Zhou X B, Schlossmann J, Hofmann F, Ruth P, Korth M
Institut für Pharmakologie und Toxikologie der Technischen Universität München, Biedersteiner Strasse 29, D-80802 Munich, Germany.
Pflugers Arch. 1998 Oct;436(5):725-34. doi: 10.1007/s004240050695.
The cloned BK channel alpha subunit from human myometrium was stably expressed in Chinese hamster ovary cells, either alone (CHOalpha cells) or in combination with the auxiliary beta subunit (CHOalpha+beta cells). We studied basic channel properties and the effects of cGMP- and cAMP-dependent protein kinases on the BK channel activity. Coexpression of alpha and beta subunits enhanced the Ca2+ and voltage sensitivity of the BK channel, and decreased the inhibitory potency of iberiotoxin. Blocking and stimulating effects on BK channel activity by charybdotoxin and nitric oxide, respectively, were independent of the beta subunit. The cGMP kinase Ialpha and cAMP kinase failed to affect BK channel activity in CHOalpha and CHOalpha+beta cells at different [Ca2+]i and voltages. In contrast, BK channels in freshly isolated myometrial cells from postmenopausal women responded to cAMP kinase and cGMP kinase with a fourfold and twofold decrease in their open probability (NPo), respectively. These effects could be reversed by alkaline phosphatase and remained unaffected by the phosphatase inhibitor okadaic acid (100 nM). In 28% of myometrial cells, however, cAMP and cGMP kinases increased NPo 2-fold and 3.5-fold, respectively. This stimulation was enhanced rather than reversed by alkaline phosphatase and was abolished by 100 nM okadaic acid. The results suggest that in stably transfected CHO cells the expressed BK channel is not regulated by cAMP kinase and cGMP kinase. However, in native myometrial cells stimulatory and inhibitory regulation of BK channels by cAMP kinase and cGMP kinase was observed, suggesting that channel regulation by the protein kinases requires factors that are not provided by CHO cells. Alternatively, failure of regulation may have been due to the primary structure of the myometrial BK channel protein used in this study.
从人子宫肌层克隆的BK通道α亚基在中华仓鼠卵巢细胞中稳定表达，既可以单独表达（CHOα细胞），也可以与辅助β亚基共同表达（CHOα+β细胞）。我们研究了基本通道特性以及环鸟苷酸（cGMP）依赖性蛋白激酶和环腺苷酸（cAMP）依赖性蛋白激酶对BK通道活性的影响。α亚基和β亚基的共表达增强了BK通道的Ca2+和电压敏感性，并降低了iberiotoxin的抑制效力。分别由蝎毒素和一氧化氮对BK通道活性的阻断和刺激作用与β亚基无关。在不同的细胞内Ca2+浓度（[Ca2+]i）和电压下，cGMP激酶Iα和cAMP激酶未能影响CHOα和CHOα+β细胞中的BK通道活性。相反，来自绝经后妇女的新鲜分离的子宫肌层细胞中的BK通道对cAMP激酶和cGMP激酶的反应分别是其开放概率（NPo）降低四倍和两倍。这些效应可被碱性磷酸酶逆转，并且不受磷酸酶抑制剂冈田酸（100 nM）的影响。然而，在28%的子宫肌层细胞中，cAMP和cGMP激酶分别使NPo增加2倍和3.5倍。这种刺激作用被碱性磷酸酶增强而非逆转，并被100 nM冈田酸消除。结果表明，在稳定转染的CHO细胞中，表达的BK通道不受cAMP激酶和cGMP激酶的调节。然而，在天然子宫肌层细胞中观察到cAMP激酶和cGMP激酶对BK通道的刺激和抑制调节，这表明蛋白激酶对通道的调节需要CHO细胞未提供的因子。或者，调节失败可能是由于本研究中使用的子宫肌层BK通道蛋白的一级结构所致。
