BBMB Research Seminars

April 24, 2008
Eduardo Perozo
University of Chicago
1414 Molecular Biology Buidling
4:10 p.m.

Structural Mechanisms for Activation and Inactivation Gating in K+ Channels

It is well established that in KcsA, proton activation leads to a widening of the inner helical bundle (IHB) and channel opening. In addition, channel activation triggers C-type inactivation at the selectivity filter (SF), a feature common among all potassium selective channels. By establishing the molecular elements responsible for proton sensitivity in KcsA, we have engineered a constitutively open-channel in which the IHB is fully open at basic pH. Additionally, we have demonstrated that deletion of the KcsA C-terminal domain enhances C-type inactivation. These two findings have given us a unique opportunity to structurally evaluate the conformational changes underlying KcsA proton-dependent activation, C- type inactivation at the SF and the conformational coupling between these two processes. By using Fab-assisted crystallization methods we have solved 17 KcsA structures "trapped" in different stages along the transition pathway, from closed to the fully open  state (open-inactivated state), including at least four classes of gating intermediates. Analysis of these structures have revealed: 1) The KcsA IHB can be stabilized with several degrees of gate opening, ranging from about 11.5 Å (Ca-Ca distances in 1K4C) to 32 Å; 2) We find no ion occupancy at the channel aqueous cavity in the fully open conformation; 3) Major conformational changes at the SF include flipping of carbonyl groups at G77-V76 and a progressive loss of ions at potassium binding sites S2 and S3. Ion occupancy at these sites is strongly correlated with the degree of opening at the IHB; 4) A small but significant conformational change of the P-loop is seen when the IHB is fully open. These structures reveal, at unprecedented detail, the molecular basis of K+ channel C-type inactivation and its coupling to activation gating.