A three-sodium-to-glycine stoichiometry shapes the structural relationships of ATB0,+ with GlyT2 and GlyT1 in the SLC6 family

ATB0,+ (SLC6A14) absorbs all neutral and cationic amino acids in the distal colon and lung epithelia, and is part of the amino acid transporter branch I of the SLC6 family with GlyT1 (SLC6A9) and GlyT2 (SLC6A5), two glycine-specific transporters coupled to 2:1 and 3:1 Na+:Cl-, respectively. However, ATB0,+ stoichiometry that specifies its driving force and electrogenicity remains unsettled. Using the reversal potential slope method, here we demonstrate that ATB0,+-mediated glycine transport is coupled to 3 Na+ and 1 Cl- and has a charge coupling of 2.1 e/glycine. ATB0,+ behaves as a unidirectional transporter with limited efflux and exchange capabilities. Analysis and computational modeling of the pre-steady-state charge movement reveal higher sodium affinity of the apo-ATB0,+, and a locking trap preventing Na+ loss at depolarized potentials. A 3 Na+/ 1 Cl- stoichiometry substantiates ATB0,+ concentrative-uptake and trophic role in cancers and rationalizes its structural proximity with GlyT2 despite their divergent substrate specificity.