title: 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 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 Vmax. 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.
authors.affiliation: Abteilung Mikrobiologie, Universität Osnabrück, D-49069 Osnabrück, Germany.
authors.forename: Nancy
authors.initials: N
authors.lastname: Tholema
authors.forename: Marc
authors.initials: M
authors.lastname: Vor der Brüggen
authors.forename: Pascal
authors.initials: P
authors.lastname: Mäser
authors.forename: Tatsunosuke
authors.initials: T
authors.lastname: Nakamura
authors.forename: Julian I
authors.initials: JI
authors.lastname: Schroeder
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kobayashi
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Uozumi
authors.forename: Evert P
authors.initials: EP
authors.lastname: Bakker
mesh_terms: D000409:Alanine / Q000737:chemistry; D000595:Amino Acid Sequence; D001224:Aspartic Acid / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry / Q000502:physiology*; D001665:Binding Sites; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D002412:Cations; D002462:Cell Membrane / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D014644:Genetic Variation; D005960:Glucosides / Q000737:chemistry; D005998:Glycine / Q000737:chemistry*; D007477:Ions; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011188:Potassium / Q000737:chemistry*; D015221:Potassium Channels / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D012694:Serine / Q000737:chemistry; D012964:Sodium / Q000737:chemistry; D013997:Time Factors; D044149:Vibrio alginolyticus / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D027682:Cation Transport Proteins; D002412:Cations; D005960:Glucosides; D007477:Ions; C113206:KtrB protein, Bacteria; D008565:Membrane Proteins; D015221:Potassium Channels; D001224:Aspartic Acid; D012694:Serine; C040358:dodecyl maltoside; D012964:Sodium; D000409:Alanine; D011188:Potassium; D005998:Glycine