Department of Bioengineering and Therapeutic Sciences, University of California, San Francisco, California 94158, USA.
J Biol Chem. 2012 Nov 2;287(45):37745-56. doi: 10.1074/jbc.M112.388157. Epub 2012 Aug 29.
The solute carrier 6 (SLC6) is a family of ion-dependent transporters that mediate uptake into the cell of osmolytes such as neurotransmitters and amino acids. Four SLC6 members transport GABA, a key neurotransmitter that triggers inhibitory signaling pathways via various receptors (e.g., GABA(A)). The GABA transporters (GATs) regulate the concentration of GABA available for signaling and are thus targeted by a variety of anticonvulsant and relaxant drugs. Here, we characterize GAT-2, a transporter that plays a role in peripheral GABAergic mechanisms, by constructing comparative structural models based on crystallographic structures of the leucine transporter LeuT. Models of GAT-2 in two different conformations were constructed and experimentally validated, using site-directed mutagenesis. Computational screening of 594,166 compounds including drugs, metabolites, and fragment-like molecules from the ZINC database revealed distinct ligands for the two GAT-2 models. 31 small molecules, including high scoring compounds and molecules chemically related to known and predicted GAT-2 ligands, were experimentally tested in inhibition assays. Twelve ligands were found, six of which were chemically novel (e.g., homotaurine). Our results suggest that GAT-2 is a high selectivity/low affinity transporter that is resistant to inhibition by typical GABAergic inhibitors. Finally, we compared the binding site of GAT-2 with those of other SLC6 members, including the norepinephrine transporter and other GATs, to identify ligand specificity determinants for this family. Our combined approach may be useful for characterizing interactions between small molecules and other membrane proteins, as well as for describing substrate specificities in other protein families.
溶质载体 6（SLC6）是一类离子依赖型转运体家族，介导神经递质和氨基酸等渗透物进入细胞。四个 SLC6 成员转运 GABA，GABA 是一种关键的神经递质，通过各种受体（例如 GABA(A)）触发抑制性信号通路。GABA 转运体（GATs）调节 GABA 的浓度，使其可用于信号传递，因此成为各种抗惊厥和松弛药物的作用靶点。在这里，我们通过构建基于亮氨酸转运体 LeuT 的晶体结构的比较结构模型，对在周围 GABA 能机制中起作用的 GAT-2 进行了表征。构建了两种不同构象的 GAT-2 模型，并通过定点突变进行了实验验证。使用计算方法筛选了来自 ZINC 数据库的 594,166 种化合物，包括药物、代谢物和片段样分子，揭示了两种 GAT-2 模型的不同配体。对 31 种小分子进行了实验测试，包括高得分化合物和与已知和预测的 GAT-2 配体化学相关的分子，在抑制测定中进行了实验测试。发现了 12 种配体，其中 6 种是化学新颖的（例如，同型高丝氨酸）。我们的结果表明，GAT-2 是一种高选择性/低亲和力转运体，对典型 GABA 能抑制剂的抑制作用具有抗性。最后，我们将 GAT-2 的结合位点与其他 SLC6 成员（包括去甲肾上腺素转运体和其他 GATs）的结合位点进行了比较，以确定该家族配体特异性决定因素。我们的综合方法可用于描述小分子与其他膜蛋白之间的相互作用，以及描述其他蛋白质家族中的底物特异性。
