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AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists

Abstract

M-type (KCNQ2/3) potassium channels are suppressed by activation of Gq/11-coupled receptors, thereby increasing neuronal excitability. We show here that rat KCNQ2 can bind directly to the multivalent A-kinase-anchoring protein AKAP150. Peptides that block AKAP150 binding to the KCNQ2 channel complex antagonize the muscarinic inhibition of the currents. A mutant form of AKAP150, AKAP(ΔA), which is unable to bind protein kinase C (PKC), also attenuates the agonist-induced current suppression. Analysis of recombinant KCNQ2 channels suggests that targeting of PKC through association with AKAP150 is important for the inhibition. Phosphorylation of KCNQ2 channels was increased by muscarinic stimulation; this was prevented either by coexpression with AKAP(ΔA) or pretreatment with PKC inhibitors that compete with diacylglycerol. These inhibitors also reduced muscarinic inhibition of M-current. Our data indicate that AKAP150-bound PKC participates in receptor-induced inhibition of the M-current.

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Figure 1: KCNQ2 can form an AKAP150 signaling complex.
Figure 2: Mapping the reciprocal binding domains of KCNQ2 and AKAP150.
Figure 3: Role of AKAP150 in receptor-mediated KCNQ2 channel regulation.
Figure 4: Loss of PKC from channel complex attenuates the agonist-induced inhibition of the M-currents in SCG neurons.
Figure 5: Effect of PKC inhibitors on the M-current inhibition.
Figure 6: PKC phosphorylation of KCNQ2 in Oxo-M treated cells.
Figure 7: Effects of mutations of potential PKC-dependent phosphorylation sites of KCNQ2 channels on receptor-induced suppression in CHO hm1 cells.

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Acknowledgements

The authors thank M. Okamura, T. Haga and T. I. Bonner for the gift of CHO hm1 cells, and T. Rafiq for help with ganglion cell cultures. This work was supported by grants to N.H. and H.H. from the Japanese Ministry of Education, Culture, Sports, Science and Technology, by grant PG7909913 to D.A.B. from the UK Medical Research Council and by National Institute of Health grant GM48231 for the support of J.D.S.

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Correspondence to Naoto Hoshi.

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Supplementary information

Supplementary Fig. 1.

Mapping the KCNQ2 binding domain within residue 1-143 of AKAP150. (a) AKAP150 interacts with KCNQ2 through multi-site contacts. Schematic diagram showing the AKAP150(1-143) deletion constructs. (b) Interaction with AKAP150 fragments were detected using the KCNQ2(321-499) overlay assay as in Fig. 2e (upper panel). Commassie stain indicates approximately equal protein loading (lower panel). (c) The amalgamation of four independent experiments is shown. (JPG 33 kb)

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Hoshi, N., Zhang, JS., Omaki, M. et al. AKAP150 signaling complex promotes suppression of the M-current by muscarinic agonists. Nat Neurosci 6, 564–571 (2003). https://doi.org/10.1038/nn1062

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