Loo D D, Brown P D, Wright E M
Department of Physiology, UCLA School of Medicine 90024-1751.
J Membr Biol. 1988 Nov;105(3):221-31. doi: 10.1007/BF01870999.
The tight-seal whole-cell recording method has been used to study Necturus choroid plexus epithelium. A cell potential of -59 +/- 2 mV and a whole cell resistance of 56 +/- 6 M omega were measured using this technique. Application of depolarizing step potentials activated voltage-dependent outward currents that developed with time. For example, when the cell was bathed in 110 mM NaCl Ringer solution and the interior of the cell contained a solution of 110 mM KCl and 5 nM Ca2+, stepping the membrane potential from a holding value of -50 to -10 mV evoked outward currents which, after a delay of greater than 50 msec, increased to a steady state in 500 msec. The voltage dependence of the delayed currents suggests that they may be currents through Ca2+-activated K+ channels. Based on the voltage dependence of the activation of Ca2+-activated K+ channels, we have devised a general method to isolate the delayed currents. The delayed currents were highly selective for K+ as their reversal potential at different K+ concentration gradients followed the Nernst potential for K+. These currents were reduced by the addition of TEA+ to the bath solution and were eliminated when Cs+ or Na+ replaced intracellular K+. Increasing the membrane potential to more positive values decreased both the delay and the half-times (t1/2) to the steady value. Increasing the pipette Ca2+ also decreased the delay and decreased t1/2. For instance, when pipette Ca2+ was increased from 5 to 500 nM, the delay and t1/2 decreased from values greater than 50 and 150 msec to values less than 10 and 50 msec. We conclude that the delayed currents are K+ currents through Ca2+-activated K+ channels. At the resting membrane potential of -60 mV, Ca2+-activated K+ channels contribute between 13 to 25% of the total conductance of the cell. The contribution of these channels to cell conductance nearly doubles with membrane depolarization of 20-30 mV. Such depolarizations have been observed when cerebrospinal fluid (CSF) secretion is stimulated by cAMP and with intracellular Ca2+. Thus the Ca2+-activated K+ channels may play a specific role in maintaining intracellular K+ concentrations during CSF secretion.
紧密封接全细胞记录方法已被用于研究美西螈脉络丛上皮细胞。使用该技术测量到细胞电位为-59±2 mV，全细胞电阻为56±6 MΩ。施加去极化阶跃电位可激活随时间发展的电压依赖性外向电流。例如，当细胞浸浴在110 mM NaCl林格溶液中且细胞内含有110 mM KCl和5 nM Ca2+的溶液时，将膜电位从-50 mV的保持值阶跃到-10 mV会诱发外向电流，在延迟大于50毫秒后，该电流在500毫秒内增加到稳态。延迟电流的电压依赖性表明它们可能是通过Ca2+激活的K+通道的电流。基于Ca2+激活的K+通道激活的电压依赖性，我们设计了一种分离延迟电流的通用方法。延迟电流对K+具有高度选择性，因为它们在不同K+浓度梯度下的反转电位遵循K+的能斯特电位。向浴液中添加TEA+可使这些电流减小，而当Cs+或Na+取代细胞内K+时，电流则被消除。将膜电位增加到更正的值会同时减少延迟和达到稳态值的半衰期（t1/2）。增加移液管中的Ca2+也会减少延迟并降低t1/2。例如，当移液管中的Ca2+从5 nM增加到500 nM时，延迟和t1/2从大于50毫秒和150毫秒的值降低到小于10毫秒和50毫秒的值。我们得出结论，延迟电流是通过Ca2+激活的K+通道的K+电流。在-60 mV的静息膜电位下，Ca2+激活的K+通道对细胞总电导的贡献在13%至25%之间。随着膜去极化20 - 30 mV，这些通道对细胞电导的贡献几乎翻倍。当脑脊液（CSF）分泌受到cAMP和细胞内Ca2+刺激时，已观察到这种去极化。因此，Ca2+激活的K+通道可能在脑脊液分泌过程中维持细胞内K+浓度方面发挥特定作用。
