(provided by candidate): Ion channel gating, the ability of channels to switch between an open conducting state and a closed non- conducting state, is the most fundamental mechanistic feature of channels throughout physiology. The overall aim of this proposal is to investigate the gating mechanisms of the MthK calcium-activated K+ ion channel. MthK is a prokaryotic K+ channel from Methanobacterium thermoautotrophicum that is representative of a family of channels activated by calcium. Eucharyotic channels of this type are known to be important for the firing behavior of neurons and the response of sensory organs such as in the inner ear. They are promising pharmaceutical targets and therefore are important topics of biophysical investigation. The principle techniques we propose using are electrophysiological recording of channels in artificial lipid bilayers and crystallography. We will test the widely-held assumption that the MthK pore-domain closes by a movement of inner-pore helices (TM2 helices) into a KcsA-like conformation, thereby preventing the entrance of K+ at the intracellular side of the pore. In the longer term, we will explore whether the K+ selective region, called the selectivity filter, may also serve as a permeation gate, alternating between open and closed configurations. Electrophysiological evidence will come from a study of the state-dependence and kinetics of block by intracellular blockers. Results from the KcsA channel will be compared with MthK in order to discern similar or unique gating mechanisms between these channels. Single channel recording in artificial bilayers will allow direct determination of the channel open probability, percent block, and gating kinetics in the presence of compounds such as tetrabutylammonium. Structural evidence for closed MthK conformations will come from x-ray crystallography. We have been optimizing crystal conditions for MthK in the absence of calcium which may reveal a closed channel conformation. We have already begun solving the structure of a mutant MthK (in the presence of calcium) that does not exhibit channel openings in the lipid bilayer and therefore may be a constitutively closed channel. These structural results will likely strengthen conclusions from the functional studies mentioned and establish the mechanism of MthK gating experimentally. Finally, in the future we will use mutational and structural studies to examine the possibility of selectivity filter gating within the MthK K+ channel.
|Posson, David J; Rusinova, Radda; Andersen, Olaf S et al. (2018) Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies. Methods Mol Biol 1684:223-235|
|Posson, David J; Rusinova, Radda; Andersen, Olaf S et al. (2015) Calcium ions open a selectivity filter gate during activation of the MthK potassium channel. Nat Commun 6:8342|
|Posson, David J; McCoy, Jason G; Nimigean, Crina M (2013) The voltage-dependent gate in MthK potassium channels is located at the selectivity filter. Nat Struct Mol Biol 20:159-66|
|Posson, David J; Thompson, Ameer N; McCoy, Jason G et al. (2013) Molecular interactions involved in proton-dependent gating in KcsA potassium channels. J Gen Physiol 142:613-24|