Tamura S, Nelson H, Tamura A, Nelson N
Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110, USA.
J Biol Chem. 1995 Dec 1;270(48):28712-5. doi: 10.1074/jbc.270.48.28712.
While the gamma-aminobutyric acid (GABA) transporter GAT1 exclusively transports GABA, GAT2, -3, and -4 also transport beta-alanine. Cross-mutations in the external loops IV, V, and VI among the various GABA transporters were performed by site-directed mutagenesis. The affinity of GABA transport as well as inhibitor sensitivity of the modified transporters was analyzed. Kinetic analysis revealed that a cross-mutation in which loop IV of GAT1 was modified to resemble GAT4 resulted in increased affinity to GABA from Km = 8.7 to 2.0 microM without changing the Vmax. A cross-mutation in loop VI, which swapped the amino acid sequence of GAT2 for GAT1, decreased the affinity to GABA (Km, 35 microM). These results suggest that loops IV and VI contribute to the binding affinity of GABA transporters. A substitution of three amino acids in loop V of GAT1 by the corresponding sequence of GAT3 resulted in beta-alanine sensitivity of its GABA uptake activity. These three amino acids in loop V seem to participate in the beta-alanine binding domain of GAT3. It is suggested that those three external loops (IV, V, and VI) form a pocket in which the substrate binds to the GABA transporters.
虽然γ-氨基丁酸（GABA）转运体GAT1仅转运GABA，但GAT2、-3和-4也转运β-丙氨酸。通过定点诱变对各种GABA转运体的外环IV、V和VI进行交叉突变。分析了修饰后转运体对GABA的转运亲和力以及抑制剂敏感性。动力学分析表明，将GAT1的环IV修饰成与GAT4相似的交叉突变导致对GABA的亲和力从Km = 8.7 μM增加到2.0 μM，而Vmax不变。环VI的交叉突变将GAT2的氨基酸序列与GAT1交换，降低了对GABA的亲和力（Km，35 μM）。这些结果表明，环IV和环VI有助于GABA转运体的结合亲和力。用GAT3的相应序列替换GAT1环V中的三个氨基酸导致其GABA摄取活性对β-丙氨酸敏感。环V中的这三个氨基酸似乎参与了GAT3的β-丙氨酸结合结构域。有人提出，这三个外环（IV、V和VI）形成一个口袋，底物在其中与GABA转运体结合。
