Chiara D C, Cohen J B
Department of Neurobiology, Harvard University School of Medicine, Boston, Massachusetts 02115, USA.
J Biol Chem. 1997 Dec 26;272(52):32940-50. doi: 10.1074/jbc.272.52.32940.
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.
d -筒箭毒碱（dTC）是一种强效的竞争性拮抗剂，可作用于电鳐烟碱型乙酰胆碱受体（nAChR），它与两个激动剂位点的结合不相等（解离常数Kd值分别为30 nM和8 μM）。当用[³H]dTC平衡的富含nAChR的膜用254 nm紫外光照射时，[³H]dTC以浓度依赖性和激动剂抑制性方式共价结合到α、γ和δ亚基中，这与高亲和力和低亲和力dTC结合位点分别位于α - γ和α - δ亚基界面的定位一致（佩德森，S. E.和科恩，J. B.（1990年）《美国国家科学院院刊》87，2785 - 2789）。我们报告了α、γ和δ亚基内作为[³H]dTC特异性光掺入位点的氨基酸。从用[³H]dTC光标记的富含nAChR的膜中分离出的亚基进行酶消化，通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳和/或反相高效液相色谱法分离含有³H标记的肽段。然后对分离出的肽段进行氨基末端序列分析，以鉴定特异性标记的残基。在α亚基内，95%的特异性掺入包含在一个从Ser - 173开始的20 kDa蛋白水解片段中，αTyr - 190是[³H]dTC光掺入的主要位点，αCys - 192和αTyr - 198的标记效率较低。在γ和δ亚基内，特异性标记分别包含在从γAla - 49和δThr - 51开始的14 kDa和21 kDa的蛋白水解片段中。γTrp - 55和δTrp - 57被鉴定为[³H]dTC特异性光掺入的位点。序列比对研究表明，γTrp - 55和δTrp - 57是同源残基，在受体亚基一级结构中的这个位置，所有nAChR（神经元型和肌肉型）都存在独特的保守模式。具体而言，所有与α亚基结合形成激动剂位点的亚基都含有一个与γTrp - 55/δTrp - 57同源的色氨酸。这种保守模式可能表明色氨酸在所有nAChR激动剂位点的该位置具有功能意义。
