Littlechild Robert, Zaidman Nathalie, Khodaverdi Darren, Mason Michael James
Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, CB2 3EG Cambridge, UK.
Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, CB2 3EG Cambridge, UK.
Cell Calcium. 2015 Feb;57(2):76-88. doi: 10.1016/j.ceca.2014.12.009. Epub 2014 Dec 23.
In the present experiments in HEL cells, we have investigated the requirement for a hyperpolarised resting membrane potential for the initial activation of the Ca(2+) activated K(+) channel, KCa3.1, following activation of the Ca(2+) release activated Ca(2+) (CRAC) entry pathway. In intact cells, fluorimetric measurements of [Ca(2+)]i following thapsigargin-mediated activation of CRAC entry revealed a sustained increase in [Ca(2+)]i. Block of KCa3.1 by application of charybdotoxin resulted in a 50% reduction in the steady-state [Ca(2+)]i, consistent with the well established role for KCa3.1-mediated hyperpolarisation in augmenting CRAC entry. Interestingly, subsequent depolarisation to 0mV by application of gramicidin resulted in a fall in steady-state Ca(2+) levels to values theoretically below that required for activation of KCa3.1. Whole cell patch clamp experiments confirmed the lack of KCa3.1 activation at 0mV following activation of the CRAC entry pathway, indicating an absolute requirement for a hyperpolarised resting membrane potential for the initial activation of KCa3.1 leading to hyperpolarisation and augmented Ca(2+) entry. Current clamp experiments confirmed the requirement for a hyperpolarised resting membrane potential in KCa3.1 activation by CRAC entry. Given the critical role played by KCa3.1 and membrane potential in general in the control of CRAC-mediated [Ca(2+)]i changes, we investigated the hypothesis that inhibition of the CRAC-mediated changes in [Ca(2+)]i observed following 2-APB addition may in part arise from direct inhibition of KCa3.1 by 2-APB. Under whole cell patch clamp, 2-APB, at concentrations typically used to block the CRAC channel, potently inhibited KCa3.1 in a reversible manner (half maximal inhibition 14.2 μM). This block was accompanied by a marked shift in the reversal potential to depolarised values approaching that set by endogenous membrane conductances. At the single channel level, 2-APB applied to the cytosolic face resulted in a significant reduction in open channel probability and a fall in the mean open time of the residual channel activity. Our data highlight the absolute requirement for a hyperpolarising resting membrane conductance for the initial activation of KCa3.1 by CRAC entry. Additionally, our results document direct inhibition of KCa3.1 by 2-APB, thus highlighting the need for caution when ascribing the site of inhibition of 2-APB exclusively to the CRAC entry pathway in experiments where membrane potential is not controlled.
在目前针对人胚肺成纤维细胞（HEL细胞）的实验中，我们研究了在钙释放激活钙（CRAC）内流途径激活后，钙激活钾通道KCa3.1的初始激活对超极化静息膜电位的需求。在完整细胞中，毒胡萝卜素介导激活CRAC内流后，通过荧光法测量细胞内钙离子浓度（[Ca(2+)]i）显示[Ca(2+)]i持续升高。应用卡律霉素阻断KCa3.1导致稳态[Ca(2+)]i降低50%，这与KCa3.1介导的超极化在增强CRAC内流中已确立的作用一致。有趣的是，随后应用短杆菌肽将膜电位去极化至0mV导致稳态钙离子水平下降至理论上低于激活KCa3.1所需的值。全细胞膜片钳实验证实，激活CRAC内流途径后，在0mV时KCa3.1未被激活，这表明KCa3.1的初始激活导致超极化和增强的钙内流绝对需要超极化的静息膜电位。电流钳实验证实了CRAC内流激活KCa3.1时对超极化静息膜电位的需求。鉴于KCa3.1和膜电位在控制CRAC介导的[Ca(2+)]i变化中通常发挥的关键作用，我们研究了以下假设：添加2-氨基乙氧基二苯硼酸（2-APB）后观察到的CRAC介导的[Ca(2+)]i变化受到抑制，可能部分源于2-APB对KCa3.1的直接抑制。在全细胞膜片钳实验中，2-APB以通常用于阻断CRAC通道的浓度，以可逆方式有效抑制KCa3.1（半数最大抑制浓度为14.2μM）。这种阻断伴随着反转电位显著向接近内源性膜电导设定值的去极化值偏移。在单通道水平，将2-APB应用于胞质面导致开放通道概率显著降低，剩余通道活性的平均开放时间缩短。我们的数据突出了CRAC内流初始激活KCa3.1时对超极化静息膜电导的绝对需求。此外，我们的结果记录了2-APB对KCa3.1的直接抑制，因此强调了在膜电位未得到控制的实验中，将2-APB的抑制位点仅归因于CRAC内流途径时需要谨慎。
