Halting KcsA'S C-Type Inactivation Gating by Controlling Water Diffusion Behind the Channel'S Selectivity Filter

Abstract

Alterations of the C-type inactivation process of several K+ channels are the underlying cause of 1) life-threatening arrhythmias, 2) episodic ataxia, 3) epilepsy, 4) chronic pain and 5) some forms of cancer. Since alterations of the C-type inactivation process of K+ channels represent a serious menace for the public health in the US, it is imperative to understand this molecular process at the molecular level. Toward this end, we have solved at atomic resolution the structure of a locked-open state of KcsA, which allowed us to generate the first atomic resolution description of a K+ channel kinetic cycle. This technological advancement allowed us to identify the novel role of structural water molecules on the onset of the C-type inactivation process. A network of water molecules behind a K+ channel selectivity filter is hydrogen bonded to its C-type inactivated conformation. The KcsA-Y82 position is located at the outer mouth of the channel and it seems to work as a “gatekeeper” regulating the diffusion of water molecules behind the channel's filter that consequently modulates C-type inactivation. Here, we report the finding of a channel mutant (Y82T) that effectively blocks the entry of water molecules into the inactivation cavity, behind the channel's selectivity filter (SF), hence precluding its collapse, as evidence by macromolecular crystallography. The mutant channel displayed robust non-inactivating macroscopic currents, which seems to indicate that the C-type inactivation process has been halted. A complete functional and thermodynamic analysis of this gating intermediate, which is represented by the closed and open state of the Y82T mutant will be presented. Support: NIH 2R01GM097159-06, Welch Foundation BI-1949.