Gating and lipid modulation in ligand-gated ion channels - ABSTRACT Members of the voltage-gated superfamily of tetrameric cation channels have central roles in human physiology. My group's long-term objective is to understand the molecular mechanisms of gating, lipid modulation and cellular function of two families of channels within this group: the large conductance and calcium activated potassium (BK) channels and the cyclic nucleotide-modulated (CNM) channels, which have been the topics of 2 distinct RO1s from NIGMS (GM088352 – K channels, and GM124451 – CNM channels). BK channels have the ability to couple intracellular Ca2+ to membrane potential variations, play major physiological roles in vascular smooth muscle tone maintenance, regulation of circadian rhythms, hearing, neurotransmitter release. CNM channels are activated by cyclic nucleotides (CNG), and hyperpolarization (HCN), and are expressed in the heart and brain where they play key roles in pacemaking, vision, olfaction. They are excellent drug targets and understanding how they gate can be therapeutically useful. BK channels can associate with tissue-specific accessory subunits, endowing the channels with different functional properties. 2 and 3 subunits induce N-type (or ball-and-chain) inactivation of the otherwise non-inactivating BK channels. The structural correlates of this process were not known. We previously determined the structural correlates of ball-and-chain inactivation in MthK, a prokaryotic homolog of BK channels from Methanotropicum thermoautotrophicum, and found it has a strong lipid dependence. We propose to determine the structural correlates of ball-and-chain inactivation in BK channels and understand how lipids modulate inactivation in both BK and MthK channels. We previously used SthK, prokaryotic homolog of CNM channels from Spirochaeta thermophila, as model to investigate CNM channel gating. We determined the mechanisms of ligand selectivity and increase in activity with anionic lipids in SthK, and our preliminary data shows that lipids modulate the temperature dependence of SthK. We propose to elucidate the molecular mechanism of this process and determine whether it is conserved in eukaryotic thermo-sensitive channels. In addition, we propose to elucidate the structure of the olfactory CNG channel, proposed to be a heteromer of CNGA2, CNGA4, and CNGB subunits and determine the mechanism of lipid modulation and ligand selectivity. Since membrane lipid composition cannot be controlled in cells, we will use a bottom-up approach of purified channels in reconstituted systems, to rigorously control lipid content. We combine state-of-the-art techniques: single-particle cryo-EM, atomic force microscopy, computational approaches, and functional assays to reach our goals. The accomplishment of these projects will provide a comprehensive picture of ball- and-chain inactivation in calcium-gated potassium channels and its lipid dependence, as well as of ligand selectivity, lipid modulation and temperature dependence in select CNM channels.
