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J. Biol. Chem., Vol. 279, Issue 11, 9944-9950, March 12, 2004

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Mechanism of Proton Gating of a Urea Channel^*

David L. Weeks, Gene Gushansky, David R. Scott, and George Sachs

From the Department of Veterans Affairs Greater Los Angeles Healthcare System, Los Angeles, California, 90073 and University of California, Los Angeles, California 90024

The size and complexity of many pH-gated channels have frustrated the development of specific structural models. The small acid-activated six-membrane segment urea channel of Helicobacter hepaticus (HhUreI), homologous to the essential UreI of the gastric pathogen Helicobacter pylori, enables identification of all the periplasmic sites of proton gating by site-directed mutagenesis. Exposure to external acidity enhances [¹⁴C]urea uptake by Xenopus oocytes expressing HhUreI, with half-maximal activity (pH_0.5) at pH 6.8. A downward shift of pH_0.5 in single site mutants identified four of six protonatable periplasmic residues (His-50 at the boundary of the second transmembrane segment TM2, Glu-56 in the first periplasmic loop, Asp-59 at the boundary of TM3, and His-170 at the boundary of TM6) that affect proton gating. Asp-59 was the only site at which a protonatable residue appeared to be essential for pH gating. Mutation of Glu-110 or Glu-114 in PL2 did not affect the pH_0.5 of gating. A chimera, where the entire periplasmic domain of HhUreI was fused to the membrane domain of Streptococcus salivarius UreI (SsUreI), retained the pH-independent properties of SsUreI. Hence, proton gating of HhUreI likely depends upon the formation of hydrogen bonds by periplasmic residues that in turn produce conformational changes of the transmembrane domain. Further studies on HhUreI may facilitate understanding of other physiologically important pH-responsive channels.

Received for publication, November 19, 2003 , and in revised form, December 22, 2003.

^* This work was supported by a Department of Veterans Affairs Greater Los Angeles Healthcare System Merit Review Award and by National Institutes of Health Grant DK58333. 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.

To whom correspondence should be addressed: Bldg. 113, Rm. 324, Wadsworth Veterans Affairs Hospital, Los Angeles, CA 90073. Fax: 310-312-9478; E-mail: gsachs{at}ucla.edu.

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