An aspartate residue in the external vestibule of GLYT2 (glycine transporter 2) controls cation access and transport coupling

Synaptic glycine levels are controlled by GLYTs (glycine transporters). GLYT1 is the main regulator of synaptic glycine concentrations and catalyses Na⁺-Cl^--glycine co-transport with a 2:1:1 stoichiometry. In contrast, neuronal GLYT2 supplies glycine to the presynaptic terminal with a 3:1:1 stoichiometry. We subjected homology models of GLYT1 and GLYT2 to molecular dynamics simulations in the presence of Na⁺. Using molecular interaction potential maps and in silico mutagenesis, we identified a conserved region in the GLYT2 external vestibule likely to be involved in Na⁺ interactions. Replacement of Asp⁴⁷¹in this region reduced Na⁺ affinity and Na⁺ co-operativity of transport, an effect not produced in the homologous position (Asp²⁹⁵) in GLYT1. Unlike the GLYT1-Asp²⁹⁵ mutation, this Asp⁴⁷¹ mutant increased sodium leakage and non-stoichiometric uncoupled ion movements through GLYT2, as determined by simultaneously measuring current and [ ³H]glycine accumulation. The homologous Asp⁴⁷¹ and Asp²⁹⁵ positions exhibited distinct cation-sensitive external accessibility, and they were involved in Na⁺ and Li ⁺-induced conformational changes. Although these two cations had opposite effects on GLYT1, they had comparable effects on accessibility in GLYT2, explaining the inhibitory and stimulatory responses to lithium exhibited by the two transporters. On the basis of these findings, we propose a role for Asp⁴⁷¹in controlling cation access to GLYT2 Na⁺ sites, ion coupling during transport and the subsequent conformational changes.