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 S4 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 S4 site of theKcsA 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 Thr75 destabilize the tetramer and alter its temperature dependence. Replacement of Thr with Ala or Cys lowers the apparent affinity ofK+, Rb+, and Cs+ for tetramer stabilization by factors ranging from 4- to 14-fold. These same mutations lower the apparent affinity of Ba2+ by approximately 10(3)- or approximately 10(4)-fold for Ala and Cys substitution, respectively,consistent with the known preference of the S4 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 S4 is important for ultrahigh K+/Na+ selectivity of K+ channel pores. Elevated temperature orCu2+ 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 S4 site engineered to bind foreign cations.