Snetkov V A, Gurney A M, Ward J P, Osipenko O N
Department of Allergy and Respiratory Medicine, United Medical School, London, UK.
Exp Physiol. 1996 Sep;81(5):743-53. doi: 10.1113/expphysiol.1996.sp003973.
Large conductance Ca(2+)-dependent K+ channels were studied in smooth muscle cells enzymatically dissociated from rabbit pulmonary artery. The current-voltage relationship of single channels recorded in cell-attached patches revealed strong inward rectification, which disappeared after patch excision. Cell permeabilization with saponin, beta-escin or equinatoxin II also removed rectification. These observations imply the existence of fast open channel block by an intracellular substance(s). Application to the cytosolic side of inside-out patches of Na+ ions, mono- di- and trinucleotides, taurine, reduced and oxidized forms of glutathione, or peptides extracted from pulmonary artery smooth muscle, did not reproduce the inward rectification. Patch treatment with either alkaline phosphatase or protein kinase A alpha-subunit, which strongly affected open state probability, was also incapable of reducing the outward single channel current. Mg2+ ions applied from the cytosolic side induced concentration- and voltage-dependent block of the outward single channel currents with a Kd of 7.9 +/- 2.3 mM, resulting in inward rectification qualitatively similar to that observed in cell-attached patches. An increase in the Mg2+ concentration of the intracellular solution induced a significant decrease in the outward whole-cell current at depolarized potentials. Another putative endogenous channel blocker, the polyamine putrescine, was not effective. However, its metabolites spermidine and spermine reduced the amplitude of the outward single channel current with Kd values of 4.9 +/- 0.6 and 1.4 +/- 0.4 mM, respectively. Pre-incubation of the cells with the irreversible inhibitor of polyamine synthesis difluoromethylornithine abolished the rectification in the cell-attached patches. These results suggest that intracellular polyamines may underlie at least part of the inward rectification of the Ca2+ activated K+ channel in this tissue, but that intracellular Mg2+ is unlikely to play a major role.
在从兔肺动脉酶解分离的平滑肌细胞中研究了大电导钙依赖性钾通道。在细胞贴附式膜片中记录的单通道电流-电压关系显示出强烈的内向整流，在膜片切除后消失。用皂角苷、β-七叶皂苷或海葵毒素II使细胞通透也消除了整流。这些观察结果表明存在一种细胞内物质对开放通道的快速阻断。将Na+离子、单核苷酸、二核苷酸和三核苷酸、牛磺酸、还原型和氧化型谷胱甘肽或从肺动脉平滑肌中提取的肽应用于内向外膜片的胞质侧，均未重现内向整流。用碱性磷酸酶或蛋白激酶Aα亚基处理膜片，这强烈影响开放状态概率，也无法降低外向单通道电流。从胞质侧施加Mg2+离子会诱导外向单通道电流的浓度和电压依赖性阻断，Kd为7.9±2.3 mM，导致内向整流，其性质与在细胞贴附式膜片中观察到的相似。细胞内溶液中Mg2+浓度的增加导致去极化电位下外向全细胞电流显著降低。另一种假定的内源性通道阻滞剂多胺腐胺无效。然而，其代谢产物亚精胺和精胺分别以4.9±0.6和1.4±0.4 mM的Kd值降低了外向单通道电流的幅度。用多胺合成不可逆抑制剂二氟甲基鸟氨酸对细胞进行预孵育，消除了细胞贴附式膜片中的整流。这些结果表明，细胞内多胺可能至少部分是该组织中钙激活钾通道内向整流的基础，但细胞内Mg2+不太可能起主要作用。
