Department of Physiology, David Geffen School of Medicine, UCLA, 10833 Le Conte Avenue, Los Angeles, CA 90095-1751, USA.
J Physiol. 2011 Mar 15;589(Pt 6):1421-42. doi: 10.1113/jphysiol.2010.202804. Epub 2011 Jan 24.
We investigated the effects of the overexpression of two enhanced green fluorescent protein (EGFP)-tagged α1sDHPR variants on Ca2+ currents (ICa), charge movements (Q) and SR Ca2+ release of muscle fibres isolated from adult mice. Flexor digitorum brevis (FDB)muscles were transfected by in vivo electroporation with plasmids encoding for EGFP-α1sDHPR-wt and EGFP-α1sDHPR-T935Y (an isradipine-insensitive mutant). Two-photon laser scanning microscopy (TPLSM) was used to study the subcellular localization of transgenic proteins, while ICa, Q and Ca2+ release were studied electrophysiologically and optically under voltage-clamp conditions. TPLSM images demonstrated that most of the transgenic α1sDHPR was correctly targeted to the transverse tubular system (TTS). Immunoblotting analysis of crude extracts of transfected fibres demonstrated the synthesis of bona fide transgenic EGFP-α1sDHPR-wt in quantities comparable to that of native α1sDHPR. Though expression of both transgenic variants of the alpha subunit of the dihydropyridine receptor (α1sDHPR) resulted in ∼50% increase in Q, they surprisingly had no effect on the maximal Ca2+ conductance (gCa) nor the SR Ca2+ release. Nonetheless, fibres expressing EGFP-α1sDHPR-T935Y exhibited up to 70% isradipine-insensitive ICa (ICa-ins) with a right-shifted voltage dependence compared to that in control fibres. Interestingly, Qand SRCa2+ release also displayed right-shifted voltage dependence in fibres expressing EGFP-α1sDHPR-T935Y. In contrast, the midpoints of the voltage dependence of gCa, Q and Ca2+ release were not different from those in control fibres and in fibres expressing EGFP-α1sDHPR-wt. Overall, our results suggest that transgenic α1sDHPRs are correctly trafficked and inserted in the TTS membrane, and that a substantial fraction of the mworks as conductive Ca2+ channels capable of physiologically controlling the release of Ca2+ from the SR. A plausible corollary of this work is that the expression of transgenic variants of the α1sDHPR leads to the replacement of native channels interacting with the ryanodine receptor 1 (RyR1), thus demonstrating the feasibility of molecular remodelling of the triads in adult skeletal muscle fibres.
我们研究了两种增强型绿色荧光蛋白（EGFP）标记的α1sDHPR 变体过表达对成年小鼠分离的肌纤维钙电流（ICa）、电荷运动（Q）和 SR Ca2+释放的影响。通过体内电穿孔将编码 EGFP-α1sDHPR-wt 和 EGFP-α1sDHPR-T935Y（一种异搏定不敏感的突变体）的质粒转染屈趾短肌（FDB）。利用双光子激光扫描显微镜（TPLSM）研究转基因蛋白的亚细胞定位，同时在电压钳条件下用电生理学和光学方法研究 ICa、Q 和 Ca2+释放。TPLSM 图像表明，大多数转基因α1sDHPR 被正确靶向到横管系统（TTS）。转染纤维的粗提物的免疫印迹分析表明，合成了与天然α1sDHPR 相当数量的真正的转基因 EGFP-α1sDHPR-wt。尽管两种二氢吡啶受体（α1sDHPR）的转基因变体的表达都导致 Q 增加约 50%，但它们对最大钙电导（gCa）和 SR Ca2+释放没有影响。尽管如此，表达 EGFP-α1sDHPR-T935Y 的纤维表现出高达 70%的异搏定不敏感的 ICa（ICa-ins），与对照纤维相比，其电压依赖性向右移位。有趣的是，在表达 EGFP-α1sDHPR-T935Y 的纤维中，Q 和 SR Ca2+释放也显示出向右移位的电压依赖性。相比之下，gCa、Q 和 Ca2+释放的电压依赖性中点与对照纤维和表达 EGFP-α1sDHPR-wt 的纤维没有不同。总的来说，我们的结果表明，转基因α1sDHPR 被正确运输并插入到 TTS 膜中，并且相当大的一部分 m 作为导电 Ca2+通道发挥作用，能够在生理上控制 SR 中 Ca2+的释放。这项工作的一个可能推论是，转基因α1sDHPR 变体的表达导致与肌质网钙释放通道 1（RyR1）相互作用的天然通道的替换，从而证明了成年骨骼肌纤维三联体的分子重塑的可行性。
