Effect of Altered Ion Occupancy on Permeation and Inactivation Gating in K+ Channel

Abstract

The selectivity filter of a K+ channel is comprised of four equally-spaced K+-binding sites. In the first three sites the main-chain carbonyl atoms provide the octa-coordination for K+ ions. In the fourth site both main-chain carbonyl oxygen atoms and threonine side-chain hydroxyl oxygen atoms contribute to K+ coordination. This threonine is highly conserved among K+ channels with the only alternative being a serine substitution. Surprisingly, this conservative mutation, which preserves the hydroxyl moiety and the side chain polarity, has been shown to produce profound changes in permeation and gating in Shaker. Indeed, mutant T442S shows a dramatic destabilization of inactivation along with an increased conductance for Rb+ over K+. Although these studies provided early insights into the role of this position in ion coordination, the structural basis for altered single-channel conductances and gating kinetics remain unclear. KcsA has served as an archetypical K+ pore providing molecular insights into understanding selectivity, ion-permeation, gating pore-blocking and C-type inactivation. Equivalent mutation in KcsA (T75S) shows remarkably similar effects as seen in Shaker, including effects on inactivation, single-channel conductance, selectivity and rectification. Combining single-channel and macroscopic current measurements along with X-ray crystallography under different ionic conditions we now provide atomistic details that underlie these divergent channel properties and the role of Thr75 in channel function.