Abstract
Excitatory amino acid transporters (EAATs) are crucial in maintaining extracellular levels of glutamate, the most abundant excitatory neurotransmitter, below toxic levels. The recent three-dimensional crystal structure of GltPh, an archaeal homolog of the EAATs, provides elegant structural details of this family of proteins, yet we know little about the mechanism of the bacterial transporter. Conflicting reports in the literature have described GltPh as an aspartate transporter driven by Na+ or a glutamate transporter driven by either Na+ or H+. Here we use purified protein reconstituted into liposomes to thoroughly characterize the ion and substrate dependence of the GltPh transport. We confirm that GltPh is a Na+-dependent transporter that is highly selective for aspartate over other amino acids, and we show that transport is coupled to at least two Na+ ions. In contrast to the EAATs, transport via GltPh is independent of H+ and K+. We propose a kinetic model of transport in which at least two Na+ ions are coupled to the cotransport of each aspartate molecule by GltPh, and where an ion- and substrate-free transporter reorients to complete the transport cycle.
Highlights
Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system; it activates a wide range of ionotropic and metabotropic receptors to generate synaptic responses
We propose a kinetic model of transport in which at least two Na؉ ions are coupled to the cotransport of each aspartate molecule by glutamate transporter family from Pyrococcus horikoshii (GltPh), and where an ion- and substrate-free transporter reorients to complete the transport cycle
In contrast to the Excitatory amino acid transporters (EAATs), which display a strict requirement for Kϩ to observe efflux [31], we find that efflux can occur in the presence of a range of substances, including Kϩ, Chϩ, and sorbitol (Fig. 5C); these results further support the contention that Kϩ is not required for the reorientation of the aspartate-free transporter
Summary
Glutamate is the predominant excitatory neurotransmitter in the mammalian central nervous system; it activates a wide range of ionotropic and metabotropic receptors to generate synaptic responses. GltPh shares about 36% amino acid identity with the EAATs. Many of the residues that have been implicated in glutamate and ion binding/translocation (10 –12) and chloride permeation [13] in the EAATs are highly conserved throughout the family, suggesting that the basic architecture of the bacterial and mammalian proteins is very similar. GltPh structure, we must first understand the basic functional properties of the transporter Those mechanistic features that are conserved between prokaryotic and eukaryotic homologs can be understood in detail by analyzing GltPh as a model structure. Boudker et al [14] reported an analysis of Naϩ dependence of aspartate binding to detergent-solubilized GltPh protein, but the dependence of transport on substrate and Naϩ concentration has not yet been analyzed. This is an important distinction, as the conditions most favorable for substrate binding to the transporter in isolation may be quite different from those required for optimal transport
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