Ca2+/calmodulin disrupts AKAP79/150 interactions with KCNQ (M-Type) K+ channels

Manjot Bal, Jie Zhang, Ciria C. Hernandez, Oleg Zaika, Mark S. Shapiro

Research output: Contribution to journalArticle

48 Scopus citations

Abstract

M-type channels are localized to neuronal, cardiovascular, and epithelial tissues, where they play critical roles in control of excitability and K + transport, and are regulated by numerous receptors via G q/11-mediated signals. One pathway shown for KCNQ2 and muscarinic receptors uses PKC, recruited to the channels by A-kinase anchoring protein (AKAP)79/150. As M-type channels can be variously composed of KCNQ1-5 subunits, andMcurrent is known to be regulated by Ca2+/calmodulin (CaM) and PIP2 , we probed the generality of AKAP79/150 actions among KCNQ1-5 channels, and the influence of Ca2+/CaM and PIP2 on AKAP79/150 actions. We first examined which KCNQ subunits are targeted by AKAP79 in Chinese hamster ovary (CHO) cells heterologously expressing KCNQ1-5 subunits and AKAP79, using fluorescence resonance energy transfer (FRET) under total internal reflection fluorescence (TIRF) microscopy, and patch-clamp analysis. Donor-dequenching FRET between CFP-tagged KCNQ1-5 and YFP-tagged AKAP79 revealed association of KCNQ2-5, but not KCNQ1, with AKAP79. In parallel with these results,CHOcells stably expressingM1 receptors studied under perforated patch-clamp showed cotransfection of AKAP79 to "sensitize" KCNQ2/3 heteromers and KCNQ2-5, but not KCNQ1, homomers to muscarinic inhibition, manifested by shifts in the dose-response relations to lower concentrations. The effect onKCNQ4was abolished by the T553A mutation of the putative PKC phosphorylation site.Wethen probed the role ofCaMand PIP 2 in these AKAP79 actions. TIRF/FRET experiments revealed cotransfection of wild-type, but not dominant-negative (DN),CaMthat cannot bind Ca2+, to disrupt the interaction of YFP-tagged AKAP791-153 with CFP-tagged KCNQ2-5. Tonic depletion of PIP2 by cotransfection of a PIP2 phosphatase had no effect, and sudden depletion of PIP 2 did not delocalize GFP-tagged AKAP79 from the membrane. Finally, patch-clamp experiments showed cotransfection of wild-type, but not DN, CaM to prevent the AKAP79-mediated sensitization of KCNQ2/3 heteromers to muscarinic inhibition. Thus, AKAP79 acts on KCNQ2-5, but not KCNQ1-containing channels, with effects disrupted by calcified CaM, but not by PIP2 depletion. Copyright

Original languageEnglish (US)
Pages (from-to)2311-2323
Number of pages13
JournalJournal of Neuroscience
Volume30
Issue number6
DOIs
StatePublished - Feb 10 2010

ASJC Scopus subject areas

  • Neuroscience(all)

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