Clustering of Individual and Diverse Ion Channels Together Into Complexes, and Their Functional Coupling, Mediated by A-Kinase A

Multi-protein complexes have emerged as a mechanism for spatiotemporal specificity and efficiency in the function and regulation of myriad cellular signals. In particular, many ion channels are clustered either with the receptors that modulate them, or with other ion channels whose activities are linked. Often, the clustering is mediated by scaffolding proteins, such as the AKAP79/150 protein that is a focus of this grant. We focus on three different channels critical to nervous function. One is the 'M-type' (KCNQ, Kv7) K+ channel that plays fundamental roles in the regulation of excitability in nerve and muscle. It is thought to associate with Gq/11- coupled receptors, protein kinases, calcineurin (CaN), calmodulin (CaM) and phosphoinositides via AKAP79/150. Another channel of focus is TRPV1, a nociceptive channel in sensory neurons that is also thought to be regulated by signaling proteins recruited by AKAP79/150. The third are L-type Ca2+ (CaV1.2) channels that are critical to synaptic plasticity, gene regulation and neuronal firing. We will probe complexes containing AKAP79/150 and these three channels using 'super-resolution' STORM imaging of primary sensory neurons and heterologously-expressed tissue-culture cells, in which individual complexes can be visualized at 10-20 nm resolution with visible light, breaking the diffraction barrier of physics. We hypothesize that AKAP79/150 brings several of these channels together to enable functional coupling, which we will examine by patch-clamp electrophysiology of the neurons. Fˆrster resonance energy transfer (FRET) will also be performed under total internal reflection fluorescence (TIRF) or confocal microscopy, further testing for complexes containing KCNQ, TRPV1 and CaV1.2 channels. Since all three of these channels bind to AKAP79/150, we hypothesize that they co-assemble into complexes in neurons, together with certain G protein-coupled receptors. Furthermore, we hypothesize these complexes to not be static, but rather to be dynamically regulated by other cellular signals, which we will examine using rapid activation of kinases or phosphatases. Several types of mouse colonies of genetically altered AKAP150 knock-out or knock-in mice will be utilized. This project breaks new ground into the physiology of signaling in neurons, using several cutting-edge, high- powered approaches that have just recently been developed. PUBLIC HEALTH RELEVANCE: Several proteins called ion channels are critical to the function of nerve cells, and have activities that are regulated by a number of cellular receptors and messenger molecules. Specificity in signaling is often achieved by scaffold molecules called A-kinase anchoring protein (AKAP)79/150. We hypothesize AKAP79/150 to be clustered together with ion channels in nerve cells, either to direct their regulation by intracellular messengers, or to bring multiple ion channels together for their function to be coupled. We will investigate this using "super-resolution" microscopy that can peer into cells at nanometer resolution, and see individual molecules, as well as electrical recording from individual living cells.