PKC activation sensitizes muscarinic suppression of the M-current via phosphorylation of S541 of KCNQ2 subunit. (A, B) Perforated patch-clamp recordings from rat SCG neurons showing muscarinic suppression of the M-current induced by 0.1 μM oxo-M is attenuated by 10 μM bisindolylmaleimide IV (BIS IV). (A) Current traces showing M-current suppression induced by oxo-M and its attenuation by BIS IV. (B) Pooled data showing muscarinic suppression of the M-current. Amplitudes of the M-currents are normalized to those at t=0. The black box indicates the presence of oxo-M. (C, D) Whole-cell patch-clamp recordings from transiently transfected CHO hm1 cells showing muscarinic suppression of wild-type KCNQ2 and its S541 mutants induced by 0.1 μM oxo-M. (C) Current traces for KCNQ2 channels and their muscarinic suppressions. (D) Pooled data showing oxo-M responses of wild-type KCNQ2, KCNQ2 (S541A), and KCNQ2 (S541D). Amplitudes of the KCNQ2 currents are normalized to those at t=0. (E) Current densities of wild-type KCNQ2, KCNQ2 (S541A), and KCNQ2 (S541D). *<0.05, ***<0.001 calculated by nonparametric ANOVA followed by t-test. (F) Cell surface biotin labelling of wild-type KCNQ2, KCNQ (S541A), and KCNQ2 (S541D) (top panels), and of cytosolic GFP (lower panels). A representative blot from four independent experiments is shown. Error bars indicate s.e.m. Figure source data can be found with the Supplementary data.