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Transmembrane Domains I and II of the -Aminobutyric Acid Transporter GAT-4 Contain Molecular Determinants of Substrate Specificity

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

The sodium- and chloride-dependent GABA transporters GABA transporter (GAT) 1 to 4 in the central nervous system enable efficient synaptic transmission by removing the neurotransmitter from the cleft. Taurine interacts only weakly with the GABA transporter GAT-4 (IC₅₀ 1.6 mM). Glutamate-61 is located in the conserved transmembrane domain I of GAT-4, whereas in the related taurine-transporter taurine transporter (TAUT), glycine occupies the equivalent position. [³H]GABA uptake by the GAT-4 E61G mutant becomes markedly more sensitive to inhibition by taurine (IC₅₀ 0.26 mM). Replacement of cysteine-94, located in the conserved transmembrane domain II of GAT-4, to its TAUT counterpart serine, results only in a modest increase in the ability of taurine to inhibit GABA uptake. However, introduction of glycine at this position decreases the IC₅₀ for taurine by approximately 8-fold (IC₅₀ 0.20 mM). The inhibitory potency of taurine is inversely correlated with the volume of the side chain of the amino acid residue introduced at positions 61 and 94. It is striking that the IC₅₀ for taurine of the E61G/C94G double mutant is decreased by approximately 35-fold (IC₅₀ 0.05 mM), and this inhibition of GABA transport is competitive. Changes in the inhibitory potency of the mutants described are also observed with -ala-nine and GABA, although they are much less pronounced. Our results suggest that determinants on transmembrane domains I and II can influence the specificity of the substrate binding pocket. The size of the side chain at positions 61 and 94 seems to determine the ability of substrate and substrate analogs to interact with the transporter.

Received October 6, 2003; accepted March 1, 2004.

Address correspondence to: Baruch I. Kanner, Department of Biochemistry, Hebrew University, Hadassah Medical School, P.O. Box 12272, Jerusalem 91120, Israel. E-mail: kannerb{at}cc.huji.ac.il

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