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

Abstract

M-type (KCNQ2/3) potassium channels are suppressed by activation of G q/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.The M-current is a low-threshold, slowly activating potassium current that exerts negative control over neuronal excitability. Activation of G q/11 -coupled receptors suppresses the Mcurrent, creating a slow excitatory postsynaptic potential, enhancing excitability and reducing spike-frequency adaptation 1,2 . The M-type K + channel is a promising therapeutic target, as the channel blocker linopirdine acts as a cognition enhancer 3,4 , and the channel activator retigabine functions as an anticonvulsant 5,6 . M-type channels are heteromeric complexes of certain KCNQ-family potassium channel subunits (KCNQ2-5) [7][8][9][10] . KCNQ2 and KCNQ3 were the first members of this family identified as M-channel forming subunits 7 . The KCNQ3 subunit is a core component that co-assembles with KCNQ2, KCNQ4 and KCNQ5 to form functional M-type channels 10 . © 2003 Nature Publishing GroupCorrespondence should be addressed to N.H. (hoshin@ohsu.edu). Note: Supplementary information is available on the Nature Neuroscience website. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. NIH Public Access Author ManuscriptNat Neurosci. Author manuscript; available in PMC 2014 March 04. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptAlthough the subunits that form M-type K + channels have been identified, the molecular details of the signaling pathways that lead to suppression of the M-currents upon receptor stimulation have not yet been fully defined 2,11 . We know that inhibition results from activation of G proteins of the G q/11 family, with the α-subunit as the active moiety 12,13 , and that a 'diffusible' messenger is involved. That is, the receptor/G protein complex can be physically remote from the channel 14,15 . Thus, closure most likely results from some product of phospholipase C activity. M-type channels can be closed by raising intracellular calcium 16 , and there is evidence that this might be a 'second messenger' for bradykinin 17 and nucleotides 18 , but not...

Distinct enzyme combinations in AKAP signalling complexes permit functional diversity

“…Although a role for PKC in this process has been elusive 22 , recent pharmacological studies imply that an anchored pool of the kinase facilitates this suppression of M current in superior cervical ganglion (SCG) neurons 10 . PKC phosphorylation of Ser 534 and Ser 541 on the KCNQ2 channel subunit has been implicated in the suppression of recombinant M currents 10 . The net result is a hyperexcitability of neurons because firing of action potentials is favoured when M currents are suppressed 18,19 .…”

“…6f). AKAP150 binds to the KCNQ2 subunit, a core component of the M current, presumably to direct PKC towards phosphorylation sites in the cytoplasmic tail of the channel 10 (Fig. 6a).…”

“…SCG neurons were isolated from 2-3-week-old rats and cultured as described previously 10 . Hippocampal neurons were isolated from 1-2-d-old rats and cultured as described previously 29 .…”

“…For experiments in AKAP150-silenced hippocampal neurons, suspensions of hippocampal cells from newborn rats were transfected with pSAKAP150i, various AKAP79 forms in pcDNA3, and either pEGFP-N1 or pDsRED2 using a Nucleofector kit (Amaxa, Koeln, Germany). Treated cells were overlaid onto a glial feeder layer and cultured for 4-7 d. For AKAP150-silenced SCG neurons, the same constructs were introduced by nuclear injection as described previously 10 and cultured for 3 d.…”

“…They are tethered to the inner face of the plasma membrane, where they can respond to the generation of second messengers, such as cAMP, calcium and phospholipid 7 . Functional studies have shown that this AKAP family controls the phosphorylation status and action of several ion channels, including AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)-type glutamate receptors, L-type calcium channels, aquaporin water channel, and M-type potassium channels [8][9][10] . One theory that accounts for this diversity of action is that unique combinations of anchored enzymes are recruited to individual ion channels.…”