Striessnig J, Knaus H G, Glossmann H
Institut für Biochemische Pharmakologie, Universität Innsbruck, Austria.
Biochem J. 1988 Jul 1;253(1):39-47. doi: 10.1042/bj2530039.
This study identifies calcium-antagonist-receptor-carrying polypeptides of calcium channels in guinea-pig hippocampus membranes. The arylazide ligands (-)-[3H]azidopine and [N-methyl-3H]LU49888 [(-)-5-[(3-azidophenethyl) [N-methyl-3H]methylamino]-2-(3,4,5-trimethoxyphenyl-2- isopropylvaleronitrile] were used to selectively label 1,4-dihydropyridine and phenylalkylamine receptors respectively. In the absence of u.v. light, both ligands reversibly bound to a single class of high-affinity receptors with a calcium-channel-typical pharmacological profile. [N-methyl-3H]LU49888 bound to the extent of 849 +/- 188 fmol/mg of protein (mean +/- S.D., n = 3) with a dissociation constant (Kd) of 1.4 +/- 0.3 nM. Under identical assay conditions (-)-[3H]azidopine labelled to the extent of 562 +/- 132 fmol/mg of protein with a Kd of 0.096 +/- 0.024 nM. After u.v. irradiation of the [N-methyl-3H]LU49888- and (-)-[3H]azidopine-labelled membranes, both photo-affinity probes were found to be incorporated specifically into a 190-195 kDa band as shown by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE). Photoincorporation occurred with a protection profile identical with that produced by reversible binding-inhibition. [N-methyl-3H]LU49888, but not (-)-[3H]-azidopine, specifically labelled an additional 265 kDa band. Both photolabelled bands had an identical electrophoretic mobility on SDS/PAGE, irrespective of pretreatment either with 10 mM-N-ethylmaleimide or 10 mM-dithiothreitol. The electrophoretic properties of the 195 kDa polypeptide and the lability of receptor-incorporated (-)-[3H]azidopine to nucleophilic agents resemble those of the previously described drug-receptor-carrying alpha 1 subunit of the purified skeletal-muscle calcium channel. The data suggest that this polypeptide carries both the high-affinity 1,4-dihydropyridine as well as the phenylalkylamine receptor of neuronal calcium channels in guinea-pig hippocampus and is a component of the L-type calcium channel.
本研究鉴定了豚鼠海马体膜中钙通道携带钙拮抗剂受体的多肽。芳基叠氮化物配体（-）-[³H]叠氮平与[N-甲基-³H]LU49888 [（-）-5-[(3-叠氮苯乙基)[N-甲基-³H]甲氨基]-2-(3,4,5-三甲氧基苯基)-2-异丙基戊腈]分别用于选择性标记1,4-二氢吡啶和苯烷基胺受体。在无紫外光的情况下，两种配体均以典型的钙通道药理学特征可逆地结合到一类高亲和力受体上。[N-甲基-³H]LU49888以849±188 fmol/mg蛋白质的量结合（平均值±标准差，n = 3），解离常数（Kd）为1.4±0.3 nM。在相同的测定条件下，（-）-[³H]叠氮平以562±132 fmol/mg蛋白质的量标记，Kd为0.096±0.024 nM。对用[N-甲基-³H]LU49888和（-）-[³H]叠氮平标记的膜进行紫外照射后，如十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）所示，两种光亲和探针均被特异性地掺入到一条190 - 195 kDa的条带中。光掺入的保护模式与可逆结合抑制产生的模式相同。[N-甲基-³H]LU49888而非（-）-[³H]叠氮平特异性地标记了一条额外的265 kDa条带。两条光标记条带在SDS/PAGE上具有相同的电泳迁移率，无论用10 mM - N - 乙基马来酰亚胺还是10 mM - 二硫苏糖醇进行预处理。195 kDa多肽的电泳特性以及受体掺入的（-）-[³H]叠氮平对亲核试剂的不稳定性类似于先前描述的纯化骨骼肌钙通道携带药物受体的α1亚基。数据表明，该多肽携带豚鼠海马体中神经元钙通道的高亲和力1,4 - 二氢吡啶以及苯烷基胺受体，并且是L型钙通道的一个组成部分。
