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J. Biol. Chem., Vol. 280, Issue 50, 41146-41154, December 16, 2005

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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 Mäser1, Tatsunosuke Nakamura¶, Julian I. Schroeder, Hiroshi Kobayashi||, Nobuyuki Uozumi**, and Evert P. Bakker2

From the Abteilung Mikrobiologie, Universität Osnabrück, D-49069 Osnabrück, Germany, the Division of Biological Sciences, Cell and Developmental Biology Section, and the Center for Molecular Genetics, University of California at San Diego, La Jolla, California 92093-0116, the ^¶Faculty of Pharmacy, Niigata University of Pharmacy and Applied Life Sciences, Niigata 950-2081, Japan, the ^||Graduate School of Pharmaceutical Sciences, Chiba University, Inage-ku, Chiba 263, Japan, and the ^**Bioscience and Biotechnology Center, Nagoya University, Nagoya 464-8601, Japan

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.

Received for publication, July 14, 2005 , and in revised form, October 5, 2005.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants Ba 713/4-1 and SFB431 (Teilprojects K5 and P6) (to E. P. B.), by Grants-in-aid for Center of Excellence Research and for Scientific Research 13660088 and 14014219 from the Ministry of Education, Science, Sports, and Culture of Japan (to N. U.), and in part by Department of Energy Grant FG02-03ER15449 (to J. I. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4 and Table S1.

¹ Present address: Inst. of Cell Biology, University of Bern, CH-3012 Bern, Switzerland.

² To whom correspondence should be addressed: Abteilung Mikrobiologie, Barabarastrae 11, D-49076 Osnabrück, Germany. Tel.: 49-541-969-3515; Fax: 49-541-969-2870; E-mail: Bakker_e{at}biologie.uni-osnabrueck.de.

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