biostudies-literatureReddy KDAmerican Heart AssociationSimons FoundationNYSTARNINDS NIH HHSNational Institutes of HealthAgouron InstituteNational Institute of General Medical SciencesNIGMS NIH HHSNIH HHSe202213131https://www.ebi.ac.uk/biostudies/studies/S-EPMC9044058biostudies-literatureUnknown154(5)Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following subThe Journal of general physiologyThe archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding.PMC9044058F32 NS10232519PRE34380215349247S10 OD019994R01NS064357R37NS085318P41 GM103310R01 NS064357GM103310F00316S10 OD019994-01R37 NS085318Reddy KDCiftci DBoudker OScopelliti AJfalseThe archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding.Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following sub2022-01-01T00:00:00Z2022 May2026-05-09T23:38:46.996Z2025-04-04T19:32:52.823ZS-EPMC90440583545209010.1085/jgp.202213131