Tyrosine 140 of the γ-Aminobutyric Acid Transporter GAT-1 Plays a Critical Role in Neurotransmitter Recognition

Abstract

The gamma-aminobutyric acid (GABA) transporter GAT-1 is located in nerve terminals and catalyzes the electrogenic reuptake of the neurotransmitter with two sodium ions and one chloride. We now identify a single tyrosine residue that is critical for GABA recognition and transport. It is completely conserved throughout the superfamily, and even substitution to the other aromatic amino acids, phenylalanine (Y140F) and tryptophan (Y140W), results in completely inactive transporters. Electrophysiological characterization reveals that both mutant transporters exhibit the sodium-dependent transient currents associated with sodium binding as well as the chloride-dependent lithium leak currents characteristic of GAT-1. On the other hand, in both mutants GABA is neither able to induce a steady-state transport current nor to block their transient currents. The nontransportable analog SKF 100330A potently inhibits the sodium-dependent transient in the wild type GAT-1 but not in the Y140W transporter. It partly blocks the transient of Y140F. Thus, although sodium and chloride binding are unimpaired in the tyrosine mutants, they have a specific defect in the binding of GABA. The total conservation of the residue throughout the family suggests that tyrosine 140 may be involved in the liganding of the amino group, the moiety common to all of the neurotransmitters.

The Family of Na+/Cl− Neurotransmitter Transporters

“…Both mutants are inactive in GABA-induced steady-state transport currents. It was concluded that Y140 may be involved in the ligand binding of the amino group of GABA and other neurotransmitters (Bismuth et al, 1997). Fleetrophysiological studies also indicate that the conserved residue W68 in the first putative TM I ofGATI may be involved in the binding of Na'' and/or CL' (Mager et al, 1996).…”

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

“…This tyrosine residue is conserved in all the family members, including the bacterial transporters (24). It has been suggested that the tyrosine may interact with a moiety common to all substrates of the family, namely the amino group (22).In this study we have modified Tyr 288 and the conserved Tyr 289 located in the TMIII of GLYT2a transporter. By using a glycine transport assay and the whole cell recording configuration of the patch-clamp technique, we have studied the functional effects of these point modifications.…”

“…Previous work from related GABA (GAT-1) and serotonin (SERT) transporters point to a tyrosine residue and its surroundings in the third transmembrane domain (TMIII) as being critical in substrate binding (22,23). This tyrosine residue is conserved in all the family members, including the bacterial transporters (24).…”

“…This tyrosine residue is conserved in all the family members, including the bacterial transporters (24). It has been suggested that the tyrosine may interact with a moiety common to all substrates of the family, namely the amino group (22).…”

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

“…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).…”