Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model
Divalent cations permeation in a Ca2+ non-conducting skeletal muscle dihydropyridine receptor mouse model
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DOI：
10.1016/j.ceca.2020.102256
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发表时间：
2020-11-01
期刊：
影响因子：
4
通讯作者：
Allard, Bruno
中科院分区：
文献类型：
--
作者：
Idoux, Romane;Fuster, Clarisse;Allard, Bruno
作为对骨骼肌纤维兴奋的响应，动作电位序列诱导锚定在管状膜上的二氢吡啶受体（DHPR）结构的改变，从而打开肌浆网膜上的Ca2+释放通道。DHPR还作为电压门控Ca2+通道，传导哺乳动物肌肉纤维中常规记录的l型Ca2+电流，其作用已经争论了40多年。最近，为了更深入地研究DHPR Ca2+内流在哺乳动物肌肉中的作用，在DHPRa1S亚基中产生了一种携带突变N617D（邻近区域II选择性过滤器E）的敲入（ki）小鼠模型（ncDHPR），该突变消除了Ca2+通过通道的渗透[Dayal等，2017]。在本研究中，Mn2+猝灭技术最初打算用于来自该小鼠的电压夹紧肌肉纤维，以确定Ca2+内流是否通过与DHPR不同的途径发生，以补偿DHPR Ca2+内流的缺失。令人惊讶的是，虽然ki小鼠的N617D DHPR肌纤维不传导Ca2+，但Mn2+的进入和随后的猝灭确实发生了，因为Mn2+能够通过N617D DHPR渗透并产生l型电流。N617D DHPR也被发现传导Ba2+， Ba2+电流被外部Ca2+强烈阻断。与野生型DHPR相比，Ba2+渗透更小，电流动力学更慢，Ca2+阻断更有效。这些结果表明，当残基N617被带负电荷的残基D取代时，残基N617合适地位于孔入口处，以捕获外部Ca2+，从而阻碍了这种渗透方式。由于Ba2+与D的结合亲和力较低，因此会产生Ba2+电流，但与通过野生型通道的Ba2+电流相比，其振幅减小。我们得出结论，在完全分化的骨骼肌环境中，位于选择性过滤器外的突变影响通道渗透和可能的通道门控。
In response to excitation of skeletal muscle fibers, trains of action potentials induce changes in the configuration of the dihydropyridine receptor (DHPR) anchored in the tubular membrane which opens the Ca2+ release channel in the sarcoplasmic reticulum membrane. The DHPR also functions as a voltage-gated Ca2+ channel that conducts L-type Ca2+ currents routinely recorded in mammalian muscle fibers, which role was debated for more than four decades. Recently, to allow a closer look into the role of DHPR Ca2+ influx in mammalian muscle, a knock-in (ki) mouse model (ncDHPR) carrying mutation N617D (adjacent to domain II selectivity filter E) in the DHPRa1S subunit abolishing Ca2+ permeation through the channel was generated [Dayal et al., 2017]. In the present study, the Mn2+ quenching technique was initially intended to be used on voltage-clamped muscle fibers from this mouse to determine whether Ca2+ influx through a pathway distinct from DHPR may occur to compensate for the absence of DHPR Ca2+ influx. Surprisingly, while N617D DHPR muscle fibers of the ki mouse do not conduct Ca2+, Mn2+ entry and subsequent quenching did occur because Mn2+ was able to permeate and produce L-type currents through N617D DHPR. N617D DHPR was also found to conduct Ba2+ and Ba2+ currents were strongly blocked by external Ca2+. Ba2+ permeation was smaller, current kinetics slower and Ca2+ block more potent than in wild-type DHPR. These results indicate that residue N617 when replaced by the negatively charged residue D is suitably located at entrance of the pore to trap external Ca2+ impeding in this way permeation. Because Ba2+ binds with lower affinity to D, Ba2+ currents occur, but with reduced amplitudes as compared to Ba2+ currents through wild-type channels. We conclude that mutations located outside the selectivity filter influence channel permeation and possibly channel gating in a fully differentiated skeletal muscle environment.