High-Resolution Crystallographic Analysis of the Kcsa Gating Cycle from Cysteine-Trapped Open Channels

Abstract

The K+ channel pore domain contains all the elements necessary to catalyze the selective permeation of K+ ions, in addition to regulate events underlying activation and inactivation gating. In KcsA, an inactivation process related to C-type inactivation in eukaryotic channels has been attributed to putative conformational changes at the selectivity filter (SF)[1]. Previously, we have provided crystallographic evidence for the conformational changes associated to C-type inactivation, albeit at relatively low-resolution [2]. Here, we have taken advantage of a cysteine-bridged locked open KcsA-mutant to study the structural changes at the selectivity filter when the activation gate (AG) is open and the filter transitions between its conductive and non-conductive conformations. We report the structures of KcsA for the non-inactivating mutantE71A at 2.1 A; the fully inactivated mutant Y82A at 2.32 A; and the non-inactivating mutant F103A at 2.64 A, where the allosteric coupling between the two gates (AG and SF) has been impaired. This set of high-resolution structures for different KcsA kinetic states represent a sharp improvement over the resolution of non-cysteine trapped mutants and will be interpreted in relation to their complementary functional characterization.1. Cordero-Morales, J.F., et al., Molecular determinants of gating at the potassium-channel selectivity filter. Nat Struct Mol Biol, 2006. 13(4): p. 311-8.2. Cuello, L.G., et al. (2008). Structural basis of K+ channel C-type inactivation: Crystal structure of KcsA in the Open/C-type inactivated conformation. 52nd Annual meeting of the Biophysical Society. Mini-symposium