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

Nancy Tholema, Marc Vor Der Brüggen, Pascal Mäser, Tatsunosuke Nakamura, Julian I. Schroeder, Hiroshi Kobayashi, Nobuyuki Uozumi, Evert P. Bakker

Research output: Contribution to journalArticle

58 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)41146-41154
Number of pages9
JournalJournal of Biological Chemistry
Volume280
Issue number50
DOIs
Publication statusPublished - 2005 Dec 16
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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