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J. Biol. Chem., Vol. 280, Issue 27, 25512-25516, July 8, 2005

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Proximity of Transmembrane Domains 1 and 3 of the -Aminobutyric Acid Transporter GAT-1 Inferred from Paired Cysteine Mutagenesis^*

Elia Zomot, Yonggang Zhou, and Baruch I. Kanner

From the Department of Biochemistry, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel

GAT-1 is a sodium- and chloride-dependent -aminobutyric acid transporter and is the first identified member of a family of transporters that maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. Because transmembrane domains 1 and 3 contain amino acid residues important for transport activity, we hypothesized that these domains may participate in the formation of the binding pocket of the transporter. Pairwise substitutions have been introduced in several predicted transmembrane domains and in the first extracellular loop of GAT-1. In the double mutant W68C/I143C, in which the cysteines were introduced at locations at the extracellular part of transmembrane domains 1 and 3, respectively, 70% inhibition of transport was observed by cadmium with an IC₅₀ of 10 µM. This inhibition was not observed in the corresponding single mutants and also not in >10 other double mutants, except for V67C/I143C, where the half-maximal effect was obtained at 50 µM. The inhibition by cadmium was only observed when the cysteine pairs were introduced in the same polypeptide. Our results suggest that transmembrane domains 1 and 3 come in close proximity within the transporter monomer.

Received for publication, April 11, 2005 , and in revised form, May 18, 2005.

^* This work was supported by The Israel Science Foundation Grant 488/03-16.1 and the Bernard Katz Minerva Center for Cellular Biophysics. 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: Dept. of Biochemistry, Hebrew University Hadassah Medical School, Jerusalem 91120, Israel. Tel.: 972-2-6758506; Fax: 972-2-6757379; E-mail: kannerb{at}cc.huji.ac.il.

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