STRUCTURAL STUDIES OF KCSA MUTANTS AND CHIMERAS

点击翻译按钮获取中文摘要

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Project 1 -- The bacterial potassium channel KcsA is gated by high concentrations of intracellular protons, allowing the channel to open at pH < 5.5. Replacing key ionizable residues from the N and C termini of KcsA with residues mimicking their protonated counterparts with respect to charge renders the channel open up to pH 9.0 (Thompson et al. 2008). We proposed that these residues function as the proton-binding sites. At neutral pH they form a complex network of inter- and intrasubunit salt bridges and hydrogen bonds near the bundle crossing, stabilizing the closed state. At acidic pH, these residues change their ionization state, thereby disrupting this network, favoring channel opening. Individual mutations of residues in this region result in modest shifts in the pH dependence of channel opening. Pair-wise mutation of certain amino acids in this region causes a marked shift in the pH dependence of channel opening suggesting significant energetic coupling between these residues. We hypothesize that the crystal structures of these mutants will shed light on the structural changes that occur at the inner helical bundle crossing upon channel gating. Project 2 -- Potassium channels allow K(+) ions to diffuse through their pores while preventing smaller Na(+) ions from permeating. Discrimination between these similar, abundant ions enables these proteins to control electrical and chemical activity in all organisms. Selection occurs at the narrow selectivity filter containing structurally identified K(+) binding sites. Selectivity is thought to arise because smaller ions such as Na(+) do not bind to these K(+) sites in a thermodynamically favorable way. We wish to examine how intracellular Na(+) and Li(+) interact with the KcsA pore and the permeant ions using X-ray crystallography.