Abstract
To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor (nAChR)-rich membranes from Torpedo electric organ with a photoactivatable analog, [(3)H]azietomidate. Based upon the inhibition of binding of the noncompetitive antagonist [(3)H]phencyclidine, azietomidate and etomidate bind with 10-fold higher affinity to nAChRs in the desensitized state (IC(50) = 70 microm) than in the closed channel state. In addition, both drugs between 0.1 and 1 mm produced a concentration-dependent enhancement of [(3)H]ACh equilibrium binding affinity, but they inhibited binding at higher concentrations. UV irradiation resulted in preferential [(3)H]azietomidate photoincorporation into the nAChR alpha and delta subunits. Photolabeled amino acids in both subunits were identified in the ion channel domain and in the ACh binding sites by Edman degradation. Within the nAChR ion channel in the desensitized state, there was labeling of alphaGlu-262 and deltaGln-276 at the extracellular end and deltaSer-258 and deltaSer-262 toward the cytoplasmic end. Within the acetylcholine binding sites, [(3)H]azietomidate photolabeled alphaTyr-93, alphaTyr-190, and alphaTyr-198 in the site at the alpha-gamma interface and deltaAsp-59 (but not the homologous position, gammaGlu-57). Increasing [(3)H]azietomidate concentration from 1.8 to 150 microm increased the efficiency of incorporation into amino acids within the ion channel by 10-fold and in the ACh sites by 100-fold, consistent with higher affinity binding within the ion channel. The state dependence and subunit selectivity of [(3)H]azietomidate photolabeling are discussed in terms of the structures of the nAChR transmembrane and extracellular domains.
Highlights
To identify binding domains in a ligand-gated ion channel for etomidate, an intravenous general anesthetic, we photolabeled nicotinic acetylcholine receptor-rich membranes from Torpedo electric organ with a photoactivatable analog, [3H]azietomidate
Etomidate and Azietomidate Inhibition of [3H]PCP and [3H]ACh Binding—We tested etomidate and azietomidate as inhibitors of the binding to Torpedo nicotinic acetylcholine receptor (nAChR)-rich membranes of [3H]PCP, a positively charged, aromatic amine noncompetitive antagonist. [3H]PCP binds with high affinity (Keq ϭ 1 M) to a single site per nAChR in the desensitized state and more weakly (Keq ϭ 7 M) in the absence of agonist [28]
For nAChRs in the desensitized state and equilibrated with agonist, at 1 M [3H]azietomidate, ϳ60% of the ␣ and 20% of the ␦ subunit labeling were inhibitable by proadifen
Summary
Materials—nAChR-enriched membranes were isolated from Torpedo californica electric organ [20]. Proteolytic Digestions—For digestion with EndoLys-C or V8 protease, acetone-precipitated subunits or subunit fragments isolated from preparative scale labelings (10 mg of protein) were resuspended in 200 l of 15 mM Tris, 0.5 mM EDTA, pH 8.1, 0.1% SDS. Because sample loading under this condition results in cleavage on the N-terminal side of Trp residues [16], when fragments beginning at ␦Thr-51, which contain a Trp in cycle 7, were sequenced (Fig. 8B and Supplemental Fig. 2B), the HPLC fractions were loaded directly onto the glass fiber disk, which was dried and treated with Biobrene. For sequencing of fragments containing ␣M2 (Fig. 7B), two-thirds went to the PTH analyzer, and one-third was collected for scintillation counting For these samples, the plotted 3H release and PTH-derivative release are those caclulated based for the usual one-sixth mass analysis and five-sixths 3H determination. Details concerning the values of I0 and R for each sequencing run as well as the cpm loaded on the filters and retained after sequencing are provided in the the legends to Supplemental Figs. 1 and 2
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