Aghdasi B, Zhang J Z, Wu Y, Reid M B, Hamilton S L
Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030, USA.
J Biol Chem. 1997 Feb 7;272(6):3739-48. doi: 10.1074/jbc.272.6.3739.
Two sulfhydryl reagents, N-ethylmaleimide (NEM), an alkylating agent, and diamide, an oxidizing agent, were examined for effects on the skeletal muscle Ca2+ release channel. NEM incubated with the channel for increasing periods of time displays three distinct phases in its functional effects on the channel reconstituted into planar lipid bilayers; first it inhibits, then it activates, and finally it again inhibits channel activity. NEM also shows a three-phase effect on the binding of [3H]ryanodine by first decreasing binding (phase 1), followed by a recovery of the binding (phase 2), and then a final phase of inhibition (phase 3). In contrast, diamide 1) activates the channel, 2) enhances [3H]ryanodine binding, 3) cross-links subunits within the Ca2+ release channel tetramer, and 4) protects against phase 1 inhibition by NEM. All diamide effects can be reversed by the reducing agent, dithiothreitol. Diamide induces intersubunit dimer formation of both the full-length 565-kDa subunit of the channel and the 400-kDa generated by endogenous calpain digestion, suggesting that the cross-link does not involve sulfhydryls within the N-terminal 170-kDa fragment of the protein. NEM under phase 1 conditions blocks the formation of the intersubunit cross-links by diamide. In addition, single channels activated by diamide are further activated by the addition of NEM. Diamide either cross-links phase 1 sulfhydryls or causes a conformational change in the Ca2+ release channel which leads to inaccessibility of phase 1 sulfhydryls to NEM alkylation. The data presented here lay the groundwork for mapping the location of one of the sites of subunit-subunit contact in the Ca2+ release channel tetramer and for identifying the functionally important sulfhydryls of this protein.
研究了两种巯基试剂对骨骼肌Ca2+释放通道的影响，一种是烷基化剂N-乙基马来酰亚胺（NEM），另一种是氧化剂二酰胺。NEM与通道孵育不同时间后，对重构于平面脂质双分子层中的通道功能产生三个不同阶段的影响：首先是抑制，然后是激活，最后再次抑制通道活性。NEM对[3H]ryanodine的结合也呈现三相效应，先是结合减少（第1阶段），接着结合恢复（第2阶段），最后是抑制的最终阶段（第3阶段）。相比之下，二酰胺：1）激活通道；2）增强[3H]ryanodine结合；3）使Ca2+释放通道四聚体内的亚基交联；4）防止NEM的第1阶段抑制作用。二酰胺的所有效应都可被还原剂二硫苏糖醇逆转。二酰胺诱导通道全长565-kDa亚基以及内源性钙蛋白酶消化产生的400-kDa亚基形成亚基间二聚体，这表明交联不涉及该蛋白N端170-kDa片段内的巯基。在第1阶段条件下，NEM可阻止二酰胺形成亚基间交联。此外，二酰胺激活的单通道在加入NEM后会进一步激活。二酰胺要么交联第1阶段的巯基，要么导致Ca2+释放通道构象改变，从而使第1阶段的巯基无法被NEM烷基化。本文提供的数据为绘制Ca2+释放通道四聚体中亚基-亚基接触位点之一的位置以及鉴定该蛋白功能上重要的巯基奠定了基础。
