapsigargin-induced ISOC was normal in PAECs transfected with scrambled peptide, it was completely blocked by the prr/4.1 peptide in eight of eleven experiments (Figure 6c). Similar to our observations in PAECs expressing TRPC4, the inward Ca2+ current was reduced to instrument noise level, and the reversal potential left-shifted to 0 mV. These results demonstrate a functional requirement for the protein 4.1 binding domain and adjacent proline-rich region located on the C terminus of TRPC4. We conclude that this region is essential for endothelial ISOC activation, likely through direct interaction between protein 4.1 and TRPC4. Collectively, these data support a regulatory role for protein 4.1 in ISOC channel activation.
Discussion
SOC entry was first described by Putney nearly two decades ago.30 Since then, the structural and functional components underlying activation of SOC entry have been aggressively studied, hotly debated, and, to date, still remain elusive. Although most would agree that the TRPCs 1, 3, 4, and 5 proteins can form SOC entry channels, no one has yet described the oligomeric state of even one endogenous SOC entry channel. Equally puzzling is the regulatory mechanism(s) linking store depletion to activation of SOC entry channels. Although these questions remain to be answered, the physiological implications of SOC entry are beginning to be understood. In the pulmonary circulation, for example, activation of the endothelial ISOC channel promotes endothelial barrier disruption,6 and as such, the ISOC channel may represent an attractive therapeutic target.
In this study, we identified protein 4.1 as a binding partner of TRPC4. This interaction likely occurs through a protein 4.1 binding domain +/eCadjacent proline-rich region found on the C terminus of TRPC4. The TRPC4eCprotein 4.1 interaction appears to be a high affinity, constitutive interaction, such that an intact TRPC4eCprotein 4.1 complex can be dissociated from the endothelial membrane skeleton. It is important that this interaction is constitutive and does not require activation of SOC entry. Thus, physical association between the ISOC channel and membrane skeleton is already established before Ca2+ store depletion. This observation supports our earlier evidence that disruption of the spectrineCprotein 4.1 interaction abolishes ISOC.17
A number of binding domains have been identified on the C terminus of TRPC4 as well as other TRPC homologues.31 Although all the domains have not yet been studied, the majority of data to date show that discrete proteineCprotein interactions serve specific functions, including modulation of SOC entry channel activation states and proper assembly within the signaling scaffold. Rosado and Sage32 demonstrated a requirement for interaction between the inositol-1,4,5-trisphosphate receptor (II) and TRPC1 for SOC entry in platelets; and Singh et al33 showed that a TRPC1eCcalmodulin interaction was important for channel inactivation in human submandibular gland cells. The proline-rich region adjacent to the protein 4.1 binding domain represents an additional site of proteineCprotein interaction. Parts of this region have already been implicated in immunophilin FKBP52 binding to TRPCs 1, 4, and 534 and Homer binding to TRPC1.35 The PSD-95/discs large protein/zonula occludens 1 (PDZ) binding domain located at the distal C terminus of TRPC4 and TRPC5 binds to the Na+/H+ exchange regulatory factor (NHERF) scaffolding protein36 (Figure 7). In addition to coupling TRPC4 to other members of the signaling cascade, such as G-proteineCcoupled receptors37 and phospholipase C,36,38 NHERF may function to indirectly link TRPC4 to the membrane skeleton. NHERF binds to members of the ezrin-radixin-moesin protein family,39 which can interact with transmembrane proteins and also bind directly to actin.40 The TRPC4eCNHERF interaction appears to be required for signalplex localization to organize signaling events into spatially41 and temporally efficient cascades.
In this study, we demonstrated a functional requirement of the protein 4.1 binding domain and adjacent proline-rich region on TRPC4 for ISOC channel activation. ISOC was abolished by deletion of the protein 4.1 binding domain and adjacent proline-rich region. Transfection of a competitive peptide to this region also prevented ISOC activation. Indeed, this is the first evidence for an ISOC regulatory mechanism. Collectively, our data suggest that a protein 4.1 bridge between TRPC4 and the membrane skeleton is an absolute requirement for the regulation of channel activation and ion permeation.
Acknowledgments
This work was supported by National Institutes of Health grants HL66299 and HL60024 to T.S. and NS42183 to M.X.Z. D.L.C. received American Heart Association predoctoral fellowship 0215100B. The authors thank Linn Ayers, Anna Penton, and Tray Hamil for assistance with cell culture and Ingrid Durcova for performing immunocytochemistry.
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