Role of endogenous TRPC6 channels in Ca2+ signal generation in A7r5 smooth muscle cells
Jonathan Soboloff1, Maria Spassova, Wen Xu
1Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Abstract:
The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bore the hallmarks of TRPC6 function; it was inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry was blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels would allow depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction.
Insights
Canonical transient receptor potential (TRPC) channels, particularly TRPC6, are crucial for smooth muscle cell signaling. TRPC6 activation leads to sodium entry and depolarization, subsequently activating L-type calcium channels for calcium influx and muscle contraction.
Area of Science:
- Molecular Biology
- Cell Physiology
- Ion Channel Function
Background:
- Canonical transient receptor potential (TRPC) ion channels are vital for calcium (Ca2+) signal generation, but their activation mechanisms and specific roles are not fully understood.
- Most research on TRPC channels involves overexpressed systems, limiting understanding of endogenous channel function in native cellular environments.
- A7r5 smooth muscle cells are a relevant model for studying endogenous TRPC channel activity in the context of cellular signaling.
Purpose of the Study:
- To investigate the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells using a combination of molecular, biochemical, and electrophysiological techniques.
- To elucidate the specific role of TRPC6 in mediating cation currents and calcium (Ca2+) entry in response to various stimuli.
- To determine the relationship between TRPC6 activity, membrane depolarization, and the subsequent activation of L-type calcium channels.
Main Methods:
- Real-time PCR and Western blot analyses were employed to determine the expression levels of various TRPC channel subtypes in A7r5 cells.
- Electrophysiological recordings (e.g., patch-clamp) were used to characterize cation currents activated by phospholipase C-coupled receptors or diacylglycerol analogues.
- RNA interference (RNAi) targeting TRPC6 was utilized to assess the functional impact of TRPC6 knockdown on ion channel activity and intracellular calcium levels.
Main Results:
- TRPC6 was identified as the predominantly expressed diacylglycerol-responsive TRPC channel in A7r5 cells, alongside abundant TRPC1, TRPC4, and TRPC5.
- TRPC6 small interfering RNA (siRNA) effectively reduced TRPC6 expression without altering other TRPC channel levels, confirming target specificity.
- TRPC6 knockdown significantly inhibited vasopressin-induced Ca2+ entry and OAG-activated cation currents, but not store-operated Ca2+ entry, indicating a specific role for TRPC6.
- OAG-induced Ca2+ entry, though unaffected by TRPC6 knockdown, exhibited characteristics of TRPC6 function, including inhibition by protein kinase C and PP2, and blockade by nimodipine.
- TRPC6 activation primarily mediates Na+ entry and cell depolarization, which in turn activates L-type Ca2+ channels for Ca2+ influx.
Conclusions:
- Endogenous TRPC6 channels in A7r5 smooth muscle cells play a critical role in mediating cation entry and subsequent depolarization.
- TRPC6-mediated depolarization is a key event that triggers the activation of L-type calcium channels, facilitating Ca2+ influx required for smooth muscle function.
- The study reveals a crucial functional coupling between TRPC6 and L-type calcium channels, essential for regulating smooth muscle cell membrane potential and contraction.
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