Lopatin A N, Nichols C G
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri.
J Pharmacol Exp Ther. 1993 May;265(2):1011-6.
DRK1 is a cloned K+ channel from rat brain with consensus sites for protein kinase-dependent phosphorylation that might be expected to be functionally regulated by phosphorylation. 2,3-Butane-dione-monoxime (BDM) chemically removes phosphate groups from many proteins, and its action on DRK1 channels was examined after expression of DRK1 cRNA in Xenopus oocytes. In two-microelectrode voltage-clamp experiments, the application of BDM to the bath inhibited DRK1 current (ki = 16.6 mM, H = 0.96) rapidly and reversibly, with a time course similar to the time course of solution change within the bath. DRK1 current was inhibited at all potentials; the time course of current activation, deactivation and inactivation were unaffected by BDM. In inside-out patch-clamp experiments, the application of BDM to the cytoplasmic surface similarly inhibited channel activity rapidly and reversibly (ki = 10.7 mM, H = 1.01) in the absence of rephosphorylating substrates. These results are inconsistent with a phosphatase effect, because such an effect should be irreversible in cell-free, ATP-free patches. Instead, the results suggest that BDM can inhibit DRK1 channels directly from inside or outside of the membrane.
DRK1是一种从大鼠大脑中克隆出来的钾离子通道，具有蛋白激酶依赖性磷酸化的共有位点，可能预期会受到磷酸化的功能调节。2,3-丁二酮一肟（BDM）能化学去除许多蛋白质上的磷酸基团，在非洲爪蟾卵母细胞中表达DRK1 cRNA后，研究了其对DRK1通道的作用。在双电极电压钳实验中，将BDM施加到浴液中会迅速且可逆地抑制DRK1电流（ki = 16.6 mM，H = 0.96），其时间进程与浴液中溶液变化的时间进程相似。在所有电位下DRK1电流均受到抑制；电流激活、失活和失活的时间进程不受BDM影响。在膜内向外膜片钳实验中，在没有再磷酸化底物的情况下，将BDM施加到细胞质表面同样会迅速且可逆地抑制通道活性（ki = 10.7 mM，H = 1.01）。这些结果与磷酸酶效应不一致，因为在无细胞、无ATP的膜片中这种效应应该是不可逆的。相反，结果表明BDM可以直接从膜的内部或外部抑制DRK1通道。
