Identification of amino acids contributing to high and low affinity d-tubocurarine sites in the Torpedo nicotinic acetylcholine receptor.
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.
Chiara
D C
DC
Department of Neurobiology, Harvard University School of Medicine, Boston, Massachusetts 02115, USA.
Cohen
J B
JB
D000581:Amidohydrolases / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D006596:Hexosaminidases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D010777:Photochemistry; D011487:Protein Conformation; D011978:Receptors, Nicotinic / Q000737:chemistry / Q000378:metabolism*; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000378:metabolism; D014101:Torpedo; D014403:Tubocurarine / Q000378:metabolism*
D000596:Amino Acids; D011978:Receptors, Nicotinic; D006596:Hexosaminidases; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D014403:Tubocurarine