Different Residues in the GABAA Receptor α1T60-α1K70 Region Mediate GABA and SR-95531 Actions

Abstract

Although ␥-aminobutyric acid type A receptor agonists and antagonists bind to a common site, they produce different conformational changes within the site because agonists cause channel opening and antagonists do not. We used the substituted cysteine accessibility method and two-electrode voltage clamping to identify residues within the binding pocket that are important for mediating these different actions. Each residue from ␣ 1 T60 to ␣ 1 K70 was mutated to cysteine and expressed with wild-type ␤ 2 subunits in Xenopus oocytes. Methanethiosulfonate reagents reacted with ␣ 1 T60C, ␣ 1 D62C, ␣ 1 F64C, ␣ 1 R66C, ␣ 1 S68C, and ␣ 1 K70C. ␥-Aminobutyric acid (GABA) slowed methanethiosulfonate modification of ␣ 1 F64C, ␣ 1 R66C, and ␣ 1 S68C, whereas SR-95531 slowed modification of ␣ 1 D62C, ␣ 1 F64C, and ␣ 1 R66C, demonstrating that different residues are important for mediating GABA and SR-95531 actions. In addition, methanethiosulfonate reaction rates were fastest for ␣ 1 F64C and ␣ 1 R66C, indicating that these residues are located in an open, aqueous environment lining the core of the binding pocket. Positively charged methanethiosulfonate reagents derivatized ␣ 1 F64C and ␣ 1 R66C significantly faster than a negatively charged reagent, suggesting that a negative subsite important for interacting with the ammonium group of GABA exists within the binding pocket. Pentobarbital activation of the receptor increased the rate of methanethiosulfonate modification of ␣ 1 D62C and ␣ 1 S68C, demonstrating that parts of the binding site undergo structural rearrangements during channel gating.

Structural Link between γ-Aminobutyric Acid Type A (GABAA) Receptor Agonist Binding Site and Inner β-Sheet Governs Channel Activation and Allosteric Drug Modulation

“…SR-95531 IC 50 experiments were performed as described previously (22). Oocytes expressing WT or mutant receptors were challenged with EC 50 GABA concentration (except for experiments involving ␤Gly2, -4, and -8, which were performed at 30 mM GABA) followed by co-application of the same concentration of GABA and a test concentration of SR-95531.…”

Charged Residues in the α1and β2Pre-M1 Regions Involved in GABAAReceptor Activation

“…The rates at which the various MTS reagents modified the engineered cysteines were determined by measuring the effect of sequential applications of low concentrations of MTS reagents on I GABA , as described previously (Holden and Czajkowski, 2002). The protocol is described as follows: EC 40 -60 GABA was applied for 10 s every 3-5 min until I GABA stabilized (Ͻ3% variance).…”

Mapping the ρ1 GABAC Receptor Agonist Binding Pocket

“…As a further test, we examined protection by a competitive antagonist. Although recent studies suggest that a competitive antagonist can also induce a conformational change beyond the binding site to stabilize the GABA receptor in the closed state (17,28,29), this antagonist-induced conformational change is distinct from that induced by the agonist (24). Thus, only when the agonist and antagonist both protect a residue from modification do we consider that residue FIG.…”

“…By testing the functional effect of a water-soluble cysteine modification reagent and blockability of this effect by agonist and antagonist, the substituted cysteine accessibility method has also been successfully used to probe the binding sites of the GABA A (6,17) and nicotinic receptors (18,19). With this approach, novel residues involved in agonist binding were identified, and the secondary structures of the newly identified domain were predicted (6,7).…”

“…In loops B, C (2), and D (5, 13), the binding residues identified by site-directed mutagenesis are almost identical. In loop D, two additional binding residues, Arg-66 and Ser-68, in the ␣ 1 subunit are recognized by the substituted cysteine accessibility method (6,17). The corresponding residue Arg-104 in the 1 subunit seems to line the binding pocket.…”