豚鼠结肠平滑肌细胞大电导钙激活钾通道特性及氧化修饰的影响

The characteristics and oxidative modulation of large-conductance calciumactivated potassium channels in guinea-pig colon smooth muscle cells

本文旨在研究豚鼠结肠平滑肌细胞大电导钙激活K+通道(BKCa)的特性及H2O2对BKCa通道的影响及其可能的作用机制,以初步揭示活性氧在调控胃肠运动方面的作用。用木瓜蛋白酶法分离豚鼠结肠平滑肌细胞,采用单通道膜片钳内面向外式记录BKCa电流,电极液及灌流液采用对称的高钾溶液。豚鼠结肠平滑肌细胞的BKCa通道具有电压依赖性、对K+的高度选择性、大电导[(223.7±9.2)pS]以及对胞浆面钙离子浓度高度敏感的特性。H2O2可降低或升高BKCa通道的开放概率,两种作用均可被半胱氨酸特异性还原剂DTT所翻转,但对BKCa通道的电导没有影响。半胱氨酸特异性氧化剂DTNB同样可降低或升高BKCa通道的开放概率,其作用同样可被DTT所翻转。以上结果显示,H2O2及DTNB通过氧化BKCa通道的半胱氨酸残基来调节BKCa通道的开放概率。

To investigate the characteristics of large-conductance calcium-activated potassium channels （BKca） and the effect of hydrogen peroxide （H202） on BKca in guinea-pig proximal colon smooth muscle cells, single smooth muscle cells of guinea-pig colon were enzymatically isolated in low calcium solution containing papain （3 mg/mL）, DTT （2 mg/mL）, and bovine serum albumin （BSA,2 mg/mL）. Tissues were incubated at 36 ~C in enzyme solution for 15 min and were then suspended in enzyme-free low calcium solution. Inside-out single channel recording technique was used to record BKca current. The intracellular （bath） and microelectrode solution both contained symmetrical high potassium. The BKCa in guinea-pig colon smooth muscle cell possesses： 1） voltage-dependence,2） high selectivity for potassium ion, 3） large conductance （223.7 pS±9.2 pS）, 4） dependence of [Ca^2＋]i. Intracelluar application of H2O2 decreased the open probability （Po） of BKCa at low concentration （〈1 mmol/L）, and increased or decreased Po of BKCa at high concentration （5 mmol/L）, without affecting the unitary conductance. The effects of H2O2 were reversed by reducing agent dithiothreitol （DTT）. Similarly, cysteine specific oxidizing agent, DTNB, also increased or decreased Po of BKca and DTT partially reversed the effect of DTNB. It is thus suggested that H2O2 and DTNB may modulate Po of BKCa via the oxidation of cysteine residue.