INACTIVATION OF THE VOLTAGE-DEPENDENT CA2+ CHANNEL CURRENT IN SMOOTH-MUSCLE CELLS ISOLATED FROM THE GUINEA-PIG DETRUSOR
INACTIVATION OF THE VOLTAGE-DEPENDENT CA2+ CHANNEL CURRENT IN SMOOTH-MUSCLE CELLS ISOLATED FROM THE GUINEA-PIG DETRUSOR
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DOI：
10.1113/jphysiol.1993.sp019893
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发表时间：
1993-11-01
影响因子：
5.5
通讯作者：
BRADING, AF
中科院分区：
文献类型：
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作者：
NAKAYAMA, S;BRADING, AF
1.采用全细胞电压钳技术对酶法分离的豚鼠膀胱平滑肌细胞进行电压钳实验。通过在较宽范围内调控细胞膜电位和改变细胞外二价阳离子浓度来检测电压依赖性钙通道电流的失活和恢复.将细胞暴露于调节电位（-100至+80 mV，以20 mV为增量）后，通过步进至0 mV测试电位估计灭活程度。在2.5 mm Ca2+存在下，电流的失活相对于条件电位呈U形，最大失活在0 mV。在0 - 8秒和5秒的调节持续时间后，最大失活分别为60%和90%。U形曲线是Ca2+依赖性失活的特征。当施加+80 mV的条件电位时，测试电位处的内向电流和随后返回保持电位时的尾电流均大于对照条件下（条件电位=保持电位时，-60 mV）.在2.5 mM Ba 2+存在下也观察到U形灭活曲线。失活是最大的约-20 mV的条件电位，失活是小于所看到的Ca2 + entry.4。成对脉冲协议被施加到检查的Ca2+内向电流的恢复的电压依赖性。在0 mV下100 ms去极化过程中，内向电流被灭活后，在-60 mV下需要700 ms才能几乎完全恢复电流。在+80 mV下也观察到恢复。当成对脉冲的电位增加到+20 mV时，当脉冲间电位为+80 mV时，观察到较少的恢复。当施加较长的去极化（3 s）时，内向电流的峰值幅度需要更长的时间才能恢复，并且在脉冲间电位的任一个下4 s后都没有完全恢复，尽管脉冲间电位为-60 mV时的恢复大于+80 mV时的恢复。在Ba 2 +.5存在下观察到类似的回收率。在一个长的去极化（8秒，0 mV），在细胞外溶液的快速变化的影响进行了检查。用Mg~（2+）替代Ca~（2+）一段时间后，内向电流部分恢复。在Ba 2+存在下未观察到这种恢复。在Ba~（2+）存在下诱发内向电流。Ca 2+取代Ba2+后，Ca 2+含量明显降低。在无Ca~（2+）溶液中，当Ca~（2+）在长去极化过程中重新进入时，产生的内向电流大于相应时间的对照电流，但小于对照电流的峰值。在长去极化过程中通过操纵二价阳离子对衰减时间过程的调制意味着Ca2+和电压依赖性失活机制分别发生。我们的研究结果表明，在Ca2+通道失活的主要机制是Ca2+依赖性失活，但也涉及电压依赖性机制，包括进入一个长期的，更缓慢的失活开放状态的Ca2+通道响应延长去极化。
1. Whole-cell voltage clamp techniques were applied to single smooth muscle cells enzymatically dissociated from guinea-pig urinary bladder. The inactivation and recovery of voltage-dependent Ca2+ channel currents were examined by manipulating the membrane potential over a wide range and by changing the extracellular divalent cation concentrations.2. After exposing the cells to conditioning potentials (- 100 to + 80 mV in 20 mV increments), the degree of inactivation was estimated by stepping to a 0 mV test potential. In the presence of 2.5 mm Ca2+, the inactivation of the current was U-shaped with respect to the conditioning potential, with maximum inactivation at 0 mV. The maximal inactivation was 60 and 90 % after conditioning durations of 0-8 and 5 s, respectively. The U-shaped curve is characteristic of Ca2+-dependent inactivation. When conditioning potentials of + 80 mV with either duration were applied, the inward current at the test potential and the subsequent tail current on returning to the holding potential were larger than in control conditions (when the conditioning potential = the holding potential, - 60 mV).3. A U-shaped inactivation curve was also observed in the presence of 2.5 mM Ba2+. The inactivation was maximal with a conditioning potential of about - 20 mV, and the inactivation was smaller than seen with Ca2+ entry.4. Paired-pulse protocols were applied to examine the voltage dependence of recovery of the Ca2+ inward current. After the inward current had been inactivated during a 100 ms depolarization at 0 mV, it took 700 ms at - 60 mV for nearly complete recovery of the current. Recovery was also observed at + 80 mV. When the potential of the paired pulses was increased to + 20 mV, less recovery was seen when the interpulse potential was at + 80 mV. When a longer (3 s) depolarization was applied, the peak amplitude of the inward current took much longer to recover, and had not completely recovered after 4 s at either of the interpulse potentials, although recovery was greater with an interpulse potential of - 60 mV than with one of + 80 mV. Similar recoveries were observed in the presence of Ba2+.5. During a long depolarization (8 s, 0 mV), the effects of rapid changes in the extracellular solution were examined. Partial recovery of the inward current occurred after a period in which Ca2+ Was replaced with Mg2+. This recovery was not observed in the presence of Ba2+. The inward current evoked in the presence of Ba2+. was markedly reduced by substitution of Ba2+ with Ca2+. In Ca2+-free solution, it Ca2+ was readmitted during a long depolarization, the resulting inward current was larger than the control current at the corresponding time, but smaller than the peak amplitude of the control. The modulation of decay time course by manipulating divalent cations during long depolarizations implies that Ca2+- and voltage-dependent inactivation mechanisms occur separately.6. Our results suggest that the dominant mechanism involved in Ca2+ channel inactivation is Ca2+-dependent inactivation, but voltage-dependent mechanisms are also involved, including the entry of the Ca2+ Channels into a long, more slowly inactivating open state in response to prolonged depolarization.