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Multiple regulation by calcium of murine homologues of transient receptor potential proteins TRPC6 and TRPC7 expressed in HEK293 cells
Author(s) -
Shi Juan,
Mori Emiko,
Mori Yasuo,
Mori Masayuki,
Li Jishuo,
Ito Yushi,
Inoue Ryuji
Publication year - 2004
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.2004.075051
Subject(s) - trpc6 , chemistry , transient receptor potential channel , intracellular , biophysics , patch clamp , hek 293 cells , trpc3 , calcium in biology , calmodulin , microbiology and biotechnology , biochemistry , receptor , biology , trpc , enzyme
We investigated, by using the patch clamp technique, Ca 2 + ‐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 Ca 2 + entry channels. With nystatin‐perforated recording, the magnitude and time courses of activation and inactivation of carbachol (CCh; 100 μ m )‐activated TRPC6 currents ( I TRPC6 ) were enhanced and accelerated, respectively, by extracellular Ca 2 + (Ca 2 o + ) whether it was continuously present or applied after receptor stimulation. In contrast, Ca 2 o + solely inhibited TRPC7 currents ( I TRPC7 ). Vigorous buffering of intracellular Ca 2 + (Ca 2 i + ) under conventional whole‐cell clamp abolished the slow potentiating (i.e. accelerated activation) and inactivating effects of Ca 2 o + , disclosing fast potentiation (EC 50 : ∼0.4 m m ) and inhibition (IC 50 : ∼4 m m ) of I TRPC6 and fast inhibition (IC 50 : ∼0.4 m m ) 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 Sr 2 + but not Ba 2 + . The activation process of I TRPC6 or its acceleration by Ca 2 o + probably involves phosphorylation by calmodulin (CaM)‐dependent kinase II (CaMKII), as pretreatment with calmidazolium (3 μ m ), coexpression of Ca 2 + ‐insesentive 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 Ca 2 i + via CaM and conversely enhanced by IP 3 . 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 Ca 2 + from both sides of the membrane through differential Ca 2 + −CaM‐dependent and ‐independent mechanisms.