Department of Pharmacological and Biomolecular Sciences, Università degli Studi di Milano, Via Trentacoste 2, 20134, Milano, Italy.
Department of Biotechnology and Life Sciences, University of Insubria, Via J.H. Dunant 3, 21100, Varese, Italy.
Biochim Biophys Acta Biomembr. 2018 May;1860(5):1179-1186. doi: 10.1016/j.bbamem.2018.01.020. Epub 2018 Jan 31.
The crystallizations of the prokaryotic LeuT and of the eukaryotic DAT and SERT transporters represent important steps forward in the comprehension of the molecular physiology of Neurotransmitter:Sodium Symporters, although the molecular determinants of the coupling mechanism and of ion selectivity still remain to be fully elucidated. The insect NSS homologue KAAT1 exhibits unusual physiological features, such as the ability to use K as the driver ion, weak chloride dependence, and the ability of the driver ion to influence the substrate selectivity; these characteristics can help to define the molecular determinants of NSS function. Two non-conserved residues are present in the putative sodium binding sites of KAAT1: Ala 66, corresponding to Gly 20 in the Na2 site of LeuT, and Ser 68, corresponding to Ala 22 in the Na1 site. Thr 67 appears also to be significant since it is not conserved among NSS members, is present as threonine only in KAAT1 and in the paralogue CAATCH1 and, according to LeuT structure, is close to the amino acid binding site. Mutants of these residues were functionally characterized in oocytes. The T67Y mutant exhibited uptake activity comparable to that of the wild type, but fully chloride-independent and with enhanced stereoselectivity. Interestingly, although dependent on the presence of sodium, the mutant showed reduced transport-associated currents, indicating uncoupling of the driver ion and amino acid fluxes. Thr 67 therefore appears to be a key component in the coupling mechanism, participating in a network that influences the cotransport of Na and the amino acid.
原核 LeuT 和真核 DAT 和 SERT 转运体的晶体结构代表了在理解神经递质：钠离子转运体的分子生理学方面的重要进展，尽管离子选择性和偶联机制的分子决定因素仍有待充分阐明。昆虫 NSS 同源物 KAAT1 具有不寻常的生理特征，例如能够使用 K+作为驱动离子、对氯离子的依赖性弱、以及驱动离子能够影响底物选择性；这些特征有助于定义 NSS 功能的分子决定因素。KAAT1 中假定的钠离子结合位点存在两个非保守残基：Ala 66，对应于 LeuT 的 Na2 位点中的 Gly 20，和 Ser 68，对应于 Na1 位点中的 Ala 22。Thr 67 似乎也很重要，因为它在 NSS 成员中不保守，仅在 KAAT1 和其旁系同源物 CAATCH1 中存在苏氨酸，并且根据 LeuT 结构，它靠近氨基酸结合位点。这些残基的突变体在卵母细胞中进行了功能表征。T67Y 突变体表现出与野生型相当的摄取活性，但完全氯离子独立，并且立体选择性增强。有趣的是，尽管依赖钠离子，但突变体显示出减少的转运相关电流，表明驱动离子和氨基酸通量的解偶联。因此，Thr 67 似乎是偶联机制中的关键组成部分，参与影响 Na 和氨基酸共转运的网络。
