Flux coupling, not specificity, shapes the transport and phylogeny of SLC6 glycine transporters

Abstract

ATB 0 , + ( SLC6A14 ) is a member of the amino acid transporter branch of the SLC6 family along with GlyT1 ( SLC6A9 ) and GlyT2 ( SLC6A5 ), two glycine-specific transporters coupled to 2:1 and 3:1 Na + :Cl − , respectively. In contrast, ATB 0 , + exhibits broad substrate specificity for all neutral and cationic amino acids, and its ionic coupling remains unsettled. Using the reversal potential slope method, we demonstrate a 3:1:1 Na + :Cl − :Gly stoichiometry for ATB 0 , + that is consistent with its 2.1 e /Gly charge coupling. Like GlyT2, ATB 0 , + behaves as a unidirectional transporter with virtually no glycine efflux at negative potentials after uptake, except by heteroexchange as remarkably shown by leucine activation of NMDARs in Xenopus oocytes coexpressing both membrane proteins. Analysis and computational modeling of the charge movement of ATB 0 , + reveal a higher affinity for sodium in the absence of substrate than GlyT2 and a gating mechanism that locks Na + into the apo-transporter at depolarized potentials. A 3:1 Na + :Cl − stoichiometry justifies the concentrative transport properties of ATB 0 , + and explains its trophic role in tumor growth, while rationalizing its phylogenetic proximity to GlyT2 despite their extreme divergence in specificity.