el-Hayek R, Lokuta A J, Arévalo C, Valdivia H H
Department of Physiology, University of Wisconsin Medical School, Madison 53706, USA.
J Biol Chem. 1995 Dec 1;270(48):28696-704. doi: 10.1074/jbc.270.48.28696.
We have used [3H]ryanodine binding experiments and single channel recordings to provide convergent descriptions of the effect of imperatoxin A (IpTxa), a approximately 5-kDa peptide from the venom of the scorpion Pandinus imperator (Valdivia, H. H., Kirby, M. S., Lederer, W. J., and Coronado, R. (1992) Proc. Ntl. Acad. Sc. U.S.A. 89, 12185-12189) on Ca2+ release channels/ryanodine receptors (RyR) of sarcoplasmic reticulum (SR). At nanomolar concentrations, IpTxa increased the binding of [3H]ryanodine to skeletal SR and, to a lesser extent, to cerebellum microsomes. The activating effect of IpTxa on skeletal SR was Ca(2+)-dependent, synergized by caffeine, and independent of other modulators of RyRs. However, IpTxa had negligible effects on tissues where the expression of skeletal-type RyR isoforms (RyR1) is small or altogether absent, i.e. cardiac, cerebrum, and liver microsomes. Thus, IpTxa may be used as a ligand capable of discriminating between RyR isoforms with nanomolar affinity. IpTxa increased the open probability (Po) of rabbit skeletal muscle RyRs by increasing the frequency of open events and decreasing the duration of the closed lifetimes. This activating effect was dose-dependent (ED50 = 10 nM), had a fast onset, and was fully reversible. Purified RyR from solubilized skeletal SR displayed high affinity for [3H]ryanodine with a KD of 6.1 nM and Bmax of approximately 30 pmol/mg of protein. IpTxa increased [3H]ryanodine binding noncompetitively by increasing Bmax to approximately 60 pmol/mg of protein. These results suggested the presence of an IpTxa-binding site on the RyR or a closely associated regulatory protein. This site appears to be distinct from the caffeine- and adenine nucleotide-regulatory sites. IpTxa may prove a useful tool to identify regulatory domains critical for channel gating and to dissect the contribution of skeletal-type RyRs to intracellular Ca2+ waveforms generated by stimulation of different RyR isoforms.
我们利用[3H]ryanodine结合实验和单通道记录，对帝王毒素A（IpTxa）——一种来自帝王蝎毒液的约5 kDa肽（瓦尔迪维亚，H. H.，柯比，M. S.，莱德勒，W. J.，和科罗纳多，R.（1992年）《美国国家科学院院刊》89，12185 - 12189）对肌浆网（SR）的Ca2+释放通道/ryanodine受体（RyR）的作用进行了趋同描述。在纳摩尔浓度下，IpTxa增加了[3H]ryanodine与骨骼肌SR的结合，对小脑微粒体的结合增加程度较小。IpTxa对骨骼肌SR的激活作用依赖于Ca(2+)，与咖啡因协同作用，且独立于RyRs的其他调节剂。然而，IpTxa对骨骼肌型RyR亚型（RyR1）表达较少或完全缺失的组织，即心脏、大脑和肝脏微粒体，影响可忽略不计。因此，IpTxa可作为一种能够以纳摩尔亲和力区分RyR亚型的配体。IpTxa通过增加开放事件的频率和缩短关闭寿命的持续时间，增加了兔骨骼肌RyRs的开放概率（Po）。这种激活作用呈剂量依赖性（ED50 = 10 nM），起效快，且完全可逆。从溶解的骨骼肌SR中纯化的RyR对[3H]ryanodine具有高亲和力，KD为6.1 nM，Bmax约为30 pmol/mg蛋白质。IpTxa通过将Bmax增加到约60 pmol/mg蛋白质，非竞争性地增加了[3H]ryanodine结合。这些结果表明在RyR或紧密相关的调节蛋白上存在一个IpTxa结合位点。该位点似乎与咖啡因和腺嘌呤核苷酸调节位点不同。IpTxa可能被证明是一种有用的工具，用于识别对通道门控至关重要的调节域，并剖析骨骼肌型RyRs对由不同RyR亚型刺激产生的细胞内Ca2+波形的贡献。
