Dual Regulation of M-Type K+ Channels by AKAP79/150 Signaling Complexes

Abstract

M-type K+ channels, comprised of KCNQ2-5 (Kv7.2-7.5) subunits, play key roles in the regulation of neuronal excitability. We study the regulation of M-channel activity and transcriptional expression by A-kinase-anchoring protein (AKAP)79/150. Our FRET and functional studies suggest AKAP79/150 action correlates with the PIP2-depletion mode of neuronal IM suppression, and is disrupted by functional calmodulin. The modulation of M current involves recruitment of PKC to the channels, their subsequent phosphorylation, and likely decreased affinity for PIP2. The complex orchestrated by AKAP79/150 also includes M1 muscarinic receptors, thus constituting a “signalosome” spanning the initiating receptor to channel target. We have discovered a novel mechanism regulating KCNQ2/3 transcriptional expression by neuronal activity, involving calcineurin and Nuclear Factor of Activated T-cells (NFAT) transcription factors, orchestrated by AKAP79/150. The signal requires Ca2+ influx through L-type Ca2+ channels and both local and global Ca2+ elevations. AKAP79/150 and the complex it organizes thus mediate activity-dependent M-channel transcription, which may potentially serve throughout the nervous system to limit over-excitability associated with disease states such as epilepsy. We are now utilizing stochastic optical reconstruction microscopy (STORM) offering sub-diffraction (∼20 nm) resolution, to directly visualize these AKAP79/150 signaling complexes and interactions between AKAP79/150, ion channels and receptors in neurons. STORM uses dyes that can cycle between a dark and a fluorescent state thousands of times, thus enabling detection of the precise localization of the center of these scattered spots given by cumulative integration of each cycle. Using multi-color STORM to simultaneously image AKAP150, KCNQ2-3 or KCNQ5 channels, and receptors, we observe AKAP150 to form signaling clusters with the channels and receptors at the single-complex level. We have also obtained evidence that AKAP79/150 links different channel types together, raising the possibility of their functional, as well as physical, coupling.