Abstract
A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.
Highlights
From the Departmentsof $Biochemistryand Molecular Biophysics, and VNeurology, Collegeof Physicians and Surgeons, Columbia University, New York, New York I0032 of acetylcholine.The dimensions of the optimal affinity labels implied that thealkylated cysteinyl residue i s 1nm from the negative subsite that binds the quaternary ammonium group (Karlin, 1969; Karlin, 1980)
Our results dem- cysteinyl residues occur in all species whosea sequences have onstrate that: 1)the binding site disulfide is between been determined (Noda et al, 1982; Devillers-Thiery et al, Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross- 1983;Noda et al, 198313;Boulter et al, 1986).The functional linked to Cys-142; and 3)under conditions that reduce roles of these four Cys residues in
A (Bodanszky and Stahl, 1974) and the y subunit of bovine transducin (Ovchinnikovet al., 1985).In thispaper, we demonstrate directly that Cys-192is disulfide-cross-linkedto CysThe binding of acetylcholine to the nicotinic acetylcholine 193. receptor initiates a cascade of conformational changes that culminate in the transient opening of a cation-specific channel
Summary
Twenty nanomoles of purified receptor (Reynolds and Kariin, 1978),containing 40 nmol of toxin binding sites and 5.6 mg opfrotein, in 0.1% Triton X-100 solution was alkylated with 1 mM NEM to block free sulfhydryls, dialyzed, and reduced with 0.2 mM &thiothreitol at pH8.0 and 25 "C for 20 min. The abbreviations used are: f3H]MBTA, 4-(N-maleimido)benz~ltri[~H]methylammoniumiodide; HPLC, high-performance liquid chromatograpy; Hse, homoserine; [I4C]NEM,N-[l-"C]ethylmaleimide; PTH, phenylthiohydantoin; toxin, principal curarimimetic toxin of Naja mja siamensis venom. 25 "C for 10 min, in a final volume of 4 ml Under these conditions, 50% of the curarimimetic toxin binding sites (Damle and Karlin, 1978) were blocked.Alkylation was terminated by dialysis at pH 6 and 4"C, ratherthan with 2-mercaptoethanol (Karlin and Cowburn, 1973), to preserve remaining disulfide bonds, Receptor subunits were separated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and recovered by electroelution
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