Tyrosine 62 of the γ-Aminobutyric Acid Type A Receptor β2 Subunit Is an Important Determinant of High Affinity Agonist Binding

Abstract

The ␥-aminobutyric acid type A receptor (GABA A R) carries both high (K D ‫؍‬ 10 -30 nM) and low (K D ‫؍‬ 0.1-1.0 M) affinity binding sites for agonists. We have used site-directed mutagenesis to identify a specific residue in the rat ␤2 subunit that is involved in high affinity agonist binding. Tyrosine residues at positions 62 and 74 were mutated to either phenylalanine or serine and the effects on ligand binding and ion channel activation were investigated after the expression of mutant subunits with wild-type ␣1 and ␥2 subunits in tsA201 cells or in The GABA A R 1 is a member of a superfamily of ligand-gated ion channels that includes the nicotinic acetylcholine receptor (nAChR), the glycine receptor, and the serotonin type 3 receptor (1). The GABA A R carries binding sites for a number of therapeutic agents including the benzodiazepines, barbiturates, neurosteroids, some general anesthetics, and possibly also alcohol (1). In brain membranes, there are at least two classes of binding sites for the endogenous neurotransmitter, which differ by more than an order of magnitude in their affinity for GABA or its structural analogues (2-4). This heterogeneity in binding was originally thought to reflect the diversity of GABA A R subtypes in brain tissue. However, the presence of both classes of sites in a stable cell line expressing a specific subtype (5) suggests that both exist in a single receptor molecule. On the basis of biochemical studies, the reasonable correlation between the concentration of agonist required to elicit ion flux and to potentiate the binding of benzodiazepine ligands suggested that the low affinity sites are important for channel gating (see Ref. 6). However, the role(s) of the high affinity binding sites in receptor function remains unclear.All members of this receptor family are believed to be pentameric complexes formed by homologous subunits assembled to form a central ion channel (7). Recent models (see Ref. 8) predict that ligand binding sites occur at subunit-subunit interfaces. This was first demonstrated in the nAChR in which the ␣-␥ and ␣-␦ interfaces were implicated in forming nonequivalent binding sites for d-tubocurarine (9). In the GABA A R, low affinity GABA sites (i.e. those that have been implicated in channel activation) are thought to be located at the interfaces between the ␤ and ␣ subunits (10 -13), whereas the benzodiazepine binding site is predicted to occur at the homologous ␣-␥ interface (14 -19). More detailed analyses of the properties of these sites have led to a "loop model" of ligand binding sites (see Ref. 20) in which amino acid residues from at least three discontinuous regions (denoted "loops" A-C) of one subunit together with residues from at least one region of the adjacent subunit ("loop" D) form the binding pocket (see Fig. 1A).In the GABA A R, evidence for the location of the high affinity agonist site(s) is derived from a number of experimental approaches. Photoaffinity labeling studies first suggested that the ␤ subunit is a major determinant of h...

GABAA Receptor Composition Is Determined by Distinct Assembly Signals within α and β Subunits

“…Consistent with the location of these assembly signals to intersubunit contact points, the ␣/␥ signals (18,19,21) are located proximal to the GABA and benzodiazepine binding sites (22,23) formed at subunit interfaces between the ␣-␤ and ␣␥ subunits, respectively, and also the ␤2 high affinity GABA site (24). Similarly, the homologous region in 1 is an important component of the GABA binding domain (25).…”

“…In keeping with other studies (18,28), these authors found that Gln-67 and Trp-69 were essential for the production of functional receptors. Moreover, this region is not only important in the ␣1 subunit contribution to GABA binding, but the high affinity GABA binding site has been shown to be produced by the homologous region in ␤2 (␤2A interface to ␣1) (24). In addition, the benzodiazepine binding site on the ␥2 subunit resides within the same homologous region, with A79 (homologous position to Arg-66 in ␣1) lining the benzodiazepine binding pocket (23).…”

Identification of a Tyrosine in the Agonist Binding Site of the Homomeric ρ1 γ-Aminobutyric Acid (GABA) Receptor That, When Mutated, Produces Spontaneous Opening

“…The residues homologous to ␣1F64 in the ␤2 (Tyr-62) and ␥2 (Phe-77) subunits do not seem to play a key role in agonist binding as mutation of ␤2Y62 and ␥2F77 resulted in a negligible shift in GABA sensitivity (12). However, a complete loss of high affinity binding was reported when ␤2Y62 was mutated to serine (13). They suggested that ␤2Y62 might be a component of the high affinity GABA binding site, but not part of the binding site linked to channel gating.…”

Acute neurosteroid modulation and subunit isolation of the gamma-aminobutyric acidA receptor in the bullfrog, Rana catesbeiana

“…The sequence of the bullfrog 1-like subunit revealed conservation of the rat 1 subunit Tyr-62 residue associated with high-affinity agonist binding (Newell et al 2000). Also, two GABA binding domains found on the rat 1 subunit (as well as subunits of other phyla) were conserved in the bullfrog 1-like subunit (rat Tyr-157-Thr-160 and Thr-202-Tyr-205; Amin & Weiss 1993).…”