Heteromultimeric TRPC6-TRPC7 Channels Contribute to Arginine Vasopressin-Induced Cation Current of A7r5 Vascular Smooth Muscle Cel
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Yoshiaki Maruyama, Yuko Nakanishi, Emma
参见附件。
The Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, Alberta, Canada.
Abstract
The molecular identity of receptor-operated, nonselective cation channels (ROCs) of vascular smooth muscle (VSM) cells is not known for certain. Mammalian homologues of the Drosophila canonical transient receptor potential channels (TRPCs) are possible candidates. This study tested the hypothesis that heteromultimeric TRPC channels contribute to ROC current of A7r5 VSM cells activated by [Arg8]-vasopressin. A7r5 cells expressed transcripts encoding TRPC1, TRPC4, TRPC6, and TRPC7. TRPC4, TRPC6, and TRPC7 protein expression was confirmed by immunoblotting and association of TRPC6 with TRPC7, but not TRPC4, was detected by coimmunoprecipitation. The amplitude of arginine vasopressin (AVP)-induced ROC current was suppressed by dominant-negative mutant TRPC6 (TRPC6DN) but not TRPC5 (TRPC5DN) mutant subunit expression. These data indicate a role for TRPC6- and/or TRPC7-containing channels and rule a more complex subunit composition including TRPC1 and TRPC4. Increasing extracellular Ca2+ concentration ([Ca2+]o) from 0.05 to 1 mmol/L suppressed currents owing to native, TRPC7, and heteromultimeric TRPC6-TRPC7 channels, but not TRPC6 current, which was slightly enhanced. The relative changes in native and heteromultimeric TRPC6-TRPC7 current amplitudes for [Ca2+]o between 0.01 and 1 mmol/L were identical, but the changes in homomultimeric TRPC6 and TRPC7 currents were significantly less and greater, respectively, compared with the native channels. Taken together, the data provide biochemical and functional evidence supporting the view that heteromultimeric TRPC6-TRPC7 channels contribute to receptor-activated, nonselective cation channels of A7r5 VSM cells.
Key Words: TRPC6 TRPC7 receptor-operated cation channel vascular smooth muscle
Introduction
Activation of G protein–coupled receptors (GPCRs) of vascular smooth muscle (VSM) cells by a variety of vasoconstrictor agonists causes an elevation in [Ca2+]i and contraction owing to depolarization and Ca2+ influx from the extracellular space, Ca2+ release from internal stores and Ca2+ sensitization of contractile filaments.1–4 The depolarization and influx of Ca2+ evoked by GPCRs is attributable in part to the activation of receptor-operated, nonselective cation channels (ROCs) by a signaling pathway involving phospholipase and diacylglycerol (reviewed previously3–6). The molecular basis of VSM ROCs is not known with certainty, but accumulating evidence suggests that transient receptor potential channel (TRPC) family subunits (TRPC1 to TRPC7)7–9 are likely involved.3–6 Moreover, ROCs owing to heterologous expression of TRPC3, TRPC6, or TRPC7 are activated by a similar mechanism involving phospholipase and diacylglycerol.10–12 Understanding the molecular basis of VSM ROCs is clearly warranted in light of their important role in control of VSM excitability and contractility4–6 and evidence of changes in TRPC expression associated with abnormal contractility and/or VSM cell proliferation.13–15
Identifying the contribution of TRPC subunits to VSM ROCs has been compromised by a lack of specific/selective pharmacological blockers. For this reason, alternative strategies involving antibodies, anti-sense or small interfering RNAs (siRNAs) were used to indicate roles for: (1) TRPC1 as store-operated channels16; (2) TRPC3 as ROCs17; (3) TRPC6 as ROCs and/or mechanosensitive channels11,18–20; (4) TRPV2 as mechanosensitive channels21; (5) TRPM4 as mechanosensitive channels22; and (6) TRPV4 in the stimulation of focal Ca2+ release ("sparks") from sarcoplasmic reticulum Ca2+ stores.23 In some cases, the biophysical and/or pharmacological properties of heterologously expressed TRPC and native VSM ROCs have been compared. It is significant, however, that the properties of homomultimeric TRPC are similar, but not necessarily identical, to those of native ROCs.4–6,11,12,14,24–26 For example, antisense and siRNA experiments indicate that TRPC6 is an important component of 1-adrenoceptor– and arginine vasopressin (AVP) V1a receptor–induced ROC currents of portal vein11 and A7r5 cultured VSM cells,18,19 respectively, as well as cation current activated by diacylglycerol in rat cerebral myocytes.20,24 However, the Ca2+-sensitivity of these channels is markedly different; specifically, increasing [Ca2+]o from 0.05 to 2 mmol/L suppressed native ROC currents11,18,25 but increased in TRPC6 current.12 Additionally, as noted by Beech et al,4 the current-voltage (I-V) relations of TRPC6-containing cation channels of portal vein,11 cerebral,20,24 and pulmonary arterial14 myocytes and A7r5 VSM cells18 are not identical. The molecular basis of these differences in functional properties is presently unknown.
Previous results indicate that multiple types of TRPC subunits are expressed at varied levels by VSM cells of different vessels in a species-dependent manner.4,6,13,14,18,19,27 Thus, VSM ROCs may have a subunit composition that varies in a vessel-dependent fashion, different agonists may activate ROCs composed of different subunits, and/or a given agonist may activate multiple ROCs with varied subunit composition.4,26 VSM ROC could also result from the heteromultimeric assembly of more than 1 type of TRPC subunit.4–6 Coassociation of different voltage-gated K+ channel subunits is an established principle, and it occurs in many cell types, including VSM cells.28,29 Expression of heteromultimeric TRPC6-containing channels with unique properties provides a viable explanation for the lack of complete functional identity of homomultimeric TRPC6 and native ROCs, as well as the varied properties of TRPC6-containing ROCs in different VSM cells.4–6 TRPC3 and TRPC6 antisense oligonucleotides both inhibit ROC currents of rat prostate smooth muscle cells,30 providing indirect evidence for the presence of heteromultimeric channels. However, direct biochemical evidence of TRPC heteromultimerization, such as that shown for heterologous expression systems and brain tissues,31–34 has not been obtained for VSM cells.
Here we tested the hypothesis that heteromultimeric TRPC channels contribute to ROCs of A7r5 VSM cells. This aortic smooth muscle cell line was specifically used because it permitted a detailed analysis of the biochemical and electrophysiological properties of the ROCs that would otherwise not be possible using native cells because of the low level of channel expression and relatively low affinity of currently available subunit-specific antibodies. TRPC expression and heteromultimerization was assessed by RT-PCR, immunoblotting, and coimmunoprecipitation. The participation of TRPC-containing ROCs was addressed using a dominant-negative strategy,32,34 and the sensitivity of the native ROCs and homo- and heteromultimeric TRPC to [Ca2+]o was compared. Our results provide the first direct evidence that heteromultimeric TRPC channels can contribute to ROCs activated by GPCRs in VSM cells.
Materials and Methods
Human embryonic kidney 293 (HEK293; passage 5 to 17) and A7r5 VSM cells (passage 10 to 22) (American Type Culture Collection, Manassas, Va) were used. Both cell types were maintained in DMEM (Invitrogen [Gibco-BRL], Burlington, Canada) supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). HEK293 cells grown on glass coverslips were transfected with cDNAs encoding 0.5 μg of angiotensin AT1 receptor, 0.5 μg of green fluorescent protein (GFP), and varied amounts of TRPC subunit cDNAs using FuGENE 6 (Roche Diagnostics Canada, Laval) and used within 24 to 48 hours. Eighty percent confluent cultures of A7r5 cells were transfected with cDNAs encoding 0.5 μg of GFP, 1.5 μg of TRPC subunit cDNAs, or equal amounts of empty vector pcDNA3 (mock transfection). The A7r5 cells were plated onto coverslips 48 hours after transfection and used within 24 hours. The methods for whole-cell voltage clamp, immunocytochemistry, immunoprecipitation, immunoblotting, and/or molecular biology were as previously described28,29 and/or as indicated in the online data supplement. All primers used are included in the online data supplement available at http://circres.ahajournals.org.
Results
TRPC Transcript Expression
TRPC message expression was analyzed by RT-PCR using subunit-specific primers and mRNA from A7r5 cells and rat brain; the latter used to confirm primer function and reaction integrity (Figure 1A). TRPC1, TRPC4, TRPC6, and TRPC7 amplicons were detected in A7r5 cell mRNA (Figure 1B) and confirmed by sequencing. TRPC3 and TRPC5 expression was not detected but was consistently apparent for rat brain (Figure 1A). Alternate primer sets for TRPC3, TRPC4/TRPC4, and TRPC7 were also used. TRPC3 (data not shown) and TRPC4 (Figure 1C) were not detected, but amplicons for TRPC4 (245 base pairs [bp]; Figure 1C) and TRPC7 (649 bp; Figure 1D) were identified (additional 300 and 400 bp amplicons in Figure 1C did not correspond to any known gene).
For comparative purposes, we also determined whether TRPC6 and TRPC7 were expressed by native VSM cells. Amplicons were detected for both TRPC subunits in mRNA samples derived from populations of 50 to 75 individually selected myocytes freshly isolated from rat middle and posterior cerebral arteries (Figure 1E).29 Expression of endothelin-1 transcript (328 bp) was not apparent (positive control for primers described previously29), indicating a lack of contamination by message derived from endothelial cells.
TRPC Protein Expression and Coassembly
We probed for TRPC1 protein expression using anti-TRPC1 and detected a 145 kDa protein (data not shown), as noted previously.35 The presence of TRPC4, TRPC6, and TRPC7 in lysates of A7r5 cells was also detected. Anti–c-myc immunoreactive bands were evident in HEK293 cells expressing full-length c-myc–tagged TRPC4 and the smaller TRPC4 between 105 and 100 kDa (Figure 2A). An immunoreactive band of 100 kDa and consistent with TRPC4 was detected with anti-TRPC4 in A7r5 (Figure 2A) and HEK293 (not shown) cell lysates. Anti-TRPC6 detected multiple bands between 100 to 130 kDa in lysates of HEK293 cells expressing TRPC6, likely corresponding to monomeric and glycosylated subunit,36 as well as protein at 125 kDa in A7r5 cell lysates (Figure 2B). These bands were not evident in lysates of non- or mock-transfected (empty vector) HEK293 cells (not shown). Anti-TRPC7 failed to detect protein in mock-transfected HEK293 cells or cells transfected with TRPC6, but multiple immunoreactive bands between 100 to 125 kDa were identified in lysates of cells expressing TRPC6 and TRPC7 (Figure 2C). An immunoreactive band of 100 kDa was detected in A7r5 cell lysates using anti-TRPC7 (Figure 2C).
Figure 2D shows the results of coimmunoprecipitation experiments using anti-TRPC6 followed by immunoblotting with antibodies against TRPC6, TRPC4, or TRPC7. TRPC6 immunoreactive protein was detected between 110 and 130 kDa in anti-TRPC6 immunoprecipitates of HEK293 cells expressing TRPC6 (Figure 2Da). No evidence of immunoreactive protein corresponding to TRPC4 at 100 kDa was identified by anti-TRPC4 in anti-TRPC6 immunoprecipitates of A7r5 cells, but nonspecific bands were apparent at 72 to 80 kDa and 130 kDa (closed circles; Figure 2Db). TRPC7 immunoreactive protein was detected at 100 kDa in anti-TRPC6 immunoprecipitates (Figure 2Dc) corresponding to the 100 kDa band present in immunoblots of A7r5 and TRPC7-expressing HEK293 cells, indicating the association of these subunits in A7r5 cells (Figure 2C). Nonspecific bands similar to those in Figure 2Db were also evident in TRPC7 immunoblots of anti-TRPC6 immunoprecipitates.
Dominant-Negative Suppression of AVP-Induced ROC Current
The contribution of TRPC channels to AVP-induced ROC current of A7r5 cells was assessed using dominant-negative pore mutants of TRPC6 (TRPC6DN) and TRPC5 (TRPC5DN) with C- and N-terminal c-myc and hemagglutinin (HA) tags, respectively. The mutants were tested for specificity in HEK293 cells (see online data supplement). Anti–c-myc immunofluorescence was evident in GFP-positive A7r5 cells transfected with TRPC6DN but not mock-transfected cells (Figure 3A). AVP caused a marked increase in N-methyl-D-glutamine (NMDG)-sensitive cation current at –60 mV in GFP-positive A7r5 cells expressing empty vector (Figure 3B) but consistently less current in GFP-positive cells of sister culture dishes expressing TRPC6DN (Figure 3D and 3G; 18.2±4.8 [n=15] versus 5.4±2.4 pA/pF [n=16]; P<0.02). In contrast, AVP-induced current amplitude was not different in GFP-positive cells of sister dishes transfected with empty vector or TRPC5DN (Figure 3F and 3G; 5.3±1.7 [n=15] versus 3.0±1.4 pA/pF [n=11]; P=0.15). The I-V relationship of AVP-induced current in mock-transfected (n=14) and in 9 TRPC6DN-transfected A7r5 cells (with >1.0 pA/pF current) was doubly rectifying (Figure 3C and 3E). Increasing [Ca2+]o from 0.05 to 1 mmol/L reduced current in mock- and TRPC6DN-transfected cells, particularly between –100 and 0 mV (Figure 3B through 3E). The relative change in current at –100 and +100 mV in these cells owing to [Ca2+]o were identical (ie, current in 1.0/0.05 mmol/L [Ca2+]o; Figure 3E).
Effect of [Ca2+]o on Native and Recombinant Nonselective Cation Currents
The effect of increasing [Ca2+]o on native ROCs was compared with that of homo- and heteromultimeric TRPC6 and TRPC7 channels. TRPC3 and TRPC3-TRPC6 channels were also considered because of the expression of TRPC3 (rather than TRPC7) with TRPC6 in some vessels.4,6,27 Ang II was used as the agonist for the recombinant channel experiments because of availability of cDNAs encoding the AT1 receptor and justified by the lack of a difference in AVP- and Ang II–induced ROC currents of A7r5 cells (see online data supplement) and previous reports that similar A7r5 cell ROC currents are activated by serotonin, PDGF, AlF4–, and OAG.18 Ang II–induced ROC currents were observed in HEK293 cells expressing TRPC3, TRPC6, and TRPC7 alone or in combination (Figure 4B through 4F). The I-V relations for homo- and heteromultimeric TRPC combinations all doubly rectified (Figure 4B through 4F), consistent with previous studies.11,12,36 Increasing [Ca2+]o caused a rapid inhibition of native current (Figure 4A), as well as currents owing to TRPC7 and TRPC3 (Figure 4C and 4D) and heteromultimeric TRPC3-TRPC6 and TRPC6-TRPC7 channels (Figure 4E and 4F). In contrast, TRPC6 current amplitude increased immediately on the change in [Ca2+]o and subsequently declined to a maintained level that was not different from that recorded before the solution change (Figure 4B). The differing effects of [Ca2+]o on current amplitude were most apparent between –100 and 0 mV (Figure 4G).
A more detailed analysis of the effects of [Ca2+]o on current amplitude was performed for native A7r5 ROCs, TRPC6, TRPC7, and TRPC6-TRPC7 channels using the approach of Helliwell and Large.25 Currents were evoked in 1 mmol/L [Ca2+]o before switching to nominally Ca2+-free, 0.05, 0.1, or 0.3 mmol/L [Ca2+]o solutions (Figure 5). TRPC7 currents showed the greatest increase in peak inward current when [Ca2+]o was lowered from 1 mmol/L (Figure 5). In contrast, TRPC6 current was only slightly enhanced by decreasing [Ca2+]o and consistently exhibited a slight increase in amplitude on subsequent reexposure to 1 mmol/L [Ca2+]o. The mean changes in current amplitude at each [Ca2+]o were determined by normalizing peak amplitude after the change in [Ca2+]o to the value in 1 mmol/L [Ca2+]o and plotted as a function of [Ca2+]o (Figure 6). The relative change in amplitude of homomultimeric TRPC6 and TRPC7 currents were less and greater, respectively, compared with that of the native ROCs (Figure 6). In contrast, the change heteromultimeric TRPC6-TRPC7 current mimicked that of the native A7r5 cell ROCs (Figure 6).
Discussion
This study provides the first direct evidence that heteromultimeric TRPC channels can contribute to ROCs of VSM cells. At least 4 distinct types of cation channels are present in smooth muscle cells, including store-operated, ROC, stretch-activated, and tonically active cation channels, but the molecular identity of each conductance is a matter of debate.4,6,37 These channels are thought to contribute to several physiological functions of VSM cells, including agonist- and stretch-induced depolarization, maintenance of SR Ca2+ store filling, and mediating Ca2+ influx in response to ligands and physical stimuli (eg, stretch).4–6 Understanding the molecular basis of VSM ROCs is of particular interest in light of the important contribution of these channels to the actions of vasoconstrictors,1–6 eg, 1-adrenoceptor agonists, Ang II, and endothelin-1, that are well known to contribute to vascular pathology and the recognition that alterations in TRP channel expression, including TRPC subunits, is associated with abnormal control of vascular contractility and proliferation.13–15 Multiple TRPC subunits, as well as members of TRPV and TRPM families, have been shown to be expressed by VSM cells in a tissue- and species-specific pattern.4,6,13,27 For example, A7r5 cells were found to express TRPC1 and TRPC618 and, more recently, TRPC3, TRPC4, TRPC5 and/or TRPC7, depending on the primers used and/or specific strain of A7r5 cells studied,19,38 a pattern of expression that is apparent in several arteries and veins.4 Here we show the expression of transcripts encoding the short TRPC4 splice variant of TRPC4, as well as TRPC1, TRPC6, and TRPC7, in A7r5 cell mRNA extracts, and the presence of these proteins was confirmed by immunoblotting. Additionally, we provide biochemical and electrophysiological evidence of coassembly of TRPC6 and TRPC7 and functional identity of native ROC and TRPC6-TRPC7 heteromultimeric channels in terms of Ca2+ sensitivity, respectively. These observations support the view that VSM ROC current may be the result of expression and association of multiple TRPC subunits.
The evidence that TRPC6 and TRPC7 coassemble and contribute to the AVP-induced ROCs of A7r5 cells may be summarized as follows. First, TRPC6 and TRPC7 message and protein expression were detected by RT-PCR and immunoblotting using subunit-specific primers and antibodies with a specificity demonstrated in previous studies33 and were confirmed here using HEK293 cells expressing TRPC6 and/or TRPC7. Second, direct biochemical evidence of TRPC subunit coassembly was obtained in experiments in which anti-TRPC7 immunoreactive protein was coimmunoprecipitated from lysates of A7r5 cells using anti-TRPC6, similar to the association previously identified to occur in synaptosomes derived from adult rat brain.33 Third, a dominant-negative strategy demonstrated the involvement of TRPC subunits in the response of A7r5 cells to AVP. The strategy is based on the assumption that mutant subunits with the conserved leucine-phenylalanine-tryptophan channel pore motif replaced with an alanine triplet (AAA) coassemble with wild-type subunits and suppress cation permeation through the resultant heteromultimeric channels.32 Using this approach, Hofmann et al32 concluded that the TRPC family divides into 2 subgroupings capable of heteromultimerization: TRPC3, TRPC6, and TRPC7 versus TRPC1, TRPC4, and TRPC5. Here we found a significant dominant-negative suppression of AVP-induced A7r5 cell ROC current by TRPC6DN but not TRPC5DN. In the absence of TRPC3 expression, this result indicates the potential involvement of homo- and/or heteromultimeric TRPC6 and TRPC7 channels. Recent observations indicate that TRPC coassembly may be more complex than that described by Hofmann et al,32 with interactions between members of the subgroups being possible.34 For example, TRPC3 or TRPC6 in combination with TRPC1, TRPC4, or TRPC5 were identified in lysates of embryonic rat brain and a critical role for TRPC1 as a mediator of cross-grouping interactions was indicated.34 In light of these findings and the expression of TRPC1 and TRPC4 by the A7r5 cells used here, the possibility that A7r5 cell ROCs may have a more complex subunit composition was considered. We probed for an association of TRPC4 with TRPC6 but were unable to detect the presence of TRPC4 protein in anti-TRPC6 immunoprecipitates. It is possible that the channel density and/or antibody affinity was too low to permit detection or that the antigenic sites on the channel protein were inaccessible when in a heteromultimeric complex.33 However, we also failed to observe a suppression of ROC current in A7r5 cells expressing TRPC5DN and suppression of TRPC1 expression by A7r5 cells using an anti-sense oligonucleotide approach has no effect on AVP-induced ROC currents (L.I. Brueggman and K.L. Byron, personal communication). Taken together, these data imply that channels containing TRPC6, TRPC7, and TRPC1 alone or with TRPC4 likely do not contribute to AVP-induced ROCs of A7r5 cells.
The fourth line of evidence supporting the view that heteromultimeric ROCs are activated by AVP in A7r5 cells was obtained in experiments that compared the effects of [Ca2+]o on A7r5 cell ROCs versus homo- and heteromultimeric TRPC channels. Previous studies using TRPC6 antisense oligonucleotides or siRNAs imply that TRPC6 is a component of portal vein, cerebral arterial, and A7r5 VSM cell ROCs, as these approaches resulted in a suppression of agonist- and/or OAG-induced induced ROC current.11,18,19,20,24 However, the previously reported 1.5 increase in TRPC6 current amplitude caused by increasing [Ca2+]o from between 0.2 to 0.5 to between 1 to 2 mmol/L11,12 contrasts markedly with the rapid inhibition observed for native VSM ROC currents of portal vein and A7r5 cells evoked by 1-adrenoceptor agonists and AVP, respectively.11,18,25 This difference in behavior cannot be attributed to variations in the [Ca2+]o sensitivity of the mechanism of channel activation, as identical responses have been reported for TRPC6, TRPC7, and native currents activated by a diverse set of compounds.6,11,12,18 Rather, the results of this study indicate that the difference in functional identity between TRPC6 and these VSM ROCs can be attributed to the presence of multiple pore-forming subunit(s) in the channel complex. Despite sharing considerable sequence identity with TRPC6 subunits, TRPC339 and TRPC712,40 channels are inhibited by changing [Ca2+]o from nominally Ca2+ free to 1 mmol/L, confirmed here by stepping [Ca2+]o from 0.05 to 1 mmol/L and from 1 mmol/L to between nominally Ca2+ free and 0.3 mmol/L. Moreover, we show for the first time that heteromultimeric TRPC3-TRPC6 and TRPC6-TRPC7 channels are also inhibited by increasing [Ca2+]o from 0.05 to 1 mmol/L. Thus, the potentiation of TRPC6 current by 1 mmol/L [Ca2+]o is lost when this subunit coassembles with TRPC3 or TRPC7 subunits. Similarly, TRPC7 exhibited a significantly greater relative inhibition by [Ca2+]o compared with heteromultimeric TRPC6-TRPC7 channels, suggesting that the properties of TRPC7 are modified by the presence of TRPC6 in the complex. Thus, heteromultimeric TRPC6-TRPC7 exhibit at least 1 property that is distinct from homomultimeric TRPC6 and TRPC7 channels, and only this heteromultimeric combination of subunits produced ROCs that showed a relative inhibition by [Ca2+]o that was consistent with that of native AVP-induced ROCs of A7r5 cells. It is unlikely that AVP activates 2 populations of ROCs because of expression of homomultimeric TRPC6 and TRPC7 channels, with the TRPC6 channels potentiated and the TRPC7 rapidly inhibited by increasing [Ca2+]o. Increasing [Ca2+]o to 1 mmol/L caused an almost complete suppression of AVP-induced native ROC current, rather than the partial inhibition that would be expected for the case of 2 populations of homomultimeric channels.
We focused here on the inhibitory effect of [Ca2+]o on native and recombinant cation current amplitude because of the marked difference in behavior of A7r5 cell ROCs and TRPC6 channels. However, modulation of VSM ROCs and TRPC channels by [Ca2+]o is complicated12,25,41 and involves additional effects not considered. These include facilitation by [Ca2+]o between <0.001 and 0.2 mmol/L (EC50 3 to 6 μmol/L)12,25 and indirect modulation by Ca2+-calmodulin–dependent and –independent kinases.12,25,41,42,43 These aspects of Ca2+ regulation of heteromultimeric TRPC channels should be considered in the future. Lack of control over subunit stoichiometry is a limitation of experiments coexpressing cDNAs, as this approach may not yield channels with an appropriate subunit stoichiometry. Because stoichiometry may be an important for appropriate regulation by Ca2+, as well as other functional properties, the use of concatemeric subunits to produce channels of known composition is warranted.
A limitation of this study is that the evidence of TRPC subunit heteromultimerization was obtained using cultured A7r5 cells rather than native VSM cells. This approach was required because, firstly, intact arteries cannot be used because of potential contamination by TRPC channels of endothelial cells. Secondly, the low level of channel expression in VSM cells complicates analysis of TRPC subunit association in denuded vessel/isolated myocyte preparations by coimmunoprecipitation using the relatively low-affinity TRPC antibodies that are currently available. Finally, in the absence of direct biochemical evidence of coassembly, the finding that native VSM ROC currents are inhibited by 1 mmol/L [Ca2+]o cannot be used to differentiate between the contribution of homo- and heteromultimeric channels containing TRPC3 and TRPC7 or these subunits associated with TRPC6. On the other hand, the use of the A7r5 cell line as a model for the study of VSM ROCs can be justified based on the pattern of TRPC subunit message expression. The set of TRPC subunits expressed by the A7r5 cells used here included TRPC1, TRPC4, TRPC6, and TRPC7. TRPCC4, TRPC6, and TRPC7 mRNAs were previously shown to be expressed together in canine pulmonary and renal arteries,27 and TRPC6 and TRPC7 were both detected in rat thoracic and cerebral arteries.13 These findings are consistent with our data showing the presence of TRPC6 and TRPC7 transcripts in mRNA derived from isolated rat cerebral myocytes, an approach that precludes false-positive identification of subunit expression attributable to contamination by message derived from endothelial cells, blood cells, and/or fibroblasts present in the vessel wall. Taken together, these findings provide molecular evidence consistent with the view that TRPC6- and TRPC7-containing ROCs may also be expressed by native VSM cells within some vessels. However, based on the reported variability in properties of native VSM ROC currents,4–6 TRPC6-TRPC7 channels may not be the only ROC channel type expressed by VSM cells; other homomultimeric (eg, uridine triphosphate-activated TRPC3 channels17) and heteromultimeric combinations of TRPC subunits (eg, TRPC1, TRPC3, and TRPC614) could also be present.
In summary, this report provides molecular, biochemical, and electrophysiological evidence that multiple TRPC subunits are expressed by A7r5 VSM cells, that TRPC6 and TRPC7 coassemble in this cell type, and that heteromultimeric TRPC6-TRPC7 channels share a functional identity with AVP-induced ROCs of A7r5 cells with respect to their I-V relation and inhibition by [Ca2+]o.
Acknowledgments
Sources of Funding
This work was supported by the Canadian Institutes for Health Research (MT-13505) and an Alberta Heritage Foundation for Medical Research postdoctoral fellowship (to Y.M.).
Disclosures
Dr Y. Maruyama was the recipient of a Pfizer Canada Young Scientist Education Award in Smooth Muscle Research.
Footnotes
Original received November 22, 2005; revision received April 21, 2006; accepted May 2, 2006.
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The Smooth Muscle Research Group, Faculty of Medicine, University of Calgary, Alberta, Canada.
Abstract
The molecular identity of receptor-operated, nonselective cation channels (ROCs) of vascular smooth muscle (VSM) cells is not known for certain. Mammalian homologues of the Drosophila canonical transient receptor potential channels (TRPCs) are possible candidates. This study tested the hypothesis that heteromultimeric TRPC channels contribute to ROC current of A7r5 VSM cells activated by [Arg8]-vasopressin. A7r5 cells expressed transcripts encoding TRPC1, TRPC4, TRPC6, and TRPC7. TRPC4, TRPC6, and TRPC7 protein expression was confirmed by immunoblotting and association of TRPC6 with TRPC7, but not TRPC4, was detected by coimmunoprecipitation. The amplitude of arginine vasopressin (AVP)-induced ROC current was suppressed by dominant-negative mutant TRPC6 (TRPC6DN) but not TRPC5 (TRPC5DN) mutant subunit expression. These data indicate a role for TRPC6- and/or TRPC7-containing channels and rule a more complex subunit composition including TRPC1 and TRPC4. Increasing extracellular Ca2+ concentration ([Ca2+]o) from 0.05 to 1 mmol/L suppressed currents owing to native, TRPC7, and heteromultimeric TRPC6-TRPC7 channels, but not TRPC6 current, which was slightly enhanced. The relative changes in native and heteromultimeric TRPC6-TRPC7 current amplitudes for [Ca2+]o between 0.01 and 1 mmol/L were identical, but the changes in homomultimeric TRPC6 and TRPC7 currents were significantly less and greater, respectively, compared with the native channels. Taken together, the data provide biochemical and functional evidence supporting the view that heteromultimeric TRPC6-TRPC7 channels contribute to receptor-activated, nonselective cation channels of A7r5 VSM cells.
Key Words: TRPC6 TRPC7 receptor-operated cation channel vascular smooth muscle
Introduction
Activation of G protein–coupled receptors (GPCRs) of vascular smooth muscle (VSM) cells by a variety of vasoconstrictor agonists causes an elevation in [Ca2+]i and contraction owing to depolarization and Ca2+ influx from the extracellular space, Ca2+ release from internal stores and Ca2+ sensitization of contractile filaments.1–4 The depolarization and influx of Ca2+ evoked by GPCRs is attributable in part to the activation of receptor-operated, nonselective cation channels (ROCs) by a signaling pathway involving phospholipase and diacylglycerol (reviewed previously3–6). The molecular basis of VSM ROCs is not known with certainty, but accumulating evidence suggests that transient receptor potential channel (TRPC) family subunits (TRPC1 to TRPC7)7–9 are likely involved.3–6 Moreover, ROCs owing to heterologous expression of TRPC3, TRPC6, or TRPC7 are activated by a similar mechanism involving phospholipase and diacylglycerol.10–12 Understanding the molecular basis of VSM ROCs is clearly warranted in light of their important role in control of VSM excitability and contractility4–6 and evidence of changes in TRPC expression associated with abnormal contractility and/or VSM cell proliferation.13–15
Identifying the contribution of TRPC subunits to VSM ROCs has been compromised by a lack of specific/selective pharmacological blockers. For this reason, alternative strategies involving antibodies, anti-sense or small interfering RNAs (siRNAs) were used to indicate roles for: (1) TRPC1 as store-operated channels16; (2) TRPC3 as ROCs17; (3) TRPC6 as ROCs and/or mechanosensitive channels11,18–20; (4) TRPV2 as mechanosensitive channels21; (5) TRPM4 as mechanosensitive channels22; and (6) TRPV4 in the stimulation of focal Ca2+ release ("sparks") from sarcoplasmic reticulum Ca2+ stores.23 In some cases, the biophysical and/or pharmacological properties of heterologously expressed TRPC and native VSM ROCs have been compared. It is significant, however, that the properties of homomultimeric TRPC are similar, but not necessarily identical, to those of native ROCs.4–6,11,12,14,24–26 For example, antisense and siRNA experiments indicate that TRPC6 is an important component of 1-adrenoceptor– and arginine vasopressin (AVP) V1a receptor–induced ROC currents of portal vein11 and A7r5 cultured VSM cells,18,19 respectively, as well as cation current activated by diacylglycerol in rat cerebral myocytes.20,24 However, the Ca2+-sensitivity of these channels is markedly different; specifically, increasing [Ca2+]o from 0.05 to 2 mmol/L suppressed native ROC currents11,18,25 but increased in TRPC6 current.12 Additionally, as noted by Beech et al,4 the current-voltage (I-V) relations of TRPC6-containing cation channels of portal vein,11 cerebral,20,24 and pulmonary arterial14 myocytes and A7r5 VSM cells18 are not identical. The molecular basis of these differences in functional properties is presently unknown.
Previous results indicate that multiple types of TRPC subunits are expressed at varied levels by VSM cells of different vessels in a species-dependent manner.4,6,13,14,18,19,27 Thus, VSM ROCs may have a subunit composition that varies in a vessel-dependent fashion, different agonists may activate ROCs composed of different subunits, and/or a given agonist may activate multiple ROCs with varied subunit composition.4,26 VSM ROC could also result from the heteromultimeric assembly of more than 1 type of TRPC subunit.4–6 Coassociation of different voltage-gated K+ channel subunits is an established principle, and it occurs in many cell types, including VSM cells.28,29 Expression of heteromultimeric TRPC6-containing channels with unique properties provides a viable explanation for the lack of complete functional identity of homomultimeric TRPC6 and native ROCs, as well as the varied properties of TRPC6-containing ROCs in different VSM cells.4–6 TRPC3 and TRPC6 antisense oligonucleotides both inhibit ROC currents of rat prostate smooth muscle cells,30 providing indirect evidence for the presence of heteromultimeric channels. However, direct biochemical evidence of TRPC heteromultimerization, such as that shown for heterologous expression systems and brain tissues,31–34 has not been obtained for VSM cells.
Here we tested the hypothesis that heteromultimeric TRPC channels contribute to ROCs of A7r5 VSM cells. This aortic smooth muscle cell line was specifically used because it permitted a detailed analysis of the biochemical and electrophysiological properties of the ROCs that would otherwise not be possible using native cells because of the low level of channel expression and relatively low affinity of currently available subunit-specific antibodies. TRPC expression and heteromultimerization was assessed by RT-PCR, immunoblotting, and coimmunoprecipitation. The participation of TRPC-containing ROCs was addressed using a dominant-negative strategy,32,34 and the sensitivity of the native ROCs and homo- and heteromultimeric TRPC to [Ca2+]o was compared. Our results provide the first direct evidence that heteromultimeric TRPC channels can contribute to ROCs activated by GPCRs in VSM cells.
Materials and Methods
Human embryonic kidney 293 (HEK293; passage 5 to 17) and A7r5 VSM cells (passage 10 to 22) (American Type Culture Collection, Manassas, Va) were used. Both cell types were maintained in DMEM (Invitrogen [Gibco-BRL], Burlington, Canada) supplemented with 10% FCS, 100 U/mL penicillin, and 100 μg/mL streptomycin (Invitrogen). HEK293 cells grown on glass coverslips were transfected with cDNAs encoding 0.5 μg of angiotensin AT1 receptor, 0.5 μg of green fluorescent protein (GFP), and varied amounts of TRPC subunit cDNAs using FuGENE 6 (Roche Diagnostics Canada, Laval) and used within 24 to 48 hours. Eighty percent confluent cultures of A7r5 cells were transfected with cDNAs encoding 0.5 μg of GFP, 1.5 μg of TRPC subunit cDNAs, or equal amounts of empty vector pcDNA3 (mock transfection). The A7r5 cells were plated onto coverslips 48 hours after transfection and used within 24 hours. The methods for whole-cell voltage clamp, immunocytochemistry, immunoprecipitation, immunoblotting, and/or molecular biology were as previously described28,29 and/or as indicated in the online data supplement. All primers used are included in the online data supplement available at http://circres.ahajournals.org.
Results
TRPC Transcript Expression
TRPC message expression was analyzed by RT-PCR using subunit-specific primers and mRNA from A7r5 cells and rat brain; the latter used to confirm primer function and reaction integrity (Figure 1A). TRPC1, TRPC4, TRPC6, and TRPC7 amplicons were detected in A7r5 cell mRNA (Figure 1B) and confirmed by sequencing. TRPC3 and TRPC5 expression was not detected but was consistently apparent for rat brain (Figure 1A). Alternate primer sets for TRPC3, TRPC4/TRPC4, and TRPC7 were also used. TRPC3 (data not shown) and TRPC4 (Figure 1C) were not detected, but amplicons for TRPC4 (245 base pairs [bp]; Figure 1C) and TRPC7 (649 bp; Figure 1D) were identified (additional 300 and 400 bp amplicons in Figure 1C did not correspond to any known gene).
For comparative purposes, we also determined whether TRPC6 and TRPC7 were expressed by native VSM cells. Amplicons were detected for both TRPC subunits in mRNA samples derived from populations of 50 to 75 individually selected myocytes freshly isolated from rat middle and posterior cerebral arteries (Figure 1E).29 Expression of endothelin-1 transcript (328 bp) was not apparent (positive control for primers described previously29), indicating a lack of contamination by message derived from endothelial cells.
TRPC Protein Expression and Coassembly
We probed for TRPC1 protein expression using anti-TRPC1 and detected a 145 kDa protein (data not shown), as noted previously.35 The presence of TRPC4, TRPC6, and TRPC7 in lysates of A7r5 cells was also detected. Anti–c-myc immunoreactive bands were evident in HEK293 cells expressing full-length c-myc–tagged TRPC4 and the smaller TRPC4 between 105 and 100 kDa (Figure 2A). An immunoreactive band of 100 kDa and consistent with TRPC4 was detected with anti-TRPC4 in A7r5 (Figure 2A) and HEK293 (not shown) cell lysates. Anti-TRPC6 detected multiple bands between 100 to 130 kDa in lysates of HEK293 cells expressing TRPC6, likely corresponding to monomeric and glycosylated subunit,36 as well as protein at 125 kDa in A7r5 cell lysates (Figure 2B). These bands were not evident in lysates of non- or mock-transfected (empty vector) HEK293 cells (not shown). Anti-TRPC7 failed to detect protein in mock-transfected HEK293 cells or cells transfected with TRPC6, but multiple immunoreactive bands between 100 to 125 kDa were identified in lysates of cells expressing TRPC6 and TRPC7 (Figure 2C). An immunoreactive band of 100 kDa was detected in A7r5 cell lysates using anti-TRPC7 (Figure 2C).
Figure 2D shows the results of coimmunoprecipitation experiments using anti-TRPC6 followed by immunoblotting with antibodies against TRPC6, TRPC4, or TRPC7. TRPC6 immunoreactive protein was detected between 110 and 130 kDa in anti-TRPC6 immunoprecipitates of HEK293 cells expressing TRPC6 (Figure 2Da). No evidence of immunoreactive protein corresponding to TRPC4 at 100 kDa was identified by anti-TRPC4 in anti-TRPC6 immunoprecipitates of A7r5 cells, but nonspecific bands were apparent at 72 to 80 kDa and 130 kDa (closed circles; Figure 2Db). TRPC7 immunoreactive protein was detected at 100 kDa in anti-TRPC6 immunoprecipitates (Figure 2Dc) corresponding to the 100 kDa band present in immunoblots of A7r5 and TRPC7-expressing HEK293 cells, indicating the association of these subunits in A7r5 cells (Figure 2C). Nonspecific bands similar to those in Figure 2Db were also evident in TRPC7 immunoblots of anti-TRPC6 immunoprecipitates.
Dominant-Negative Suppression of AVP-Induced ROC Current
The contribution of TRPC channels to AVP-induced ROC current of A7r5 cells was assessed using dominant-negative pore mutants of TRPC6 (TRPC6DN) and TRPC5 (TRPC5DN) with C- and N-terminal c-myc and hemagglutinin (HA) tags, respectively. The mutants were tested for specificity in HEK293 cells (see online data supplement). Anti–c-myc immunofluorescence was evident in GFP-positive A7r5 cells transfected with TRPC6DN but not mock-transfected cells (Figure 3A). AVP caused a marked increase in N-methyl-D-glutamine (NMDG)-sensitive cation current at –60 mV in GFP-positive A7r5 cells expressing empty vector (Figure 3B) but consistently less current in GFP-positive cells of sister culture dishes expressing TRPC6DN (Figure 3D and 3G; 18.2±4.8 [n=15] versus 5.4±2.4 pA/pF [n=16]; P<0.02). In contrast, AVP-induced current amplitude was not different in GFP-positive cells of sister dishes transfected with empty vector or TRPC5DN (Figure 3F and 3G; 5.3±1.7 [n=15] versus 3.0±1.4 pA/pF [n=11]; P=0.15). The I-V relationship of AVP-induced current in mock-transfected (n=14) and in 9 TRPC6DN-transfected A7r5 cells (with >1.0 pA/pF current) was doubly rectifying (Figure 3C and 3E). Increasing [Ca2+]o from 0.05 to 1 mmol/L reduced current in mock- and TRPC6DN-transfected cells, particularly between –100 and 0 mV (Figure 3B through 3E). The relative change in current at –100 and +100 mV in these cells owing to [Ca2+]o were identical (ie, current in 1.0/0.05 mmol/L [Ca2+]o; Figure 3E).
Effect of [Ca2+]o on Native and Recombinant Nonselective Cation Currents
The effect of increasing [Ca2+]o on native ROCs was compared with that of homo- and heteromultimeric TRPC6 and TRPC7 channels. TRPC3 and TRPC3-TRPC6 channels were also considered because of the expression of TRPC3 (rather than TRPC7) with TRPC6 in some vessels.4,6,27 Ang II was used as the agonist for the recombinant channel experiments because of availability of cDNAs encoding the AT1 receptor and justified by the lack of a difference in AVP- and Ang II–induced ROC currents of A7r5 cells (see online data supplement) and previous reports that similar A7r5 cell ROC currents are activated by serotonin, PDGF, AlF4–, and OAG.18 Ang II–induced ROC currents were observed in HEK293 cells expressing TRPC3, TRPC6, and TRPC7 alone or in combination (Figure 4B through 4F). The I-V relations for homo- and heteromultimeric TRPC combinations all doubly rectified (Figure 4B through 4F), consistent with previous studies.11,12,36 Increasing [Ca2+]o caused a rapid inhibition of native current (Figure 4A), as well as currents owing to TRPC7 and TRPC3 (Figure 4C and 4D) and heteromultimeric TRPC3-TRPC6 and TRPC6-TRPC7 channels (Figure 4E and 4F). In contrast, TRPC6 current amplitude increased immediately on the change in [Ca2+]o and subsequently declined to a maintained level that was not different from that recorded before the solution change (Figure 4B). The differing effects of [Ca2+]o on current amplitude were most apparent between –100 and 0 mV (Figure 4G).
A more detailed analysis of the effects of [Ca2+]o on current amplitude was performed for native A7r5 ROCs, TRPC6, TRPC7, and TRPC6-TRPC7 channels using the approach of Helliwell and Large.25 Currents were evoked in 1 mmol/L [Ca2+]o before switching to nominally Ca2+-free, 0.05, 0.1, or 0.3 mmol/L [Ca2+]o solutions (Figure 5). TRPC7 currents showed the greatest increase in peak inward current when [Ca2+]o was lowered from 1 mmol/L (Figure 5). In contrast, TRPC6 current was only slightly enhanced by decreasing [Ca2+]o and consistently exhibited a slight increase in amplitude on subsequent reexposure to 1 mmol/L [Ca2+]o. The mean changes in current amplitude at each [Ca2+]o were determined by normalizing peak amplitude after the change in [Ca2+]o to the value in 1 mmol/L [Ca2+]o and plotted as a function of [Ca2+]o (Figure 6). The relative change in amplitude of homomultimeric TRPC6 and TRPC7 currents were less and greater, respectively, compared with that of the native ROCs (Figure 6). In contrast, the change heteromultimeric TRPC6-TRPC7 current mimicked that of the native A7r5 cell ROCs (Figure 6).
Discussion
This study provides the first direct evidence that heteromultimeric TRPC channels can contribute to ROCs of VSM cells. At least 4 distinct types of cation channels are present in smooth muscle cells, including store-operated, ROC, stretch-activated, and tonically active cation channels, but the molecular identity of each conductance is a matter of debate.4,6,37 These channels are thought to contribute to several physiological functions of VSM cells, including agonist- and stretch-induced depolarization, maintenance of SR Ca2+ store filling, and mediating Ca2+ influx in response to ligands and physical stimuli (eg, stretch).4–6 Understanding the molecular basis of VSM ROCs is of particular interest in light of the important contribution of these channels to the actions of vasoconstrictors,1–6 eg, 1-adrenoceptor agonists, Ang II, and endothelin-1, that are well known to contribute to vascular pathology and the recognition that alterations in TRP channel expression, including TRPC subunits, is associated with abnormal control of vascular contractility and proliferation.13–15 Multiple TRPC subunits, as well as members of TRPV and TRPM families, have been shown to be expressed by VSM cells in a tissue- and species-specific pattern.4,6,13,27 For example, A7r5 cells were found to express TRPC1 and TRPC618 and, more recently, TRPC3, TRPC4, TRPC5 and/or TRPC7, depending on the primers used and/or specific strain of A7r5 cells studied,19,38 a pattern of expression that is apparent in several arteries and veins.4 Here we show the expression of transcripts encoding the short TRPC4 splice variant of TRPC4, as well as TRPC1, TRPC6, and TRPC7, in A7r5 cell mRNA extracts, and the presence of these proteins was confirmed by immunoblotting. Additionally, we provide biochemical and electrophysiological evidence of coassembly of TRPC6 and TRPC7 and functional identity of native ROC and TRPC6-TRPC7 heteromultimeric channels in terms of Ca2+ sensitivity, respectively. These observations support the view that VSM ROC current may be the result of expression and association of multiple TRPC subunits.
The evidence that TRPC6 and TRPC7 coassemble and contribute to the AVP-induced ROCs of A7r5 cells may be summarized as follows. First, TRPC6 and TRPC7 message and protein expression were detected by RT-PCR and immunoblotting using subunit-specific primers and antibodies with a specificity demonstrated in previous studies33 and were confirmed here using HEK293 cells expressing TRPC6 and/or TRPC7. Second, direct biochemical evidence of TRPC subunit coassembly was obtained in experiments in which anti-TRPC7 immunoreactive protein was coimmunoprecipitated from lysates of A7r5 cells using anti-TRPC6, similar to the association previously identified to occur in synaptosomes derived from adult rat brain.33 Third, a dominant-negative strategy demonstrated the involvement of TRPC subunits in the response of A7r5 cells to AVP. The strategy is based on the assumption that mutant subunits with the conserved leucine-phenylalanine-tryptophan channel pore motif replaced with an alanine triplet (AAA) coassemble with wild-type subunits and suppress cation permeation through the resultant heteromultimeric channels.32 Using this approach, Hofmann et al32 concluded that the TRPC family divides into 2 subgroupings capable of heteromultimerization: TRPC3, TRPC6, and TRPC7 versus TRPC1, TRPC4, and TRPC5. Here we found a significant dominant-negative suppression of AVP-induced A7r5 cell ROC current by TRPC6DN but not TRPC5DN. In the absence of TRPC3 expression, this result indicates the potential involvement of homo- and/or heteromultimeric TRPC6 and TRPC7 channels. Recent observations indicate that TRPC coassembly may be more complex than that described by Hofmann et al,32 with interactions between members of the subgroups being possible.34 For example, TRPC3 or TRPC6 in combination with TRPC1, TRPC4, or TRPC5 were identified in lysates of embryonic rat brain and a critical role for TRPC1 as a mediator of cross-grouping interactions was indicated.34 In light of these findings and the expression of TRPC1 and TRPC4 by the A7r5 cells used here, the possibility that A7r5 cell ROCs may have a more complex subunit composition was considered. We probed for an association of TRPC4 with TRPC6 but were unable to detect the presence of TRPC4 protein in anti-TRPC6 immunoprecipitates. It is possible that the channel density and/or antibody affinity was too low to permit detection or that the antigenic sites on the channel protein were inaccessible when in a heteromultimeric complex.33 However, we also failed to observe a suppression of ROC current in A7r5 cells expressing TRPC5DN and suppression of TRPC1 expression by A7r5 cells using an anti-sense oligonucleotide approach has no effect on AVP-induced ROC currents (L.I. Brueggman and K.L. Byron, personal communication). Taken together, these data imply that channels containing TRPC6, TRPC7, and TRPC1 alone or with TRPC4 likely do not contribute to AVP-induced ROCs of A7r5 cells.
The fourth line of evidence supporting the view that heteromultimeric ROCs are activated by AVP in A7r5 cells was obtained in experiments that compared the effects of [Ca2+]o on A7r5 cell ROCs versus homo- and heteromultimeric TRPC channels. Previous studies using TRPC6 antisense oligonucleotides or siRNAs imply that TRPC6 is a component of portal vein, cerebral arterial, and A7r5 VSM cell ROCs, as these approaches resulted in a suppression of agonist- and/or OAG-induced induced ROC current.11,18,19,20,24 However, the previously reported 1.5 increase in TRPC6 current amplitude caused by increasing [Ca2+]o from between 0.2 to 0.5 to between 1 to 2 mmol/L11,12 contrasts markedly with the rapid inhibition observed for native VSM ROC currents of portal vein and A7r5 cells evoked by 1-adrenoceptor agonists and AVP, respectively.11,18,25 This difference in behavior cannot be attributed to variations in the [Ca2+]o sensitivity of the mechanism of channel activation, as identical responses have been reported for TRPC6, TRPC7, and native currents activated by a diverse set of compounds.6,11,12,18 Rather, the results of this study indicate that the difference in functional identity between TRPC6 and these VSM ROCs can be attributed to the presence of multiple pore-forming subunit(s) in the channel complex. Despite sharing considerable sequence identity with TRPC6 subunits, TRPC339 and TRPC712,40 channels are inhibited by changing [Ca2+]o from nominally Ca2+ free to 1 mmol/L, confirmed here by stepping [Ca2+]o from 0.05 to 1 mmol/L and from 1 mmol/L to between nominally Ca2+ free and 0.3 mmol/L. Moreover, we show for the first time that heteromultimeric TRPC3-TRPC6 and TRPC6-TRPC7 channels are also inhibited by increasing [Ca2+]o from 0.05 to 1 mmol/L. Thus, the potentiation of TRPC6 current by 1 mmol/L [Ca2+]o is lost when this subunit coassembles with TRPC3 or TRPC7 subunits. Similarly, TRPC7 exhibited a significantly greater relative inhibition by [Ca2+]o compared with heteromultimeric TRPC6-TRPC7 channels, suggesting that the properties of TRPC7 are modified by the presence of TRPC6 in the complex. Thus, heteromultimeric TRPC6-TRPC7 exhibit at least 1 property that is distinct from homomultimeric TRPC6 and TRPC7 channels, and only this heteromultimeric combination of subunits produced ROCs that showed a relative inhibition by [Ca2+]o that was consistent with that of native AVP-induced ROCs of A7r5 cells. It is unlikely that AVP activates 2 populations of ROCs because of expression of homomultimeric TRPC6 and TRPC7 channels, with the TRPC6 channels potentiated and the TRPC7 rapidly inhibited by increasing [Ca2+]o. Increasing [Ca2+]o to 1 mmol/L caused an almost complete suppression of AVP-induced native ROC current, rather than the partial inhibition that would be expected for the case of 2 populations of homomultimeric channels.
We focused here on the inhibitory effect of [Ca2+]o on native and recombinant cation current amplitude because of the marked difference in behavior of A7r5 cell ROCs and TRPC6 channels. However, modulation of VSM ROCs and TRPC channels by [Ca2+]o is complicated12,25,41 and involves additional effects not considered. These include facilitation by [Ca2+]o between <0.001 and 0.2 mmol/L (EC50 3 to 6 μmol/L)12,25 and indirect modulation by Ca2+-calmodulin–dependent and –independent kinases.12,25,41,42,43 These aspects of Ca2+ regulation of heteromultimeric TRPC channels should be considered in the future. Lack of control over subunit stoichiometry is a limitation of experiments coexpressing cDNAs, as this approach may not yield channels with an appropriate subunit stoichiometry. Because stoichiometry may be an important for appropriate regulation by Ca2+, as well as other functional properties, the use of concatemeric subunits to produce channels of known composition is warranted.
A limitation of this study is that the evidence of TRPC subunit heteromultimerization was obtained using cultured A7r5 cells rather than native VSM cells. This approach was required because, firstly, intact arteries cannot be used because of potential contamination by TRPC channels of endothelial cells. Secondly, the low level of channel expression in VSM cells complicates analysis of TRPC subunit association in denuded vessel/isolated myocyte preparations by coimmunoprecipitation using the relatively low-affinity TRPC antibodies that are currently available. Finally, in the absence of direct biochemical evidence of coassembly, the finding that native VSM ROC currents are inhibited by 1 mmol/L [Ca2+]o cannot be used to differentiate between the contribution of homo- and heteromultimeric channels containing TRPC3 and TRPC7 or these subunits associated with TRPC6. On the other hand, the use of the A7r5 cell line as a model for the study of VSM ROCs can be justified based on the pattern of TRPC subunit message expression. The set of TRPC subunits expressed by the A7r5 cells used here included TRPC1, TRPC4, TRPC6, and TRPC7. TRPCC4, TRPC6, and TRPC7 mRNAs were previously shown to be expressed together in canine pulmonary and renal arteries,27 and TRPC6 and TRPC7 were both detected in rat thoracic and cerebral arteries.13 These findings are consistent with our data showing the presence of TRPC6 and TRPC7 transcripts in mRNA derived from isolated rat cerebral myocytes, an approach that precludes false-positive identification of subunit expression attributable to contamination by message derived from endothelial cells, blood cells, and/or fibroblasts present in the vessel wall. Taken together, these findings provide molecular evidence consistent with the view that TRPC6- and TRPC7-containing ROCs may also be expressed by native VSM cells within some vessels. However, based on the reported variability in properties of native VSM ROC currents,4–6 TRPC6-TRPC7 channels may not be the only ROC channel type expressed by VSM cells; other homomultimeric (eg, uridine triphosphate-activated TRPC3 channels17) and heteromultimeric combinations of TRPC subunits (eg, TRPC1, TRPC3, and TRPC614) could also be present.
In summary, this report provides molecular, biochemical, and electrophysiological evidence that multiple TRPC subunits are expressed by A7r5 VSM cells, that TRPC6 and TRPC7 coassemble in this cell type, and that heteromultimeric TRPC6-TRPC7 channels share a functional identity with AVP-induced ROCs of A7r5 cells with respect to their I-V relation and inhibition by [Ca2+]o.
Acknowledgments
Sources of Funding
This work was supported by the Canadian Institutes for Health Research (MT-13505) and an Alberta Heritage Foundation for Medical Research postdoctoral fellowship (to Y.M.).
Disclosures
Dr Y. Maruyama was the recipient of a Pfizer Canada Young Scientist Education Award in Smooth Muscle Research.
Footnotes
Original received November 22, 2005; revision received April 21, 2006; accepted May 2, 2006.
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