All Four Putative Selectivity Filter Glycine Residues in KtrB Are Essential for High Affinity and Selective K super(+) Uptake by the KtrAB System from Vibrio alginolyticus

The subunit KtrB of bacterial Na super(+)-dependent K super(+)-translocating KtrAB systems belongs to a superfamily of K super(+) 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 super(+) channels. We investigated whether these glycines also form a selectivity filter in KtrB. The single residues Gly super(70), Gly super(185), Gly super(290), and Gly super(402) from p-loops P sub(A) to P sub(D) of Vibrio alginolyticus KtrB were replaced with alanine, serine, or aspartate. The three alanine variants KtrB sub(A70), KtrB sub(A185), and KtrB sub(A290) maintained a substantial activity in KtrAB-mediated K super(+) uptake in Escherichia coli. This activity was associated with a decrease in the affinity for K super(+) by 2 orders of magnitude, with little effect on V sub(max). Minor activities were also observed for three other variants: KtrB sub(A402), KtrB sub(S70), and KtrB sub(D185). With all of these variants, the property of Na super(+) dependence of K super(+) transport was preserved. Only the four serine variants mediated Na super(+) uptake, and these variants differed considerably in their K super(+)/Na super(+) selectivity. Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na super(+)-independent, slow, high affinity, and mutation-specific K super(+) uptake as well as K super(+)-independent Na super(+) uptake. These data demonstrate that KtrB contains a selectivity filter for K super(+) 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.