Transmembrane Domain III Plays an Important Role in Ion Binding and Permeation in the Glycine Transporter GLYT2

Abstract

The neuronal glycine transporter GLYT2 takes up glycine from the extracellular space by an electrogenic process where this neurotransmitter is co-transported with sodium and chloride ions. We report in this paper that tyrosine at position 289 of GLYT2a is crucial for ion coupling, glycine affinity and sodium selectivity, stressing the essential role played by this residue of transmembrane domain III in the mechanism of transport. Substitution to tryptophan (Y289W), phenylalanine (Y289F), or serine (Y289S), renders transporters unable to catalyze glycine uptake. Measurements of glycine evoked steady-state currents in transfected HEK-293 cells reveal EC 50 values for glycine 17-fold (Y289F) and 45-fold (Y289S) higher than that of the wild type transporter. Sodium dependence is severely altered in tyrosine 289 mutants, both at the level of apparent affinity and cooperativity, with the more dramatic change corresponding to the less conservative substitution (Y289S). Accordingly, sodium selectivity is gradually lost in Y289F and Y289S mutants, and chloride dependence of glycine evoked currents is markedly decreased in Y289F and Y289S mutants. In the absence of threedimensional information from these transporters, these results provide experimental evidence supporting the hypothesis of transmembrane domain III being part of a common permeation pathway for substrate and cotransported ions.Glycine is a major inhibitory neurotransmitter in the spinal cord and the brain stem of vertebrates, where it participates in a variety of motor and sensory functions. In addition, glycine positively modulates the action of glutamate, the main excitatory neurotransmitter in the brain, on postsynaptic NMDA 1 receptors. The re-uptake of glycine into presynaptic nerve terminals or the surrounding fine glial processes plays a major role in the maintenance of low synaptic levels of the transmitter (1-3). It is now well demonstrated that hypofunction of glutamatergic (NMDA receptor-mediated) neurotransmission is involved in symptoms of schizophrenia (4). It is also likely that a decrease in the glycinergic inputs is involved in pathologies of the muscle tone regulation (5). Therefore, compounds able to potentiate glycinergic neurotransmission may find applications in the treatment of psychiatric diseases and spasticity (6).Glycine transporters are members of the Na ϩ -and Cl Ϫ -dependent neurotransmitter transporter family (7-9), a group of integral glycoproteins (10, 11) that share a common structure with 12 transmembrane domains (12). Several neurotransmitter uptake systems, including those for glycine, present an unexpected molecular heterogeneity.By now, two glycine transporters, GLYT1 and GLYT2, have been cloned (13-17). GLYT1 presents three isoforms (GLYT1a, GLYT1b, and GLYT1c) that differ in their amino-terminal sequences and are generated both by alternative promoter usage and by alternative splicing (13,15,17,18). Recently, a second GLYT2 isoform (GLYT2b) has been isolated, cloned, and characterized in our laboratory (19). The GLYT...

The Second Intracellular Loop of the Glycine Transporter 2 Contains Crucial Residues for Glycine Transport and Phorbol Ester-induced Regulation

“…4). This points out that Ile-292 is a more critical residue for glycine transport than A223C, which is in agreement with our previous observations (20). In addition, the reduction in MTSET accessibility from single (outward facing) to double (inward facing) mutants was lower for I292C than A223C mutants, according to the higher mobility of EL1 during the transport-associated conformational changes (23 lationship connecting IL2 residues and the substrate-binding site shows a gradient of intensity increasing from the N-to C-terminal end of the loop with minimum connection at position 415 and maximum connection at 422, which also points toward a higher relevance of Lys-422 within a series of possible regulatory residues in IL2.…”

“…Although little is known about the three-dimensional structure of these transporters (16), site-directed mutagenesis approaches have permitted the identification of critical residues for the transport function in TMI, -III, and -IV (17)(18)(19)(20)(21)(22), which are candidates to contribute to the binding of substrates. In addition, conformationally active residues have been identified in several hydrophilic loops (23)(24)(25)(26)(27)(28)(29)(30), and it has been suggested that IL2-IL3-IL4 might be part of an intracellular "gating" domain, because a mutation of three residues in those loops of DAT seems to disrupt intramolecular interactions stabilizing the conformation able to bind extracellular substrate (31)(32)(33).…”

Comparative Molecular Field Analysis Using Selectivity Fields Reveals Residues in the Third Transmembrane Helix of the Serotonin Transporter Associated with Substrate and Antagonist Recognition

“…In a previous study, Bismuth et al (1997) determined that the GABA transporter (GAT) residue Tyr140, a residue conserved in the third TMH throughout the sodium/chloride-dependent transporter family, is critical for GABA recognition and transport. Furthermore, residue Tyr289 of the neuronal glycine transporter GLYT2 has an important role in glycine binding and transport, ion dependence, and sodium selectivity (Ponce et al, 2000). GAT Tyr140 and GLYT2 Tyr289 are both homologous to Tyr176 in the hSERT.…”

Proximity of Transmembrane Domains 1 and 3 of the γ-AminobutyricAcid Transporter GAT-1 Inferred from Paired CysteineMutagenesis

“…Tyrosine 140, located one turn of a putative ␣-helix below position 143, plays a crucial role in GABA binding (16), and similar evidence exists for such a role of the conserved tyrosine in the glycine transporter GlyT (38) and in SERT (17). Because this tyrosine is invariant among all family members, we have suggested that it interacts with the amino group of the substrate, the moiety common to amino acids and biogenic amines (16).…”