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- W2066959649 abstract "KCNQ2-5 subunits encode the M-current, a K+ conductance that controls neuronal excitability. Stimulation of Gq/11-coupled receptors depresses M current via multiple intracellular signals, including depletion of PIP2, generation of Ca2+/calmodulin, and phosphorylation of KCNQ2 by PKC, recruited to the channels by AKAP79/150. We examined the interplay between these signals via FRET measurements performed under total internal reflection fluorescence (TIRF) microscopy, in which mostly membrane events are isolated. CHO cells were transfected with CFP-tagged KCNQ2-4, and a YFP-tagged construct containing the first 153 residues of AKAP79 (AKAP791-153) shown to be sufficient for binding to KCNQ2, PKC and receptors (Hoshi et al., Nat Cell Biol. 7:1066-73). We found significant FRET between all KCNQ2-4 subunits and AKAP791-153 (13 ± 1.3%, 7.1 ± 2.0% and 10.1 ± 2.0% for KCNQ2-4, respectively). Since Ca2+/calmodulin binding not only inhibits M channels (Gamper and Shapiro, JGP 122:17-31), but also acts on AKAP79/150 (Faux and Scott, JBC 272:17038-17044), we asked whether calmodulin alters KCNQ channel-AKAP79/150 interactions. Indeed, FRET between all CFP-KCNQ2-4 subunits and YFP-AKAP791-153 was much less in cells also co-transfected with wild-type calmodulin, but not when dominant-negative calmodulin was used that cannot bind Ca2+. FRET was also substantial between the CFP-KCNQ2 (R345E) mutant that cannot bind calmodulin and YFP-AKAP791-153, which was however not reduced by calmodulin co-expression. Furthermore, the FRET between KCNQ2-4 and AKAP791-153 was also not reduced when the cells were depleted of PIP2 by co-expression of a PIP2 phosphatase. We conclude that calcified, but not apo, calmodulin interferes with KCNQ subunit-AKP79/150 interactions by binding to the channels, likely reducing their affinity for AKAP79/150, and that M channel-AKAP79/150 interactions may serve to anchor PKC to M-channel containing microdomains, where it stands poised to phosphorylate the channels upon stimulation of appropriate Gq/11-coupled receptors." @default.
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- W2066959649 date "2009-02-01" @default.
- W2066959649 modified "2023-09-28" @default.
- W2066959649 title "Disruption Of Interactions Between AKAP79/150 And KCNQ K+ Channels By Ca2+/Calmodulin Observed Using TIRF/FRET" @default.
- W2066959649 doi "https://doi.org/10.1016/j.bpj.2008.12.3677" @default.
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