Insertion of alpha1S II-III loop and C terminal sequences into alpha1H fails to restore excitation-contraction coupling in dysgenic myotubes.
Insertion of alpha1S II-III loop and C terminal sequences into alpha1H fails to restore excitation-contraction coupling in dysgenic myotubes.
复制标题
将 alpha1S II-III 环和 C 末端序列插入 alpha1H 无法恢复发育不良肌管中的兴奋-收缩耦合。
DOI：
10.1023/a:1024830132118
复制
发表时间：
2003
影响因子：
2.7
通讯作者：
Beam,KurtG
中科院分区：
文献类型：
--
作者：
Wilkens,ChristinaM;Beam,KurtG
骨骼肌 (α1S) 中的 L 型 Ca2+ 通道对于兴奋-收缩 (EC) 耦合至关重要。先前的研究使用由 α1Stogether 与 α1Cor α1M 组成的嵌合体证明了 α1SII-III 环和较小的子结构域（残基 720-764；“ECC”）在骨骼 EC 耦合中的重要性。然而，这些嵌合体未能测试 II-III 环之外区域的重要性，这些区域在 α1S 和 α1C 之间高度保守。因此，我们将编码α1S和高度分化的T型Ca2+通道α1H之间的嵌合体的cDNA注射到发育不良（缺乏α1S）肌管中。嵌合体由 GFP 标记的 α1H 组成，具有以下一种或多种取代：α1SII-III 环残基 720-764（“ECC”）、α1SC 末端的推定靶向结构域（“目标”；残基 1543-1662）或整个 α1SC 末端（“Cterm”；残基 1382-1873）。靶标或 Cterm 的存在影响从发育不良的肌肉细胞和 tsa-201 细胞记录的全细胞电流的表达和/或动力学。重要的是，单独将 ECC 替换为 GFP-α1H (GFP-α1H+ ECC)，或与目标 (GFP-α1H+ ECC + 目标) 或 Cterm (GFP-α1H+ ECC + Cterm ) 一起替换，不足以恢复电诱发收缩。 GFP-α1H+ ECC、GFP-α1H+ ECC + 靶标或 GFP-α1H+ ECC + Cterm 的去极化诱导的荧光瞬态对膜电压具有钟形依赖性（与骨骼 EC 耦合不一致），并且也非常小（与心脏 EC 耦合不同）。这些嵌合体缺乏 EC 偶联，这增加了 ECC 和靶标之外的 α1S 区域对于提供允许这两个结构域分别在 EC 偶联和靶向中发挥作用的背景所必需的可能性。此外，嵌合体的膜密度不足可能导致偶联的缺乏。
The L-type Ca2+channel in skeletal muscle (α1S) is essential for excitation–contraction (EC) coupling. Previous studies using chimeras composed of α1Stogether with α1Cor α1Mdemonstrated the importance of the α1SII–III loop and of a smaller subdomain (residues 720–764; ‘ECC’) in skeletal EC coupling. However, these chimeras failed to test the significance of regions outside the II–III loop, which are highly conserved between α1Sand α1C. Therefore, we have injected dysgenic (α1S-lacking) myotubes with cDNAs encoding chimeras between α1Sand the highly divergent T-type Ca2+channel, α1H. The chimeras consisted of GFP-tagged α1Hwith one or more of the following substitutions: α1SII–III loop residues 720–764 (‘ECC’), a putative targeting domain of the α1SC terminus (‘target’; residues 1543–1662) or the entire α1SC terminus (‘Cterm’; residues 1382–1873). The presence of eithertargetorCtermaffected the expression and/or kinetics of whole-cell currents recorded from both dysgenic muscle cells and tsa-201 cells. Importantly, substitution ofECCalone into GFP-α1H(GFP-α1H+ ECC), or together with eithertarget(GFP-α1H+ ECC + target) orCterm(GFP-α1H+ ECC + Cterm ), was insufficient to restore electrically evoked contractions. Depolarization-induced fluorescence transients for GFP-α1H+ ECC, GFP-α1H+ ECC + target or GFP-α1H+ ECC + Cterm had a bell shaped dependence upon membrane voltage (inconsistent with skeletal EC coupling) and were also exceedingly small (unlike cardiac EC coupling). The absence of EC coupling for these chimeras raises the possibility that regions of α1Soutside ofECCandtargetare necessary for providing the context that allows these two domains to function in EC coupling and targeting, respectively. Additionally, an inadequate membrane density of the chimeras may have contributed to the lack of coupling.