Thermal stability of the K+ channel tetramer:: Cation interactions and the conserved threonine residue at the innermost site (S4) of the KcsA selectivity filter

The selectivity filter of most K+ channels contains a highly conserved Thr residue that uniquely forms the S-4 binding site for K+ by dual coordination with the backbone carbonyl oxygen and side chain hydroxyl of the same residue. This study examines the effect of mutations of Thr75 in the S-4 site of the KcsA K+ channel on the cation dependence of the thermal stability of the tetramer, a phenomenon that reflects the structural role of cations in the filter. Conservative mutations of Thr-75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity of K+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by similar to 10(3)- or similar to 10(4)-fold for Ala and Cys substitution, respectively, consistent with the known preference of the S-4 site for Ba2+. In contrast, substitution of Ala or Cys at T75 anomalously enhances the ability of Na+ to stabilize the tetramer, suggesting that the native Thr residue at S-4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature or Cu2+ cation catalyzes formation of covalent dimers of the T75C mutant of KcsA via formation of disulfide bonds between Cys residues of adjacent subunits. Thiophilic cations such as Hg2+ and Ag+ specifically protect the T75C tetramer against heat-induced dimer formation, demonstrating the contribution of cation interactions to tetramer stability in a channel with a non-native S-4 site engineered to bind foreign cations.