Abstract
We reported previously that tyrosine 62 of the beta2 subunit of the gamma-aminobutyric acid, type A (GABA(A)) receptor is an important determinant of high affinity agonist binding and that recombinant alpha1beta2gamma2(L) receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. We have now examined the effects of disrupting these sites on the macroscopic desensitization properties of receptors expressed in Xenopus oocytes. Desensitization was measured by the ability of low concentrations of bath-perfused agonist to reduce the current responses elicited by subsequent challenges with saturating concentrations of GABA. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50 approximately 0.7 microm, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Receptors carrying the beta2 Y62S and Y62F mutations desensitized at slightly higher (2-7-fold) agonist concentrations. However, at low perfusate concentrations, the Y62S-containing receptor recovered from the desensitized state even in the continued presence of agonist. The characteristics of desensitization in the wild-type and mutant receptors lead us to suggest that the major role of the high affinity agonist-binding site(s) of the GABA(A) receptor is not to induce desensitization but rather to stabilize the desensitized state once it has been formed.
Highlights
We reported previously that tyrosine 62 of the 2 subunit of the ␥-aminobutyric acid, type A (GABAA) receptor is an important determinant of high affinity agonist binding and that recombinant ␣12␥2L receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol
We have recently identified an amino acid residue (Tyr-62) of the 2 subunit of the GABAAR that is important for high affinity agonist binding [10]
The identification of molecular domains that contribute to receptor activation and desensitization is a primary goal in elicited current in the Y62S mutant receptor as a function of time following application of 175 M GABA in the absence (G) or presence of 3 M GABA (E)
Summary
We reported previously that tyrosine 62 of the 2 subunit of the ␥-aminobutyric acid, type A (GABAA) receptor is an important determinant of high affinity agonist binding and that recombinant ␣12␥2L receptors carrying the Y62S mutation lack measurable high affinity sites for [3H]muscimol. Wild-type receptors were desensitized by pre-perfused muscimol with an IC50 ϳ0.7 M, which correlates well with the lower affinity sites for this agonist that are measured in direct binding studies. Members of a receptor gene family (see Ref. 4) that includes the nicotinic acetylcholine receptors (nAChRs), glycine receptors, and the serotonin type 3 receptor These receptors are structurally and functionally homologous and desensitize in the continued presence of agonist [5, 6]. Because higher concentrations of muscimol are required to activate this receptor subtype (EC50 of 7.1 M when expressed in Xenopus oocytes), sites of still lower affinity may be present (see below). The roles of these multiple sites in receptor function require clarification, it is generally accepted that the high affinity binding that is measured under equilibrium conditions reflects binding to a desensitized conformation(s) of the receptor (see Ref. 14). It is assumed that agonist occupancy of these sites in the resting state of the protein induces the conformational changes that lead to this equilibrium desensitized state
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