Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52428 Jülich, Germany; Institut für Neurophysiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany; Computational Biomolecular Dynamics Group, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
Institute of Complex Systems, Zelluläre Biophysik (ICS-4), Forschungszentrum Jülich, 52428 Jülich, Germany.
Cell. 2015 Jan 29;160(3):542-53. doi: 10.1016/j.cell.2014.12.035.
Excitatory amino acid transporters (EAATs) are essential for terminating glutamatergic synaptic transmission. They are not only coupled glutamate/Na(+)/H(+)/K(+) transporters but also function as anion-selective channels. EAAT anion channels regulate neuronal excitability, and gain-of-function mutations in these proteins result in ataxia and epilepsy. We have combined molecular dynamics simulations with fluorescence spectroscopy of the prokaryotic homolog GltPh and patch-clamp recordings of mammalian EAATs to determine how these transporters conduct anions. Whereas outward- and inward-facing GltPh conformations are nonconductive, lateral movement of the glutamate transport domain from intermediate transporter conformations results in formation of an anion-selective conduction pathway. Fluorescence quenching of inserted tryptophan residues indicated the entry of anions into this pathway, and mutations of homologous pore-forming residues had analogous effects on GltPh simulations and EAAT2/EAAT4 measurements of single-channel currents and anion/cation selectivities. These findings provide a mechanistic framework of how neurotransmitter transporters can operate as anion-selective and ligand-gated ion channels.
兴奋性氨基酸转运体（EAATs）对于终止谷氨酸能突触传递至关重要。它们不仅与谷氨酸/Na(+)/H(+)/K(+)转运体偶联，而且还作为阴离子选择性通道发挥作用。EAAT 阴离子通道调节神经元兴奋性，这些蛋白质的功能获得性突变导致共济失调和癫痫。我们结合了分子动力学模拟和原核同源物 GltPh 的荧光光谱以及哺乳动物 EAATs 的膜片钳记录，以确定这些转运体如何传导阴离子。虽然外向和内向构象的 GltPh 是无导电性的，但谷氨酸转运结构域从中介转运体构象的横向移动导致形成阴离子选择性传导途径。插入色氨酸残基的荧光猝灭表明阴离子进入该途径，并且同源孔形成残基的突变对 GltPh 模拟以及 EAAT2/EAAT4 对单通道电流和阴离子/阳离子选择性的测量具有类似的影响。这些发现为神经递质转运体如何作为阴离子选择性和配体门控离子通道发挥作用提供了一个机制框架。
