Reconstitution of Potassium-Coupled Substrate Transport in an Archaeal Homologue of Glutamate Transporters

Abstract

Excitatory neurotransmitter glutamate is packaged into vesicles in presynaptic neurons and is released into the synaptic cleft via exocytosis upon arrival of the action potential. Following activation of the receptors on postsynaptic neurons, glutamate is rapidly removed from the cleft to prevent excitotoxicity. Excitatory amino acid transporters (EAATs) uptake glutamate into the cytoplasm of glial cells and neurons via a process coupled to symport of three Na+ ions and a proton and antiport of one K+ ion. Studies on an archaeal homologue GltPh elucidated key mechanistic aspects of Na+ symport, including the location of Na+ binding sites and the mechanisms of Na+-mediated transporter gating and coupled binding of the substrate. In contrast, the mechanism of coupling to K+ antiport remains largely unknown. Previous studies on K+ coupling were largely based on ‘loss-of-function’ mutations of EAATs and appeared inconclusive because a surprising number of mutations disrupted coupling. To elucidate K+ coupling mechanism, we pursued a ‘gain-of-function’ approach where we aimed to reconstitute coupling in K+-independent GltPh. Toward this end, we engineered “humanized” GltPh variants bearing several modifications to closer resemble EAATs. One of the variants showed substrate uptake in proteoliposomes that was strongly dependent on the presence of internal K+. Using isothermal titration calorimetry, we further show that K+ ions inhibit substrate binding to this variant but not to the wild type GltPh. Taken together with our previous structural studies, these data allow us to propose a mechanism of K+ coupling in mammalian glutamate transporters.