Abstract
Mutagenesis of recombinant rho1 gamma-aminobutyric acid (GABA) receptors has previously identified five residues in the amino terminal extracellular domain that play an important role in GABA binding. Here, we present evidence that the tyrosine at position 102 of the rho1 receptor is also associated with the agonist binding site. Wild-type and mutant rho1 receptors were expressed in Xenopus laevis oocytes and examined using the two-electrode voltage clamp. When Tyr-102 was mutated to cysteine, serine, tryptophan, or glycine the EC(50) increased 31-, 214-, 664-, and 8752-fold, respectively. An increase in the IC(50) was also observed for the competitive antagonist 3-APMPA, but not for the non-competitive antagonist picrotoxin. Y102C was accessible to modification by methanethiosulfonate, and this modification was prevented by both GABA and 3-APMPA. An interesting characteristic of the Y102S mutant receptor was that, in the absence of GABA, there was an unusually high oocyte resting conductance that was blocked by both 3-APMPA and picrotoxin, indicating spontaneously opening GABA receptors. It appears that mutation of Tyr-102 perturbs the binding site and gates the pore. We conclude that Tyr-102 is a component of the GABA binding domain and speculate that Tyr-102 might be important for coupling agonist binding to channel opening.
Highlights
Is their distribution in the central nervous system
Mutagenesis and photoaffinity labeling studies of the ␣1 subunit of the GABAA receptor have identified a phenylalanine at position 64, which appears to be involved in agonist binding [11, 12]
Via site-directed mutagenesis, oocyte expression, and the two-electrode voltage clamp technique, we examined the effect of mutating Tyr-102 on the activation properties of the GABAC receptor
Summary
Site-directed Mutagenesis and in Vitro Transcription—The human 1 cDNA was cloned into the pGEMHE vector [14] and site-directed mutagenesis was achieved by the polymerase chain reaction overlap extension method [15]. pGEMHE-1 was linearized with NheI, and cRNA synthesis was carried out using standard in vitro transcription procedures as previously described [16]. Oocyte Isolation and cRNA Injection—Female Xenopus laevis (Xenopus I, Ann Arbor, MI) were anesthetized by 0.2% MS-222 (3-aminobenzoic acid ethyl ester, methanesulfonate salt), and oocytes were surgically removed from the frog and placed in calcium-free oocyte Ringers-2 (OR2) incubation solution consisting of 92.5 mM NaCl, 2.5 mM KCl, 5. For GABA dose-response experiments, oocytes expressing 1 wild-type and Y102C receptors were treated for 10 min in 2.5 mM MTSEA, washed for 5 min with OR2, and returned to the recording chamber. A similar recovery has been previously described for MTSEA modification of the acetylcholine receptor [17]
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