DelPrincipe F, Egger M, Niggli E
Department of Physiology, University of Bern, Buhlplatz 5, CH-3012 Bern, Switzerland.
J Physiol. 2000 Sep 15;527 Pt 3(Pt 3):455-66. doi: 10.1111/j.1469-7793.2000.00455.x.
In the present study Ca2+ entry via different voltage-dependent membrane channels was examined with a fluorescent Ca2+ indicator before and after beta-adrenergic stimulation. To clearly distinguish between Ca2+ influx and Ca2+ release from the sarcoplasmic reticulum the Ca2+ store was blocked with 0.1 microM thapsigargin and 10 microM ryanodine. Omitting Na+ from the pipette filling solution minimized Ca2+ entry via Na+-Ca2+ exchange. Individual guinea-pig ventricular myocytes were voltage clamped in the whole-cell configuration of the patch-clamp technique and different membrane currents were activated using specific voltage protocols. The intracellular Ca2+ concentration was simultaneously recorded with a laser-scanning confocal microscope using fluo-3 as a Ca2+ indicator. Ca2+ entry pathways were discriminated using pharmacological blockers under control conditions and during beta-adrenergic stimulation with 1 microM isoproterenol (isoprenaline) in the bathing solution or 100 microM cAMP in the patch-clamp pipette. Isoproterenol or cAMP potentiated the Ca2+ influx signals recorded during L-type Ca2+ current activation but, more interestingly, also during Na+ current (INa) activation. The Ca2+ influx signal arising from L-type Ca2+ current activation was usually blocked by 50 microM Cd2+. However, the Ca2+ influx signal elicited by the Na+ current activation protocol was only curtailed to 56.4 +/- 28.2 % by 100 microM Ni2+ but was reduced to 17.9 +/- 15.1 % by 50 microM Cd2+ and consistently eliminated by 5 mM Ni2+. The pronounced Cd2+ and moderate Ni2+ sensitivity of the Ca2+ influx signals suggested that the predominant source of Ca2+ influx during the Na+ current activation - before and during beta-adrenergic stimulation - was a spurious activation of the L-type Ca2+ current, presumably due to voltage escape during Na+ current activation. Calculations based on the relationship between Ca2+ current and fluorescence change revealed that, on average, we could reliably detect rapid Ca2+ concentration changes as small as 5.4 +/- 0.7 nM. Thus, we can estimate an upper limit for the Ca2+ permeability of the phosphorylated TTX-sensitive Na+ channels which is less than 0.04:1 for Ca2+ ions flowing through Na+ channels via the proposed 'slip-mode' Ca2+ conductance. Therefore the slip-mode Ca2+ conductance of Na+ channels does not contribute noticeably to the Ca2+ signals observed in our experiments.
在本研究中，在β - 肾上腺素能刺激前后，使用荧光Ca²⁺指示剂检测了通过不同电压依赖性膜通道的Ca²⁺内流。为了清楚区分Ca²⁺内流和肌浆网释放的Ca²⁺，用0.1微摩尔毒胡萝卜素和10微摩尔雷诺丁阻断Ca²⁺储存库。从移液管填充溶液中省略Na⁺可使通过Na⁺ - Ca²⁺交换的Ca²⁺内流最小化。将单个豚鼠心室肌细胞在膜片钳技术的全细胞配置下进行电压钳制，并使用特定的电压方案激活不同的膜电流。使用fluo - 3作为Ca²⁺指示剂，通过激光扫描共聚焦显微镜同时记录细胞内Ca²⁺浓度。在对照条件下以及在浴液中用1微摩尔异丙肾上腺素（异丙基肾上腺素）或膜片钳移液管中用100微摩尔环磷酸腺苷（cAMP）进行β - 肾上腺素能刺激期间，使用药理学阻滞剂区分Ca²⁺内流途径。异丙肾上腺素或cAMP增强了在L型Ca²⁺电流激活期间记录的Ca²⁺内流信号，但更有趣的是，在Na⁺电流（INa）激活期间也增强了。由L型Ca²⁺电流激活产生的Ca²⁺内流信号通常被50微摩尔Cd²⁺阻断。然而，由Na⁺电流激活方案引发的Ca²⁺内流信号仅被100微摩尔Ni²⁺减少到56.4±28.2%，但被50微摩尔Cd²⁺减少到17.9±15.1%，并被5毫摩尔Ni²⁺持续消除。Ca²⁺内流信号对Cd²⁺的明显敏感性和对Ni²⁺的中等敏感性表明，在Na⁺电流激活期间（在β - 肾上腺素能刺激之前和期间），Ca²⁺内流的主要来源是L型Ca²⁺电流的假性激活，可能是由于Na⁺电流激活期间的电压逃逸。基于Ca²⁺电流与荧光变化之间关系的计算表明，平均而言，我们能够可靠地检测到低至5.4±0.7纳摩尔的快速Ca²⁺浓度变化。因此，我们可以估计磷酸化的对河豚毒素敏感的Na⁺通道的Ca²⁺通透性上限，对于通过所提出的“滑模”Ca²⁺电导流经Na⁺通道的Ca²⁺离子，该上限小于0.04:1。因此，Na⁺通道的滑模Ca²⁺电导对我们实验中观察到的Ca²⁺信号没有明显贡献。
