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

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 AKAP79_1-153 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