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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。
This article aims to study the characteristics of large-conductance calcium-activated K + channel (BKCa) and the possible mechanism of H2O2 on BKCa channel in guinea pig colonic smooth muscle cells so as to reveal the role of ROS in the regulation of gastrointestinal motility. The guinea pig colon smooth muscle cells were isolated by papain method and the BKCa current was recorded by the single-channel patch-clamp in vitro. The electrode solution and perfusate were symmetrically potassium-rich solution. BKCa channels in guinea pig colon smooth muscle cells were voltage-dependent, highly selective for K +, and large conductance [(223.7 ± 9.2) pS] and highly sensitive to cytosolic Ca 2+ concentration. H2O2 can reduce or increase the open probability of BKCa channel, both of which can be reversed by DTT of cysteine-specific reductant, but have no effect on the conductance of BKCa channel. Cysteine-specific oxidant DTNB can also reduce or increase the open probability of BKCa channels, the role of DTNB can also be reversed. The above results show that H2O2 and DTNB regulate the open probability of BKCa channel through oxidation of cysteine residues in BKCa channel.