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

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 P_A to P_D of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB_A70, KtrB_A185, 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 V_max. Minor activities were also observed for three other variants: KtrB_A402, KtrB_S70, and KtrB_D185. 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.