Closed State Structure of the Pore Revealed by Uncoupled Shaker K+ Channel

Voltage gated potassium (Kv) channels play key roles in numerous physiological processes from cellular excitability to immune response and are among the most important pharmaceutical targets. Despite recent advances in the structural determination of Kv channels, the closed state structure of strictly coupled Kv1 family remains elusive. Here, we captured the closed state structure of the pore in the Shaker potassium by uncoupling the voltage sensor domains from the pore domains. Ionic current, gating current and fluorescence measurements show that a conserved isoleucine residue in the S4-S5 linker region, plays a key role controlling the strength of the electromechanical coupling and the channel activation-deactivation equilibrium. Structural determination of completely uncoupled I384R mutant by single particle cryoEM revealed a fully closed pore in the presence of fully activated but non-relaxed voltage sensors. The putative conformational transitions from a fully open pore domain indicates a "roll and turn" movement along the whole length of the pore-forming S6 helices in sharp contrast to canonical gating models based on limited movements of S6. The rotational and translational movement posits two hydrophobic residues, one at inner cavity and the other at the bundle crossing region, directly at the permeation pathway, limiting the pore radius to less than 0.7[A]. Voltage clamp fluorimetry of wild type channel incorporating a fluorescent unnatural amino acid strongly supports the cryoEM structural model. Surprisingly, the selectivity filter was captured in a noncanonical state, unlike the previously described dilated or pinched filter conformations. With the present experiment results, we propose a new gating model for strictly coupled Kv1 channels and the molecular mechanism of interactions among different functional states.