Isoform-Selective Physical Coupling of TRPC3 Channels to IP3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility
Rationale: Inositol 1,4,5-trisphosphate (IP3)-induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but functional signaling mechanisms mediating this effect are unclear.
Objectives: Study mechanisms by which IP3Rs stimulate TRPC channels in myocytes of resistance-size cerebral arteries.
Methods and Results: Immunofluorescence resonance energy transfer (immuno-FRET) microscopy using isoform-selective antibodies indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels in arterial myocytes. Endothelin-1 (ET-1), a phospholipase C–coupled receptor agonist, elevated immuno-FRET between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. TRPC3, but not TRPC6, coimmunoprecipitated with IP3R1. TRPC3 and TRPC6 antibodies selectively inhibited recombinant channels, but only the TRPC3 antibody blocked IP3-induced nonselective cation current (ICat) in myocytes. TRPC3 knockdown attenuated immuno-FRET between IP3R1 and TRPC3, IP3-induced ICat activation, and ET-1 and IP3-induced vasoconstriction, whereas TRPC6 channel knockdown had no effect. ET-1 did not alter total or plasma membrane-localized TRPC3, as determined using surface biotinylation. RT-PCR demonstrated that C-terminal calmodulin and IP3R binding (CIRB) domains are present in myocyte TRPC3 and TRPC6 channels. A peptide corresponding to the IP3R N-terminal region that can interact with TRPC channels activated ICat. A TRPC3 CIRB domain peptide attenuated IP3- and ET-1–induced ICat activation and vasoconstriction.
Conclusions: IP3 stimulates direct coupling between IP3R1 and membrane-resident TRPC3 channels in arterial myocytes, leading to ICat activation and vasoconstriction. Close spatial proximity between IP3R1 and TRPC3 establishes this isoform-selective functional interaction.
Activation of plasma membrane phospholipase (PL)C-coupled receptors by vasoconstrictor agonists leads to phosphatidylinositol 4,5-bisphosphate (PIP2) hydrolysis and the generation of inositol-1,4,5-trisphosphate (IP3) and diacylglycerol.1 In vascular myocytes, diacylglycerol (DAG) activates protein kinase (PK)C, leading to the phosphorylation of a wide variety of proteins, including ion channels.2 IP3 binds to sarcoplasmic reticulum (SR) IP3 receptors (IP3Rs), resulting in SR Ca2+ release, an elevation in intracellular Ca2+ concentration ([Ca2+]i), and vasoconstriction.3 Recent evidence also indicates that IP3-induced vasoconstriction can occur independently of SR Ca2+ release and via the activation of type 1 IP3 receptors (IP3R1) and type 3 canonical transient receptor potential (TRPC) channels.4,5 However, the functional signaling mechanisms by which IP3Rs and TRPC channels communicate in arterial myocytes are unclear.
The mammalian TRP channel superfamily is encoded by at least 28 different genes that are subdivided into 7 families.6 These families encode ion channels with diverse ion selectivity, modes of regulation, and physiological functions.6 Vascular myocytes express at least 4 TRP families, including TRPC, TRPM, TRPV, and TRPP.7–10 These channels regulate arterial myocyte membrane potential, [Ca2+]i, contractility, and proliferation, and are implicated in the etiology of vascular diseases.4,8–12 Given the diversity of vascular myocyte TRP channels, it has become important to identify signaling pathways that specifically regulate individual channel isoforms and to determine whether individual TRP channel isoforms perform distinct physiological functions. For instance, arterial myocytes express multiple TRPC isoforms, including 1, 3, and 6, but whether signaling pathways specifically regulate individual members and what the mechanisms are that mediate such effects are poorly understood.9,10 In cerebral artery myocytes, vasoconstrictors activate TRPC3,4,9,13,14 whereas intravascular pressure stimulates TRPC6.9,10 Thus, TRPC3 and TRPC6 channels perform distinct physiological functions, but signaling pathways that mediate this differential regulation are unclear.
Here, we studied mechanisms by which IP3R1, the principal molecular and functional arterial myocyte IP3R isoform,5 stimulates TRPC currents in resistance-size cerebral arteries. Data suggest that IP3R1 is in close spatial proximity to, and associates with, TRPC3, but not TRPC6 or TRPM4 channels. Endothelin (ET)-1, a PLC-coupled receptor agonist, and IP3 alter the interaction between the IP3R N terminus and the TRPC3 channel C terminus, leading to channel activation and vasoconstriction. Data indicate that IP3R1 selectively couples to TRPC3 channels because of the close spatial proximity of these proteins and that this mechanism is essential for mediating ET-1 and IP3-induced vasoconstriction.
Animal protocols used were reviewed and approved by the Animal Care and Use Committee at the University of Tennessee Health Science Center. Sprague–Dawley rat (≈250 g) resistance-size cerebral arteries and myocytes from these arteries were isolated as previously described.4
Immunofluorescence Resonance Energy Transfer
Paraformaldehyde-fixed myocytes were incubated with primary antibodies: mouse monoclonal anti-IP3R1 and rabbit polyclonal anti-TRPC3, rabbit polyclonal anti-TRPC6, or rabbit polyclonal anti-TRPM4. Cells were then labeled with secondary antibodies: Cy3-conjugated donkey antimouse for IP3R1 and Cy2-conjugated goat anti-rabbit for TRPC3, TRPM4, or TRPC6. Fluorescent images, acquired using a Zeiss LSM 5 Pascal confocal microscope, were background-subtracted and normalized fluorescence resonance energy transfer (N-FRET)–calculated using the method of Xia and Liu.15
TRPC Channel Knockdown
Silencing vectors that express TRPC3- (TRPC3shV), TRPC6- (TRPC6shV), or scrambled control (scrm) short hairpin RNA were inserted into cerebral arteries using reverse permeabilization, as previously described.16
Arterial lysate was incubated with control mouse IgG or IP3R1 monoclonal antibody and then incubated with protein A sepharose beads. Protein samples were then analyzed by Western blotting using mouse monoclonal anti-IP3R1, mouse polyclonal anti-TRPC3, rabbit polyclonal anti-TRPC6, and horseradish peroxidase–conjugated secondary antibodies.
Membrane expression of TRPC3 channels was measured using surface biotinylation of intact arteries, as previously described.17
Cell Culture and Transfection
HEK293 cells were transfected with vectors encoding recombinant TRPC3 or TRPC6, kindly provided by Dr James Putney (National Institute of Environmental Health Sciences) and Dr Jochen Reiser (University of Miami, Fla), respectively. Electrophysiology and Western blotting experiments were performed 36 to 72 hour after transfection.
Polymerase Chain Reaction
RT-PCR was performed on pure populations of ≈100 selected cerebral artery myocytes, as described previously.18
Membrane cation currents were measured in arterial myocytes and HEK293 cells using the conventional whole cell patch-clamp configuration, as performed previously.4,5
Pressurized Artery Diameter Measurement
Arterial diameter was measured using pressurized artery myography, as described previously.18
Data are expressed as mean±standard error of the mean. Statistical significance was calculated by using Student’s t-tests for paired or unpaired data or ANOVA followed by Student–Newman–Keuls test for multiple data sets. P<0.05 was considered significant.
An expanded Methods section is available in the Online Data Supplement at http://circres.ahajournals.org.
IP3R1 Is in Close Proximity to TRPC3 Channels in Cerebral Artery Myocytes
IP3R1 is the principal molecular and functional IP3R isoform that is expressed in myocytes of cerebral arteries and aorta.5,19 To explore spatial proximity between IP3R1 and 3 different TRP channel isoforms in cerebral artery myocytes, we used immunofluorescence resonance energy transfer. Specifically, spatial localization between IP3R1 and TRPC3, TRPC6, or TRPM4 channels was examined. Cy3- and Cy2-labeled secondary antibodies bound to primary antibodies targeting IP3R1 (monoclonal) and TRPC3 (polyclonal), respectively, produced mean N-FRET of ≈25% (Figure 1A and 1B). In contrast, fluorescent antibodies bound to IP3R1 and TRPM4 (polyclonal), or IP3R1 and TRPC6 (polyclonal), generated mean N-FRET of ≈11% and ≈12%, respectively (Figure 1A and 1B). ET-1 increased mean N-FRET between IP3R1 and TRPC3-bound antibodies to ≈34%. In contrast, N-FRET between IP3R1 and TRPC3-bound antibodies was not altered by membrane depolarization with 30 mmol/L K+ (Figure 1B). ET-1 did not change N-FRET between antibodies bound to IP3R1 and TRPC6 or IP3R1 and TRPM4 (Figure 1A and 1B). Antigenic peptides for TRPC3, TRPC6, and TRPM4 specifically blocked immunofluorescence produced by each antibody, but did not alter immunofluorescence detection by the other 2 antibodies (Online Figure I, A).
Next, N-FRET was measured in myocytes in which TRPC3 or TRPC6 expression was reduced using short hairpin RNA and quantified using Western blotting. The selectivity of antibodies for Western blotting experiments was examined. Whole Western blots for IP3R1, TRPC3 and TRPC6 antibodies on arterial lysate are illustrated in Online Figure I (B). The monoclonal IP3R1 antibody detected only 1 band corresponding to IP3R1 (Online Figure I, B), and this band is reduced by IP3R1 knockdown, indicating specificity.5 Antigenic peptides abolished appropriate size bands for each TRPC isoform but did not alter detection of the other TRPC protein (Online Figure I, C). The TRPC3 antibody detected recombinant TRPC3 expressed in HEK293 cells, but did not detect recombinant TRPC6 (Online Figure II, A). Similarly, the TRPC6 antibody detected recombinant TRPC6, but did not detect recombinant TRPC3 (Online Figure II, A). Endogenous TRPC3 and TRPC6 proteins were detected in HEK293 cells, consistent with a previous report.20 Taken together, these data further indicate that the TRPC3 and TRPC6 antibodies are selective for their respective TRPC channel isoforms.
Western blotting indicated that TRPC3shV and TRPC6shV reduced mean TRPC3 and TRPC6 protein by ≈40 and 43%, respectively, of that in arteries treated with scrm (Figure 1C and 1D). TRPC3shV did not alter TRPC6 and IP3R1 expression and TRPC6shV did not alter TRPC3 and IP3R1 expression (Figure 1C and 1D). TRPC3 knockdown reduced control N-FRET and the ET-1–induced elevation in N-FRET between IP3R1 and TRPC3 (Figure 1E). In contrast, TRPC6 knockdown had no effect on N-FRET between IP3R1 and TRPC6 in control or ET-1 (Figure 1E), indicating that this signal represents background N-FRET.
Coimmunoprecipitation (co-IP) was used to examine whether IP3R1 physically associates with TRPC3 and TRPC6 channels in small cerebral arteries. Because of the small size of the resistance-size vessels used in this study, arteries collected from ≈20 rats were required for each experiment. The IP3R1 monoclonal antibody coimmunoprecipitated both IP3R1 and TRPC3 channels from lysate (Figure 2). In contrast, TRPC6 was not detected in the same IP3R1 immunoprecipitate (Figure 2).
In summary, data indicate that TRPC3, but not TRPM4 or TRPC6, is in close spatial proximity to, and associates with, IP3R1 channels in myocytes of resistance-size cerebral arteries. Data also demonstrate that ET-1 modifies the molecular relationship between IP3R1 and TRPC3.
TRPC3 Mediates IP3-Induced ICat Activation in Cerebral Artery Myocytes
The contribution of TRPC3 and TRPC6 channels to IP3-induced ICat was examined. The TRPC3 antibody blocked whole-cell currents generated by recombinant TRPC3 expressed in HEK293 cells (Figure 3A and 3B; Online Figure II, B). In contrast, the TRPC3 antibody did not alter currents produced by recombinant TRPC6 channels. Similarly, the TRPC6 antibody blocked recombinant TRPC6 channel currents, but did not alter TRPC3 currents (Figure 3A and 3B; Online Figure II, B). Therefore, the antibodies selectively inhibit their respective TRPC currents. The TRPC3 antibody reduced mean IP3-induced ICat density by ≈57% in arterial myocytes (Figure 3C and 3D). In contrast, the TRPC6 antibody had no effect on IP3-induced ICat density in arterial myocytes (Figure 3C and 3D).
IP3-induced ICat activation and vasoconstriction were studied in myocytes and arteries in which TRPC3 or TRPC6 expression was reduced using short hairpin RNA. TRPC3 knockdown reduced IP3-induced ICat density by ≈41%. In contrast, TRPC6 knockdown did not alter IP3-induced ICat density (Figure 4A and 4B). TRPC6 knockdown reduces myogenic tone at intravascular pressures >40 mm Hg.10 Therefore, diameter regulation was measured in arteries pressurized to 20 mm Hg to avoid differences in baseline tone between arteries in which TRPC3 and TRPC6 were knocked down. TRPC3 knockdown attenuated IP3- and ET-1–induced vasoconstriction by ≈43 and 49%, respectively (Figure 4C through 4E). In contrast, TRPC6 knockdown had no effect on IP3- and ET-1–induced vasoconstriction (Figure 4C through 4E). These data indicate that TRPC3, but not TRPC6, channels mediate IP3-induced ICat activation and contraction in cerebral artery myocytes.
ET-1 Does Not Increase Surface Expression of TRPC3 Channels in Myocytes
An increase in ion channel activity (NPo) can occur because of both an elevation in the number of channels (N) and an increase in channel open probability (Po). To investigate whether ET-1 alters plasma membrane TRPC3 channel protein, we used surface biotinylation.17
Confirmation that biotin specifically labels surface proteins was obtained using biotin-treated arterial segments exposed to Texas Red-conjugated streptavidin (Online Figure III). In addition, heat shock protein 90, an intracellular protein, was detected only in the nonbiotinylated (cytosolic) fraction (Figure 5A). Biotinylation indicated that in control, ≈30% of total TRPC3 protein was present in the arterial plasma membrane (Figure 5B and 5C). ET-1 did not alter the percentage of plasma membrane-localized TRPC3 (Figure 5B and 5C). These data indicate that a significant proportion of arterial TRPC3 is cytosolic and that ET-1 elevates myocyte ICat by stimulating plasma membrane-resident TRPC3 channels.
Calmodulin and IP3R Binding Domains Are Present in Myocyte TRPC3 and TRPC6 Channels
Recombinant IP3R N-termini contain a conserved amino acid sequence that can interact with a conserved C-terminal CIRB (calmodulin and IP3R binding) domain found on all on TRPC isoforms.21,22 TRPM4 does not contain a CIRB domain (GenBank accession no. XM_574447). Our data indicate that isoform-selective association of TRPC3 channels to IP3R1 occurs in arterial myocytes (Figures 1 through 4⇑⇑⇑). Therefore, we used RT-PCR to investigate whether myocyte TRPC channels contain CIRB domains. Primers used were selective for TRPC3 and TRPC6 CIRB domains (Online Figure IV). RT-PCR was performed on manually selected arterial myocytes to prevent message contamination from other vascular wall cell types.18 Data indicated that the C-termini of arterial myocyte TRPC3 and TRPC6 channels contain CIRB domains (Figure 6A).
TRPC3-IP3R1 Physical Coupling Is Required for ET-1– and IP3-Induced ICat Activation
We tested the hypothesis that IP3-induced ICat activation in arterial myocytes occurs through an interaction between the conserved IP3R interaction domain and the TRPC CIRB domain. Synthetic peptides corresponding to a region in the TRPC3 CIRB domain (QIMKRLIKRYVLKAQVDKEND, CIRBP),21–24 and a scrambled control peptide (RDLKKAQNLVEIQKKYDRMVI, CIRBPscrm) were generated. CIRBP and CIRBPscrm alone did not alter mean baseline ICat density (pA/pF: no peptide, −3.5±0.5, n=5; CIRBPscrm, −3.7±0.9, n=10; CIRBP, −2.9±0.6, n=11; P>0.05 for each) in arterial myocytes. In contrast, CIRBP reduced IP3 and ET-1–induced ICat activation by ≈63% and 60%, respectively, when compared with CIRBPscrm (Figure 6B through 6D).
To study physiological functions of the IP3R N terminus, a peptide was constructed corresponding to the IP3R N-terminal sequence containing the conserved TRPC-binding region (EEVWLFWRD, IP3RntP). IP3RntP increased mean ICat density by ≈260% (Figure 7A and 7B). Gadolinium (Gd3+), a nonselective cation channel blocker, had no significant effect on baseline currents, but reduced mean IP3RntP-induced ICat density by ≈70% (Figure 7A and 7B). These data indicate that physical association between the IP3R1 N terminus and the TRPC C terminus CIRB domain leads to ICat activation in arterial myocytes.
CIRBP Attenuates ET-1– and IP3-Induced Vasoconstriction
The physiological function of molecular coupling between IP3R and TRPC3 channels was investigated by measuring diameter regulation of pressurized (60 mm Hg) arteries. Membrane-permeant CIRBP (CIRBP-TAT) and scrambled control (CIRBPscrm-TAT) peptides were each generated by conjugation to a HIV-1 TAT sequence (RRRQRRKKRGY).25 CIRBP-TAT reduced vasoconstriction caused by Bt-IP3, a membrane permeant IP3 analog, and ET-1 by ≈78% and ≈65%, respectively, when compared with CIRBPscrm-TAT (Figure 8A through 8D). These data indicate that interaction between the IP3R1 N terminus and the TRPC3 channel CIRB domain contributes to IP3 and ET-1–induced vasoconstriction.
Here, we identify a novel vasoregulatory signaling mechanism whereby IP3Rs couple to plasma membrane TRPC3 channels in arterial myocytes (Online Figure V). Novel findings of this study are that in cerebral artery myocytes: (1) IP3R1 is located in close proximity to TRPC3, but not TRPC6 or TRPM4 channels; (2) ET-1 elevates FRET between fluorescent antibodies bound to IP3R1 and TRPC3 channels but does not alter FRET between IP3R1 and TRPC6 or TRPM4 channels; (3) TRPC3 channels co-IP with IP3R1, whereas TRPC6 channels do not; (4) TRPC3, but not TRPC6, channels underlie IP3-induced ICat; (5) ET-1 does not alter total or plasma membrane-localized TRPC3 channel protein; (6) TRPC3 and TRPC6 both contain CIRB domains; (7) a TRPC3 CIRB domain peptide reduces IP3- and ET-1–induced ICat activation and a peptide corresponding to the IP3R1 N-terminal sequence that interacts with the TRPC CIRB domain activates ICat; and (8) CIRBP attenuates IP3- and ET-1–induced vasoconstriction. Taken together, these data indicate that IP3R1 activation causes physical coupling of the IP3R1 N terminus to the TRPC3 channel C terminus, leading to ICat activation and vasoconstriction. Physical coupling between all IP3R and TRPC channel isoforms occurs with recombinant channels,21–23,26 To our knowledge, this is the first time that molecular coupling between IP3R1 and TRPC channels has been demonstrated to occur in a native cell type, to perform a physiological function, and to exhibit TRPC channel isoform selectivity. Our data also suggest that close spatial proximity of IP3R1 to TRPC3 channels establishes selective molecular coupling of these proteins. In contrast, although TRPC6 contains a CIRB domain, spatial separation appears to prevent this channel from coupling to IP3R1 in arterial myocytes. Our study demonstrates that arterial myocyte IP3R channels, previously considered to function solely by releasing SR Ca2+, directly activate TRPC3 channels, thereby regulating arterial contractility. This study also illustrates that manipulating physical coupling between IP3R1 and TRPC3 channels is a novel mechanism that could be exploited to regulate arterial contractility.
Rat cerebral artery myocytes express several TRP isoforms, including TRPC1, -3, -6, TRPM4, and TRPV4.7,9–11 Mammalian TRPC channel isoforms share >30% amino acid sequence identity,27 with all TRPC channel isoforms containing a C-terminal CIRB domain.21,22 Cerebral artery myocytes express all 3 IP3R isoforms, with IP3R1 being the principal molecular and functional isoform.5 All recombinant IP3R isoforms contain an N-terminal TRPC channel interaction domain.21,22 Coupling of the IP3R TRPC binding domain to the TRPC channel CIRB domain displaces inhibitory calmodulin, leading to channel activation.22 The CIRB domain may also be a TRPC channel trafficking sequence.24 These previous studies were performed primarily by studying recombinant channels. It has been proposed that the physical interaction between IP3Rs and TRPC channels may have occurred in response to TRPC channel overexpression.21–23,26 Therefore, it was unclear whether physical coupling between IP3Rs and TRPC channels would occur in native cell types. Here, we demonstrate that this coupling mechanism does occur in a native cell type, selectively regulates TRPC3 currents in arterial myocytes, and leads to vasoconstriction.
In cerebral artery myocytes, IP3R stimulation leads to the activation of a ICat to which TRPC3 channels contribute.4,9 IP3R-mediated ICat activation leads primarily to Na+ influx, resulting in membrane depolarization, voltage-dependent Ca2+ channel activation, an [Ca2+]i elevation, and vasoconstriction.4 The mechanism by which IP3Rs stimulate TRPC3 channels occurs independently of SR Ca2+ release and appears to be a major mechanism by which PLC-coupled receptor agonists cause vasoconstriction.4 IP3R1 and TRPC3 channels do not contribute to the cerebral artery myogenic response.4,9 In contrast, TRPC6 channels do not contribute to ET-1 or UTP-induced cerebral artery constriction, but are essential for the myogenic response.4,9,10 The different mechanisms by which vasoconstrictors and IP3 activate TRPC3, whereas intravascular pressure stimulates TRPC6 were unclear. Fluorescent antibodies bound to endogenous IP3R1 and TRPC3 generated FRET that was elevated by ET-1 and higher than for TRPC6 or TRPM4. TRPC3 knockdown reduced basal and ET-1–induced IP3R1/TRPC3 FRET. The calculated Förster distance between Cy2 and Cy3 is 5 to 6 nm, supporting other evidence in this study that IP3R1 and TRPC3 are close enough to physically interact. In contrast, the FRET signal between IP3R1 and TRPC6 and TRPM4 was consistent with background,28 was not elevated by ET-1, and was not altered by TRPC6 knockdown. These data indicate that that TRPC6 and TRPM4 are not in close proximity to IP3R1. Mechanisms that position TRPC3 nearby IP3R1, but TRPC6 and TRPM4 away from IP3R1, are unclear. Several possibilities exist, including differential localization by local lipid environments, including caveolae. It is also possible that IP3R1 and TRPC3 are maintained by scaffolding proteins in a macromolecular complex. Scaffolding proteins, including Homer, establish close proximity of TRPC channels to other proteins, including IP3Rs.29 The ET-1–induced elevation in immunofluorescence resonance energy transfer may occur because of an IP3-induced conformational change in IP3R1 that locates the Cy3-tagged antibody closer to Cy2-bound TRPC3. Alternatively, ET-1 may cause IP3R clustering,30 thereby locating more Cy3-tagged IP3R1 channels in the vicinity of Cy2-bound TRPC3. The FRET method cannot differentiate between these potential mechanisms, but these possibilities deserve future investigation.
Co-IP data indicated that IP3R1 and TRPC3 are located in the same arterial macromolecular complex that does not contain TRPC6. The small arteries used for co-IP experiments inevitably contained endothelium. Complementary data, including those obtained using FRET, patch-clamp electrophysiology, RNA interference, antibodies and peptides, indicate that close spatial and functional interaction occurs between IP3R1 and TRPC3 in isolated myocytes. Arterial myocyte TRPC3 and TRPC6 channels both contained CIRB domains. Thus, although both TRPC3 and TRPC6 could interact molecularly with IP3R1, spatial proximity determines their ability to communicate. The CIRBP sequence used here as a competitive antagonist is identical to the sequence in TRPC3, and between 11% (TRPC1) and 89% (TRPC7) homologous to sequences in other TRPC channel isoforms.24 The CIRBP peptide did not alter ICat or diameter in the absence of ET-1 or IP3, but reduced ET-1 and IP3-induced ICat activation and vasoconstriction. Additional support that the CIRB domain is functional in arterial myocytes was data indicating that IP3RntP stimulated a Gd3+-sensitive ICat. The IP3RntP should activate all TRP channels that contain a CIRB domain, including TRPC3, regardless of their proximity to IP3Rs. However, TRPC isoforms also have variable affinities for the IP3R N terminus.21 Taken together our data indicate that: (1) IP3R1 and TRPC3 proteins are maintained in close proximity in the absence of receptor agonists; (2) physical interaction between the IP3R1 N terminus and TRPC3 channel C terminus is weak in the absence of IP3, indicating that the interaction domains do not maintain the close proximity; and 3) IP3 enhances physical interaction between IP3R1 and TRPC3. Although IP3R1 did not activate TRPC6, IP3R1 may activate TRPC isoforms other than TRPC3 in arterial myocytes. Given that there are seven TRPC isoforms and 3 IP3R isoforms, it was beyond the scope of the current study to determine which of the potential twenty-one interactions may occur molecularly and functionally in arterial myocytes. TRPC channel heteromultimers have been reported in cultured vascular myocytes and A7r5 cells,31 but it is unclear whether TRPC channels that couple to IP3R1 in arterial myocytes are homomultimers or heteromultimers. TRPC3 knockdown reduced TRPC3 protein by ≈40% and attenuated IP3-induced ICat by ≈41%. A TRPC3-selective antibody also similarly inhibited recombinant TRPC3 currents and IP3-induced ICat in arterial myocytes. Therefore, any IP3-induced current mediated by channels other than TRPC3 would be small in arterial myocytes. In other native cell types, IP3Rs and TRPC channels may exhibit different isoform-dependent coupling patterns, including promiscuous coupling similar to that with recombinant channels.
Several stimuli, including receptor agonists, increase plasma membrane trafficking of recombinant TRPC3 channels.32 Biotinylation indicated that over the same time course that ET-1 activated ICat in arterial myocytes, total TRPC3 or plasma membrane-localized TRPC3 did not change. These data indicate that ET-1 elevates ICat by increasing the Po of membrane-resident TRPC3 channels. Biotinylation also indicated that a large proportion of TRPC3 protein is intracellular. One explanation for these data are that TRPC3 channels may be retained, for example by RNF24,33 and that signaling pathways other than those activated by acute ET-1 application may stimulate plasma membrane insertion. TRPC3 channels are also present on intracellular organelle membranes.29 Given that FRET was detected at the plasma membrane and intracellularly, our data indicate that intracellular TRPC3 channels are also in close proximity to IP3R1. Protein complexes containing both IP3R1 and TRPC3 may also form before plasma membrane insertion of TRPC3.29
PLC not only generates IP3, but also elevates DAG, which activates PKC. CIRBP reduced ET-1– and IP3-induced ICat activation by >60%. These data indicate that ET-1 and IP3 stimulate a ICat primarily through molecular coupling of IP3Rs to TRPC channels, rather than through DAG and PKC activation. This conclusion is consistent with previous evidence that IP3R1 antibodies and IP3R1 knockdown also inhibit UTP-induced ICat activation in arterial myocytes.5 DAG and PKC can regulate TRPC channels, but in vascular myocytes isolated from a variety of different blood vessels, regulation is complex with studies reporting PKC-mediated activation, inhibition, or no effect.14,34–36 In cerebral artery myocytes, PKC stimulates a ICat by elevating TRPM4 channel apparent micromolar Ca2+ sensitivity.37,38 Here, in patch-clamp experiments, free intracellular Ca2+ was strongly buffered using EGTA and ET-1–induced PKC activation would not stimulate TRPM4 channels.37,38 Conceivably, a proportion of the CIRBP-insensitive ET-1–induced vasoconstriction may occur because of PKC activation of other TRP channels, including TRPM4. Agonists also stimulate vasoconstriction via additional mechanisms, including activation of Ca2+ waves and voltage-dependent Ca2+ channels, and elevation of myofilament Ca2+ sensitivity.2,3
In summary, we describe a novel mechanism by which IP3Rs regulate arterial diameter. Our data indicate that ET-1 and IP3 activate membrane-resident TRPC3 channels in arterial myocytes by causing an interaction between the IP3R1 N-terminal TRPC channel interaction sequence and the C-terminal TRPC3 channel CIRB domain. We also provide a mechanism by which IP3R1 activation selectively stimulates TRPC3, but not TRPC6, channels and indicate that this occurs because of the close spatial proximity of these proteins which allows physical interaction. Physical coupling between IP3R1 and TRPC3 channels could be manipulated therapeutically to regulate arterial contractility and, thus, brain blood flow.
We thank Dr Lidia A. Gardner for technical advice with FRET measurements.
Sources of Funding
Supported by National Heart, Lung, and Blood Institute grants R01 HL67061 and HL77678 (to J.H.J.) and K01 HL096411 (to A.A.). D.N. is a recipient of an American Heart Association Predoctoral Fellowship.
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Novelty and Significance
What Is Known?
Phospholipase C–coupled receptor agonists elevate inositol 1,4,5-trisphosphate (IP3) in arterial smooth muscle cells, leading to the activation of sarcoplasmic reticulum (SR) type 1 IP3 receptors (IP3Rs), an increase in intracellular calcium ([Ca2+]i) concentration, and vasoconstriction.
Canonical transient receptor potential (TRPC) channels are molecularly and functionally diverse proteins that regulate smooth muscle cell plasma membrane cation influx and vascular contractility.
The conventional view has been that IP3Rs stimulate vasoconstriction by releasing SR Ca2+, although recent evidence indicates that IP3Rs also induce vasoconstriction via an SR Ca2+ release-independent mechanism that involves TRPC channel activation; the mechanism is unidentified.
What New Information Does This Article Contribute?
Type 1 IP3 receptors are located in very close spatial proximity to plasma membrane TRPC3 channels in arterial smooth muscle cells.
Vasoconstrictor agonists and IP3 induce physical interaction between the IP3R1 N terminus and the TRPC3 channel C-terminal calmodulin and IP3R binding domain, leading to TRPC3 channel activation and vasoconstriction.
In arterial smooth muscle cells, IP3R1 coupling to TRPC3 channels is isoform-selective because IP3R1 does not activate spatially separated TRPC6 channels.
Many vasoconstrictors elevate intracellular IP3, but the mechanisms by which this second messenger stimulates vasoconstriction are poorly understood. We used a combination of molecular, cellular, and physiological approaches to examine IP3R regulation of TRPC channels in cerebral artery smooth muscle cells. We found that IP3R1, the predominant functional IP3R isoform in arterial smooth muscle cells, physically interacts with and directly activates plasma membrane TRPC3, but not TRPC6, channels. To our knowledge, this is the first demonstration that: (1) native IP3Rs physically interact with and activate native TRPC channels and (2) IP3Rs directly regulate the activity of a plasma membrane ion channel in arterial smooth muscle cells. Physical coupling of IP3R1 to TRPC3 channels occurs due close spatial proximity of these proteins. In contrast, TRPC6 channels are not located nearby IP3R1 and do not couple. Our findings identify a novel mechanism of arterial contractility regulation whereby agonist-induced IP3 stimulates coupling of IP3Rs to TRPC3 channels, resulting in plasma membrane cation influx, and vasoconstriction. We also show that manipulating the interaction between IP3R1 and TRPC3 channels is a novel mechanism that could be exploited to regulate arterial contractility.
Original received July 15, 2009; resubmission received January 8, 2010; revision received March 22, 2010; accepted March 29, 2010.