doi: 10.1161/01.RES.0000231255.32630.df
© 2006 American Heart Association, Inc.
Editorials |
Molecular Diversity of Receptor Operated Channels in Vascular Smooth Muscle
A Role for Heteromultimeric TRP Channels?
From the Department of Biomedical Sciences, Colorado State University, Fort Collins.
Correspondence to Scott Earley, PhD, Department of Biomedical Sciences, Colorado State University, 1680 Campus Delivery, Fort Collins, CO 80523-1680. E-mail Scott.Earley{at}Colostate.edu
See related article, pages 1520–1527
Key Words: TRP channels • vascular smooth muscle • receptor-operated channel
The mammalian transient receptor potential (TRP) superfamily of cation channels includes at least 29 individual genes divided into 6 subfamilies based on sequence homology.1 Like many ion channel proteins, TRP subunit polypeptide sequences encode 6 transmembrane domains, with a putative pore-forming loop between the fifth and sixth membrane spanning region. Functional ion-permeable channels are formed from the assembly of 4 TRP polypeptide subunits as a tetrameric complex. Typically, biophysical properties and regulation of TRP channels have been elucidated by studying cultured cells overexpressing a single cloned TRP subunit gene. Under these conditions, the overexpressed protein is the primary TRP subunit present, and channels are predominately formed from 4 identical TRP subunits, ie, "homomeric" TRP channels. Although this experimental approach has yielded abundant valuable information, it does not accurately reflect the in vivo state because native cells generally express a number of individual TRP channel genes, allowing the formation of ion channels composed of 2 or more TRP subunits, ie, "heteromultimeric" TRP channels. The existence of heteromultimeric TRP channels was first demonstrated when coexpression of the Drosophila TRP and TRPL genes in cultured cells gave rise to whole-cell currents that were distinct from those of cells expressing TRP or TRPL homomeric channels, and physical interaction between these 2 proteins in Drosophila photoreceptor cells was demonstrated by coimmunoprecipitation.2 Heteromultimeric TRP channels in native mammalian cells were first reported when subunits TRPC1 and TRPC5 from the canonical TRP (TRPC) subfamily were shown to colocalize in the rat hippocampus and, using coimmunoprecipitation, were found to form a molecular complex in brain microsomes.3 A whole-cell current was recorded from HEK cells coexpressing TRPC1 and TRPC5 that was distinct from recordings obtained from cells expressing either TRPC1 or TRPC5 alone, providing further evidence that TRPC1 and TRPC5 subunits can form functional heteromultimeric ion channels. Although several recent studies suggest important functional roles for TRP channels expressed by vascular smooth muscle cells (VSMCs),4–9 to date none have examined the significance of TRP subunit heteromultimerization in this tissue. To begin to address this void, Maruyama et al, in the current issue of Circulation Research, provide convincing evidence for a functional role for heteromultimeric complexes composed of TRPC6 and TRPC7 subunits as receptor operated channels (ROCs) in a VSMC-derived cell line.10 It is well known that ROCs contribute to arterial tone regulation in response to vasoactive agonists. Phospholipase activity is increased after activation of G protein–coupled receptors by vasoconstrictors such as endothelin-1, angiotensin II (Ang II), and 
-adrenoceptor agonists, causing the release of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (Ins(1,4,5)P3). DAG (and perhaps Ins(1,4,5)P3) directly stimulates cation influx.11 Identification of the molecular entities responsible for the formation of ROCs could have significant clinical importance because ROC-activating vasoconstrictor agonists are known to contribute to vascular pathologies, such as systemic and pulmonary hypertension. Thus, the novel findings reported by Maruyama and coworkers10 bring the much discussed, but poorly understood, issue of TRP subunit heteromultimerization to the forefront for investigators working in this area.
To understand the functional implications of heteromultimeric TRP channels in vivo, 2 central questions need to be addressed: (1) what is the relative abundance of TRP subunits in a specific cell type? and (2) what are the rules governing subunit combination? The first question was formally addressed in a recent study by Yang et al that systematically investigated the expression profile and relative mRNA levels of vanilloid TRP (TRPV) and melastain TRP (TRPM) subunits in pulmonary artery and aortic VSMCs, and reported that both cell types express 6 of the 8 TRPM subunits (M2, M3, M4, M5, M7, and M8) and 4 of the 6 TRPV proteins (V1, V2, V3, V4).12 Cerebral artery smooth muscle cells express at least 5 of the 7 TRPC subunits (C1, C2, C3, C4, and C6) (unpublished observations). These findings highlight the fact that most, if not all, cells express multiple TRP subunits, and suggest that heteromultimeric channels may be ubiquitous. In the absence of preferred paring schemes, a potentially large number of channels with distinct biophysical properties could result from promiscuous grouping of TRP subunits. Moreover, functionally distinct mRNA splice variants have been reported for a number of TRP channels,13 further increasing the potential molecular complexity of the superfamily. Fortunately, a few studies have attempted to restore order and rules governing TRP channel interactions have been proposed. In an elegant series of experiments, Hofmann et al, using a combination of fluorescence resonance energy transfer and coimmunoprecipitation, identified 2 distinct subfamilies of TRPC subunits allowing the formation of heteromultimers: TRPC3, C6, and C7 belong to one subfamily and the second comprising TRPC1, C4, and C5.14 However, the scheme proposed by Hofmann et al is challenged by a report of heteromultimeric channels consisting of a TRPC1 subunit plus a TRPC4 or TRPC5 subunit and either a TRPC3 or TRPC6 subunit present in embryonic rat brain.15 There have been fewer reports concerning the formation of heteromultimeric channels for the other TRP subfamilies. According to one study, TRPV1, V2, V3, and V4 subunits preferentially form homomeric channels, whereas TRPV5 and V6 subunits can form heteromultimeric channels,16 although another found that TRPV1/TRPV2 heteromultimeric channels were present in rat dorsal root ganglia.17 The only report of a heteromultimeric channel from the TRPM subfamily demonstrated that TRPM6 and TRPM7 subunits form a heteromultimeric channel involved in epithelial Mg2+ reabsorption.18 There have been no reports to date of heteromultimeric channels composed of subunits from different TRP subfamilies.
The findings of Maruyama et al reported in this issue10 provide compelling evidence that heteromultimeric TRP channels can form ROCs in the A7r5 smooth muscle derived cell line. After probing the TRPC expression profile in these cells, and finding that TRPC1, C4, C6, and C7 (and not C2, C3, and C5) were present, the Cole group demonstrated physical interaction between TRPC6 and C7 using coimmunoprecipitation. The authors went on to show that expression of a dominant-negative form of TRPC6 significantly attenuated Ang II–induced currents in A7r5 cells. To further characterize the molecular identity of agonist-induced currents in these cells, Maruyama and coworkers took advantage of differences in sensitivity to extracellular Ca2+ among various TRPC homomeric and heteromultimeric channels as a biophysical fingerprint of ROC subunit composition. Agonist-induced currents recorded from A7r5 cells and from HEK cells expressing TRPC7 or coexpressing TRPC6 and TRPC7 were attenuated when extracellular Ca2+ was increased from 0.05 mmol/L to 1.0 mmol/L, but were slightly increased for HEK cells expressing TRPC6. Furthermore, changes in agonist-induced current amplitude (relative to those recorded when extracellular Ca2+ was 1 mmol/L) when extracellular Ca2+ was incrementally reduced to 0.01 mmol/L were similar for A7r5 cells and HEK cells coexpressing TRPC6 and TRPC7, whereas these relative changes differed from A7r5 for HEK cells expressing TRPC6 or TRPC7 alone. Thus, in terms of sensitivity to inhibition by extracellular Ca2+, ROCs present in A7r5 cells are very similar to those present in HEK cells coexpressing TRPC6 and TRPC7 and are different from those present in HEK cells expressing TRPC6 or TRPC7 alone. An important consideration in evaluating the significance of these findings concerns the use of the A7r5 cell line, rather than native VSMCs, for these studies. Phenotypic changes occur rapidly in cultured smooth muscle cells, and it remains possible that TRPC6/C7 heteromultimeric channels may be ROCs in A7r5 cells but not in native VSMCs. However, because very large amounts of starting material are required for the biochemical experiments used to demonstrate the presence of heteromultimeric channels, it would be extremely difficult to perform the current study using freshly-isolated VSMCs. In support of a physiological role for heteromultimeric TRPC6/C7 in the vasculature, the authors demonstrate expression of mRNA for TRPC6 and C7 in freshly-isolated rat cerebral artery myocytes. Further evidence in support of heteromultimeric TRP channels as ROCs in native VSMCs awaits the development of more sensitive techniques for detecting multimolecular complexes in this tissue.
The current findings from the Cole laboratory are of particular interest when the pathophysiological consequences of altered TRP expression are considered. Because of the potential importance of these channels in understanding vascular dysfunction, considerable effort has been focused on elucidating the molecular identities of ROCs in VSMCs, and it appears that there is significant diversity in the composition of these channels between vascular beds. For example, evidence supporting a role for TRPC6 as an 1-adrenoceptor–activated ROC in portal vein smooth muscle7 and TRPC3 as a purinergic receptor activated ROC in cerebral arterial myocytes8 has been reported. In the absence of selective pharmacology, both of these studies relied on antisense technology to suppress TRP gene expression, leaving open the possibility that the TRP subunit targeted for knockdown was part of a heteromultimeric channel required for ROC activity. Thus, the findings of Maryuyama et al are consistent with the possibility that potential plasticity in TRP expression and alteration of TRP subunit stoichiometry in VSMCs during disease states could have a significant impact on the formation of heteromultimeric TRP channels and the sensitivity to specific vasoconstrictor agonists.
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Acknowledgments |
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Source of Funding
This work was supported by the American Heart Association (AHA 0535226N).
Disclosures
None.
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Footnotes |
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The opinions expressed in this editorial are not necessarily those of the editors or of the American Heart Association.
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Related Article:
Circ. Res. 2006 98: 1520-1527.
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