All four putative selectivity filter glycine residues in KtrB are essential for high affinity and selective K+ uptake by the KtrAB system from Vibrio alginolyticus

Abstract

The subunit KtrB of bacterial Na+-dependent K +-translocating KtrAB systems belongs to a superfamily of K + transporters. These proteins contain four repeated domains, each composed of two transmembrane helices connected by a putative pore loop (p-loop). The four p-loops harbor a conserved glycine residue at a position equivalent to a glycine selectivity filter residue in K+ channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly70, Gly185, Gly290, and Gly402 from p-loops PA to PD of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrBA70, KtrBA185, and KtrB A290 maintained a substantial activity in KtrAB-mediated K + uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K+ by 2 orders of magnitude, with little effect on Vmax. Minor activities were also observed for three other variants: KtrBA402, KtrBS70, and KtrBD185. With all of these variants, the property of Na+ dependence of K + transport was preserved. Only the four serine variants mediated Na+ uptake, and these variants differed considerably in their K +/Na+ selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na+-independent, slow, high affinity, and mutation-specific K+ uptake as well as K+-independent Na+ uptake. These data demonstrate that KtrB contains a selectivity filter for K+ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.