Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells.
We investigated, by using the patch clamp technique, Ca2+-mediated regulation of heterologously expressed TRPC6 and TRPC7 proteins in HEK293 cells, two closely related homologues of the transient receptor potential (TRP) family and molecular candidates for native receptor-operated Ca2+ entry channels. With nystatin-perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 microM)-activated TRPC6 currents (I(TRPC6)) were enhanced and accelerated, respectively, by extracellular Ca2+ (Ca2+(o)) whether it was continuously present or applied after receptor stimulation. In contrast, Ca2+(o) solely inhibited TRPC7 currents (I(TRPC7)). Vigorous buffering of intracellular Ca2+ (Ca2+(i)) under conventional whole-cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca2+(o), disclosing fast potentiation (EC50: approximately 0.4 mM) and inhibition (IC50: approximately 4 mM) of I(TRPC6) and fast inhibition (IC50: approximately 0.4 mM) of I(TRPC7). This inhibition of I(TRPC6) and I(TRPC7) seems to be associated with voltage-dependent reductions of unitary conductance and open probability at the single channel level, whereas the potentiation of I(TRPC6) showed little voltage dependence and was mimicked by Sr2+ but not Ba2+. The activation process of I(TRPC6) or its acceleration by Ca2+(o) probably involves phosphorylation by calmodulin (CaM)-dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 microM), coexpression of Ca2+-insensitive mutant CaM, and intracellular perfusion of the non-hydrolysable ATP analogue AMP-PNP and a CaMKII-specific inhibitory peptide all effectively prevented channel activation. However, this was not observed for TRPC7. Instead, single CCh-activated TRPC7 channel activity was concentration-dependently suppressed by nanomolar Ca2+(i) via CaM and conversely enhanced by IP3. In addition, the inactivation time course of I(TRPC6) was significantly retarded by pharmacological inhibition of protein kinase C (PKC). These results collectively suggest that TRPC6 and 7 channels are multiply regulated by Ca2+ from both sides of the membrane through differential Ca2+-CaM-dependent and -independent mechanisms.
Shi
Juan
J
Department of Pharmacology, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Mori
Emiko
E
Mori
Yasuo
Y
Mori
Masayuki
M
Li
Jishuo
J
Ito
Yushi
Y
Inoue
Ryuji
R
D000818:Animals; D002118:Calcium / Q000494:pharmacology / Q000502:physiology*; D015220:Calcium Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002413:Cations, Divalent; D002460:Cell Line; D006801:Humans; D007473:Ion Channels / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D040681:Structural Homology, Protein; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; D050053:TRPM Cation Channels; D014162:Transfection
D015220:Calcium Channels; D002413:Cations, Divalent; D007473:Ion Channels; D008565:Membrane Proteins; D050052:TRPC Cation Channels; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; C498501:TRPC7 protein, human; D050053:TRPM Cation Channels; C121934:TRPM2 protein, human; D002217:Carbachol; D002118:Calcium