Prevarskaya N B, Skryma R N, Vacher P, Daniel N, Djiane J, Dufy B
Laboratory of Neurophysiology, University of Bordeaux II, CNRS URA 1200, France.
J Biol Chem. 1995 Oct 13;270(41):24292-9. doi: 10.1074/jbc.270.41.24292.
Chinese hamster ovary (CHO) cells, stably transfected with the long form of the prolactin (PRL) receptor (PRL-R) cDNA, were used for PRL-R signal transduction studies. Patch-clamp technique in whole cell and cell-free configurations were employed. Exposure of transfected CHO cells to 5 nM PRL led to the increase of Ca(2+)- and voltage-dependent K+ channel (KCa) activity. The effect was direct as it was observed also in excised patch experiments. A series of tyrosine kinase inhibitors was studied to investigate the possible involvement of protein tyrosine kinases in KCa functioning and its stimulation by PRL. Genistein, lavendustin A, and herbimycin A decreased in a concentration and time-dependent manner the amplitude of the KCa current in whole cell and the open probability of KCa channels in cell-free experiments. The subsequent application of PRL was ineffective. The protein tyrosine phosphatase inhibitor orthovanadate (1 mM) stimulated KCa channel activity in excised patches, indicating that channels can be modulated in opposite directions by protein tyrosine kinase and protein tyrosine phosphatase. Moreover, in whole cell experiments as well as in excised patch recordings, anti-JAK2 tyrosine kinase antibody decreased the KCa conductance and the open probability of the KCa channels. Subsequent application of PRL was no longer able to stimulate KCa conductance. Immunoblotting studies using the same anti-JAK2 antibody, revealed the constitutive association of JAK2 kinase with PRL-R. Preincubation of anti-JAK2 antibody with the JAK2 Immunizing Peptide abolished the effects observed using anti-JAK2 antibody alone in both electrophysiological and immunoblotting studies. We conclude from these findings that these KCa channels are regulated through tyrosine phosphorylation/dephosphorylation; JAK2 tyrosine kinase, constitutively associated with PRL-R, is implicated in PRL stimulation of KCa channels.
将催乳素（PRL）受体（PRL-R）cDNA长形式稳定转染的中国仓鼠卵巢（CHO）细胞用于PRL-R信号转导研究。采用全细胞和无细胞配置的膜片钳技术。将转染的CHO细胞暴露于5 nM PRL会导致钙依赖性和电压依赖性钾通道（KCa）活性增加。在切除膜片实验中也观察到这种效应，表明它是直接作用。研究了一系列酪氨酸激酶抑制剂，以研究蛋白酪氨酸激酶可能参与KCa功能及其受PRL刺激的情况。染料木黄酮、拉文杜斯汀A和赫伯霉素A以浓度和时间依赖性方式降低了全细胞中KCa电流的幅度以及无细胞实验中KCa通道的开放概率。随后施加PRL无效。蛋白酪氨酸磷酸酶抑制剂原钒酸盐（1 mM）刺激了切除膜片中KCa通道的活性，表明通道可被蛋白酪氨酸激酶和蛋白酪氨酸磷酸酶以相反方向调节。此外，在全细胞实验以及切除膜片记录中，抗JAK2酪氨酸激酶抗体降低了KCa电导和KCa通道的开放概率。随后施加PRL不再能够刺激KCa电导。使用相同的抗JAK2抗体进行免疫印迹研究，揭示了JAK2激酶与PRL-R的组成性结合。在电生理和免疫印迹研究中，抗JAK2抗体与JAK2免疫肽预孵育消除了单独使用抗JAK2抗体时观察到的效应。我们从这些发现中得出结论，这些KCa通道通过酪氨酸磷酸化/去磷酸化进行调节；与PRL-R组成性结合的JAK2酪氨酸激酶参与了PRL对KCa通道的刺激。
