Role of endogenous TRPC6 channels in Ca2+ signal generation in A7r5 smooth muscle cells.
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.
Soboloff
Jonathan
J
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Spassova
Maria
M
Xu
Wen
W
He
Li-Ping
LP
Cuesta
Natalia
N
Gill
Donald L
DL
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry; D002412:Cations; D017931:DNA Primers / Q000737:chemistry; D004075:Diglycerides / Q000494:pharmacology; D004594:Electrophysiology; D018274:Electroporation; D016257:Fura-2 / Q000494:pharmacology; D007477:Ions; D008564:Membrane Potentials; D008954:Models, Biological; D032389:Myocytes, Smooth Muscle / Q000166:cytology*; D009553:Nimodipine / Q000494:pharmacology; D009841:Oligonucleotides / Q000737:chemistry; D018408:Patch-Clamp Techniques; D011743:Pyrimidines / Q000494:pharmacology; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D017483:Receptors, Vasopressin / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D012964:Sodium / Q000737:chemistry; D050052:TRPC Cation Channels / Q000737:chemistry* / Q000378:metabolism; D000074287:TRPC6 Cation Channel; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D010738:Type C Phospholipases / Q000737:chemistry; D014667:Vasopressins / Q000494:pharmacology
C412373:AG 1879; D015220:Calcium Channels; D002412:Cations; D017931:DNA Primers; D004075:Diglycerides; D007477:Ions; D009841:Oligonucleotides; D011743:Pyrimidines; D034741:RNA, Small Interfering; D017483:Receptors, Vasopressin; D050052:TRPC Cation Channels; C489571:TRPC4 ion channel; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C503500:Trpc5 protein, rat; C096351:transient receptor potential cation channel, subfamily C, member 1; D014667:Vasopressins; D009553:Nimodipine; D019284:Thapsigargin; C038237:1-oleoyl-2-acetylglycerol; D012964:Sodium; D010738:Type C Phospholipases; D002118:Calcium; D016257:Fura-2