Diversity of transport mechanisms in the SLC1 transporter family

SLC1转运体表现出令人印象深刻的功能多样性，从哺乳动物EAATs中Na+/K+/H+偶联的次级活性谷氨酸运输和复杂的运输化学计量学，Na+偶联的原核同源物（如GltPh或GltTk）对天冬氨酸的摄取，到中性氨基酸交换器（ASCTs）中的氨基酸交换。我们将结合原子分子动力学模拟、膜片钳电生理学和荧光光谱来研究Na+和氨基酸结合动力学以及离子-底物耦合机制的异构体特异性差异。SLC1蛋白是典型的升降机转运蛋白，其底物运输是通过转运结构域跨膜的大规模刚体运动来完成的。最近GltPh和人类ASCT2的实验结构定义了这种共享转运机制的起点和终点。为了建立不同同工异构体中转运速率适应的分子原理，我们将定量研究跨膜易位的能量学和动力学以及这些同工异构体中蛋白质-脂质相互作用的功能含义。最后，谷氨酸转运体是潜在的重要药理靶点，但在临床上尚未使用，以减轻谷氨酸在各种脑部疾病中的兴奋性毒性。最近发现了几种正变构调节剂，它们可以增加EAAT的转运活性，并具有神经保护作用。我们将使用分子模拟、低温电镜和电生理学来研究这些化合物如何调节谷氨酸易位，以建立变构性EAAT激活剂的机制框架。

SLC1 transporters exhibit an impressive functional diversity ranging from Na+/K+/H+-coupled secondary active glutamate transport with complex transport stoichiometry in the mammalian EAATs, Na+-coupled aspartate uptake by prokaryotic homologues (e.g., GltPh or GltTk), to amino acid exchange in the neutral amino acid exchangers (ASCTs). We will combine atomistic molecular dynamics simulations, patch-clamp electrophysiology, and fluorescence spectroscopy to investigate isoform-specific differences in Na+- and amino acid-binding kinetics and ion-substrate coupling mechanisms. SLC1 proteins are prototypic elevator transporters, whereby substrate transport is accomplished via large-scale rigid-body movements of the transport domain across the membrane. Recent experimental structures of both GltPh and human ASCT2 define the start and endpoints of this shared transport mechanism. To establish the molecular principles underlying the adaptation of transport rates in the different isoforms, we will quantitatively investigate the energetics and kinetics of transmembrane translocation and the functional implications of protein–lipid interactions in these isoforms. Finally, glutamate transporters are potentially important—yet clinically unused—pharmacological targets to mitigate glutamate excitotoxicity in various brain diseases. Recently, several positive allosteric modulators were identified, which increase EAAT transport activity and exhibit neuroprotective effects. We will investigate how such compounds modulate glutamate translocation using molecular simulations, cryo-EM, and electrophysiology to establish a mechanistic framework for allosteric EAAT activators.