Molecular mechanisms of gating and selectivity in the potassium channel KCSA.

摘要

To traverse cell membranes, ions must move from a polarizable milieu and pass through the non-polarizable membrane core, a feat that, if not catalyzed would require significant energy (-20 kcal mol-1). Protein channels in the membrane allow ions to surmount this energy barrier by providing an aqueous path. Full understanding of the physiological function of these channels requires understanding of their basic biophysical properties. This dissertation seeks to elucidate mechanisms of opening and closing (i.e., "gating") and selective ion permeation in K+ channels.;The bacterial potassium channel KcsA is gated by intracellular protons. Despite prior attempts to determine the mechanism responsible for pH-gating, the proton sensor has remained elusive. We have constructed a pH insensitive KcsA channel mutant by replacing key ionizable residues from the N- and C-termini with residues mimicking their protonated counterparts with respect to charge. We propose that these residues play crucial roles in proton gating. At neutral pH they form a complex network of inter- and intra-subunit salt bridges and hydrogen bonds near the bundle crossing, stabilizing the closed state. In our model, these residues change their ionization state at acidic pH, disrupting this network, modifying the electrostatic landscape near the channel gate, and favoring channel opening.;Potassium channels allow K+ ions to diffuse through their pores while preventing Na+ ions from permeating. This selection process 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 K+ sites in a thermodynamically favorable way. We examined how intracellular Na + and Li+ interact with the pore and the permeant ions using electrophysiology, molecular dynamics simulations, and X-ray crystallography. Our results suggest that these small cations have at least one binding site within the K+ selectivity filter, albeit a different one than the K+ sites. We propose that selective permeation from the intracellular side is achieved mainly by a large energy barrier blocking filter entry for Na+ and Li+ in the presence of K +.