Physiological Laboratory, University of Cambridge, Downing Street, Cambridge, CB2 3EG, United Kingdom.
Faculty of Health and Medical Sciences, University of Surrey, GU2 7AL, Guildford, Surrey, United Kingdom.
Sci Rep. 2019 Feb 13;9(1):1927. doi: 10.1038/s41598-018-36386-0.
We investigated effects of pharmacological triggering of exchange protein directly activated by cyclic-3',5'-adenosine monophosphate (Epac) on Nav1.4 currents from intact murine (C67BL6) skeletal muscle fibres for the first time. This employed a loose patch clamp technique which examined ionic currents in response to superimposed 10-ms V steps to varying degrees of depolarisation, followed by V steps to a fixed, +100 mV depolarisation relative to resting membrane potential following 40 mV hyperpolarising prepulses of 50 ms duration. The activation and inactivation properties of the resulting Na membrane current densities revealed reduced maximum currents and steepnesses in their voltage dependences after addition of the Epac activator 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (1 µM) to the bathing Krebs-Henseleit solutions. Contrastingly, voltages at half-maximal current and timecourses of currents obtained in response to the V depolarising steps were unchanged. These effects were abolished by further addition of the RyR-inhibitor dantrolene (10 µM). In contrast, challenge by dantrolene alone left both currents and their parameters intact. These effects of Epac activation in inhibiting skeletal muscle, Nav1.4, currents, complement similar effects previously reported in the homologous Nav1.5 in murine cardiomyocytes. They are discussed in terms of a hypothesis implicating Epac actions in increasing RyR-mediated SR Ca release resulting in a Ca-mediated inhibition of Nav1.4. The latter effect may form the basis for Ca-dependent Na channel dysregulation in SCN4A channelopathies associated with cold- and K-aggravated myotonias.
我们首次研究了环磷酸腺苷（cAMP）直接激活交换蛋白（Epac）的药理学触发对完整的鼠（C67BL6）骨骼肌纤维中 Nav1.4 电流的影响。采用宽松的膜片钳技术，检测在不同程度去极化时，叠加的 10ms V 步骤下的离子电流，然后在 40mV 超极化预脉冲后，在相对于静息膜电位的+100mV 固定去极化 V 步骤下，检测 Na 膜电流密度的激活和失活特性。加入 Epac 激活剂 8-（4-氯苯基硫代）腺苷-3'，5'-环单磷酸（1μM）后，所得 Na 膜电流密度的最大电流和电压依赖性的陡度降低。相反，加药后 V 去极化步骤引起的半最大电流和电流时程不变。这些作用被 RyR 抑制剂丹曲林（10μM）进一步加入所消除。相反，丹曲林单独作用不会改变两种电流及其参数。这些 Epac 激活抑制骨骼肌 Nav1.4 电流的作用，补充了先前在鼠心肌细胞中同源 Nav1.5 中报道的类似作用。它们根据 Epac 作用增加 RyR 介导的 SR Ca 释放从而导致 Ca 介导的 Nav1.4 抑制的假说进行了讨论。后者可能是与冷和 K 加重性肌强直相关的 SCN4A 通道病中 Ca 依赖性 Na 通道失调的基础。
