Identification and characterization of major ionic currents in isolated smooth muscle cells using the voltage-clamp technique.
Identification and characterization of major ionic currents in isolated smooth muscle cells using the voltage-clamp technique.
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使用电压钳技术识别和表征分离的平滑肌细胞中的主要离子电流。
DOI：
10.1007/bf00581336
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发表时间：
1987
期刊：
影响因子：
--
通讯作者：
Singer,JJ
中科院分区：
文献类型：
--
作者：
WalshJr,JV;Singer,JJ
在新鲜分离的中华大蟾胃肌单细胞上进行了电压钳实验。使用传统的双微电极方法，从而避免了胞浆的快速透析。当电压从负保持水平跳到更正的水平时，确定了四个主要的电流阶段。电流的第一阶段是初始的内向电流。这一电流被外部Mn2+阻断，并且具有正确的幅度，可以解释在这些细胞中发现的钙依赖的、TTX非依赖的动作电位的上升阶段。在这个初始的内向钙电流之后，观察到了一个大的外向电流，它在数百毫秒的时间内达到峰值，然后在几秒钟的时间内衰减到稳定水平。峰值外向电流和稳态外向电流构成第二和第三大电流。峰值外向电流是观察到的最大电流，其大小可达数十纳安，而内向电流最多约为1纳安。在外加TEA的存在下，最大外向电流减小了10倍以上。当使用外部Mn2+或更小的负保持电位来减小或消除先前的内向电流时，它也被减小或消除。通过这种方式，峰值外向电流被认为是一种钙激活的K+电流，其缓慢衰减被认为是由于细胞机制在内向电流导致[Ca~(2+)]i最初上升后，通过细胞机制清除了内部钙离子。第四个主要电流是早期的瞬时外向电流，当用较少的负保持电位或外部Mn2+消除内向电流时，电压跃升到更正的电位时最明显。对于内向电流，建立了经典的稳态失活关系，它是膜电位的函数。这种失活曲线的很大一部分位于内向电流激活的表观阈值的负电位，这表明真正的电压依赖失活。尽管不能排除其他依赖钙离子的失活，也没有找到证据证明这一点。
Voltage-clamp experiments were carried out on freshly dissociated single vertebrate smooth muscle cells from the stomach muscularis ofBufo marinus. Conventional two-microelectrode methodology was used, thus avoiding rapid dialysis of the cytosol. Four major phases of current were identified upon voltage jumps from negative holding levels to more positive levels. The first phase of current was an initial, inward current. This current was blocked by external Mn2+and was of the correct magnitude to account for the rising phase of the Ca2+-dependent, TTX-independent action potentials found in these cells. Following this initial, inward Ca2+current, a large outward current was observed which reached its peak over a period of hundreds of milliseconds and then decayed over a period of seconds to a steady-state level. The peak outward current and the steady-state outward current constitute the second and third major currents. The peak outward current was the largest current observed, with a magnitude as large as tens of nanoamps whereas the inward current was at most about one nanoamp. The peak outward current was reduced more than tenfold in the presence of external TEA. It was also decreased or abolished when the preceding inward current was diminished or eliminated by using external Mn2+or less negative holding potentials. In this way the peak outward current was identified as a Ca2+-activated K+current whose slow decay was hypothesized to result from removal of internal Ca ions by cellular mechanisms following the initial rise in [Ca2+]iresulting from the inward current. A fourth major current was an early transient outward current observed most clearly upon voltage jumps to more positive potentials when the inward current was eliminated by using less negative holding potentials or external Mn2+. A classical steady-state inactivation relationship as a function of membrane potential was constructed for the inward current. A substantial portion of this inactivation curve lies at potentials negative to the apparent threshold for activation of inward current, suggesting a true voltage-dependent inactivation. Although additional Ca2+-dependent inactivation could not be ruled out, neither could evidence for it be found.