ABSTRACT Potassium (K+) channels are major determinants of cell excitability and play crucial roles in physiological processes. Large conductance and Ca2+-activated K+ (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. BK channels can associate with tissue-specific accessory subunits, endowing the channels with different functional properties. b2 and b3 subunits induce N-type (or ball-and-chain) inactivation of the otherwise non-inactivating BK channels. BK channel dysfunction has been associated with many pathophysiological conditions, so understanding how they gate can have major therapeutic consequences. In the previous grant cycle, we determined the structural correlates of gating and ball-and-chain inactivation in K+ channels, using a prokaryotic homolog of BK channels, MthK, from Methanotropicum thermoautotrophicum. We also found that lipid bilayer thickness strongly affects MthK inactivation. Unlike BK, MthK channels inactivate using an intrinsic N-terminal “ball” domain, similar to Shaker K+ channels. The overall objective of this grant is to determine the structural correlates of ball-and-chain inactivation in BK channels and understand how lipids modulate inactivation in both MthK and BK. 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 composition. We will use structural, functional, and computational analysis (the last with a collaborator, see letter) on both MthK and BK channels to reach these goals. Our first aim is to determine the mechanism by which lipids modulate ball-and- chain inactivation in MthK. Our hypothesis, predicted by MD simulations, is that thicker bilayers bind the amphipathic ball domain better than thinner bilayers, which will be tested with mutagenesis of the ball domain, constructs with different chain lengths, and membrane manipulations. This aim was proposed in the previous grant cycle, we made considerable progress but the mechanism was more complex than anticipated. Hence, this aim still needs investigation. During the last cycle, we also treated another unexpected finding from the structure of closed MthK, still in the scope of the grant but not explicitly proposed, which was just published. Our second aim is to determine the structural correlates of ball-and-chain inactivation in BK channels as well as how bilayer-thickness and other bilayer properties modulate these channels. We will use b2 and a chimeric b4-b2 subunit for both structural studies and electrophysiology in HEK cells and liposomes. We will investigate lipid-dependence of BK channel gating with and without b subunits with stopped-flow assays and single- channel recordings of purified BK channels reconstituted in liposomes. Channel structur...