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
Autor(en): | Tholema, N Vor der Bruggen, M Maser, P Nakamura, T Schroeder, JI Kobayashi, H Uozumi, N Bakker, EP |
Stichwörter: | AMINO-ACID SUBSTITUTIONS; Biochemistry & Molecular Biology; CARRIER TRK2P; CHANNEL; HKT TRANSPORTERS; ION CONDUCTION; KDPA SUBUNIT; KDPFABC COMPLEX; MUTATIONS; NA+; POTASSIUM UPTAKE | Erscheinungsdatum: | 2005 | Herausgeber: | AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC | Journal: | JOURNAL OF BIOLOGICAL CHEMISTRY | Volumen: | 280 | Ausgabe: | 50 | Startseite: | 41146 | Seitenende: | 41154 | Zusammenfassung: | 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 Gly(70), Gly(185), Gly(290), and Gly(402) 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. |
ISSN: | 00219258 | DOI: | 10.1074/jbc.M507647200 |
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geprüft am 02.06.2024