Abstract
d-Tubocurarine (dTC) is a potent competitive antagonist of the Torpedo nicotinic acetylcholine receptor (nAChR) that binds non-equivalently to the two agonist sites (Kd values of 30 nM and 8 microM). When nAChR-rich membranes equilibrated with [3H]dTC are irradiated with 254 nm UV light, [3H]dTC is covalently incorporated into the alpha-, gamma-, and delta-subunits in a concentration-dependent and agonist-inhibitable manner, consistent with the localization of the high and low affinity dTC binding sites at the alpha-gamma- and alpha-delta-subunit interfaces, respectively (Pedersen, S. E. and Cohen, J. B. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2785-2789). We report on the amino acids within alpha-, gamma-, and delta-subunits that are the sites of specific photoincorporation of [3H]dTC. Subunits isolated from nAChR-rich membranes photolabeled with [3H]dTC were subjected to enzymatic digestion, and peptides containing 3H were isolated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and/or reversed-phase high performance liquid chromatography. Isolated peptides were then subjected to NH2-terminal sequence analysis to identify specifically labeled residues. Within the alpha-subunit, 95% of specific incorporation was contained within a 20-kDa proteolytic fragment beginning at Ser-173, with alphaTyr-190 the primary site of [3H]dTC photoincorporation and alphaCys-192 and alphaTyr-198 labeled at lower efficiency. Within gamma- and delta-subunits, specific labeling was contained within proteolytic fragments of 14 and 21 kDa, respectively, beginning at gammaAla-49 and deltaThr-51. gammaTrp-55 and deltaTrp-57 were identified as the sites of specific [3H]dTC photoincorporation. Sequence alignment studies reveal gammaTrp-55 and deltaTrp-57 to be homologous residues at whose position in receptor subunit primary structure a unique pattern of conservation exists in all nAChR (neuronal and muscle). Specifically, all subunits that associate with an alpha-subunit to form an agonist site contain a tryptophan homologous to gammaTrp-55/deltaTrp-57. This pattern of conservation may indicate a functional significance for tryptophan at that location in all nAChR agonist sites.
Highlights
The two agonist binding sites were first associated with the ␣-subunits by affinity labeling [4, 5] and ␣-bungarotoxin binding [6]. Both agonist and competitive antagonist affinity and photoaffinity reagents have been utilized to identify amino acids within the ␣-subunit that are involved in the structure of the agonist site
In Torpedo nicotinic acetylcholine receptor (nAChR), ␦Asp-180 has been identified by chemical cross-linking as a residue within 9 Å of ␣Cys-192–193 [13], and charge neutralizing mutations at that position in mouse nAChR as well as the homologous ␥Asp-174 affect both agonist and competitive antagonist binding [14]
This pattern of photolabeling differed from that seen for another antagonist, [3H]-p-(dimethylamino)benzenediazonium fluoroborate (DDF), which was incorporated into , ␥, and ␦-subunits at Ͻ10% of its ␣ labeling [18] and for the agonist [3H]nicotine, which was photoincorporated into ␥-subunit at ϳ25% the level of ␣, with no agonist-inhibitable incorporation in ␦-subunit detected [19]
Summary
Materials nAChR-rich membranes were isolated from the electric organs of Torpedo californica (Marinus, Westchester, CA) or Torpedo nobiliana (Biofish Associates, Georgetown, MA) using the method described by Pedersen et al [24] with the addition to the homogenization buffer of calpain inhibitors I and II (10 mg/liter, Boehringer Mannheim). The final membrane suspensions in 38% sucrose, 0.02% NaN3 were stored at Ϫ80 °C and contained 0.5–1.7 nmol of ACh binding sites/mg of protein as determined by an ultracentrifugation binding assay using [3H]ACh [25]. As determined by analytical HPLC, radiochemical specific activities of 30 and 20 Ci/mmol were obtained in two syntheses. Analytical grade endoproteinase Lys-C was obtained from Boehringer Mannheim, Staphylococcal aureus glutamyl endopeptidase (V8 protease) was obtained from ICN Biochemicals, and trypsin (L-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) was obtained from Worthington
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