Ko Jae Hong, Park Won Sun, Earm Yung E
Department of Physiology and National Research Laboratory for Cellular Signaling, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea.
Biochem Biophys Res Commun. 2005 Apr 8;329(2):531-7. doi: 10.1016/j.bbrc.2005.01.156.
A whole-cell patch recording was used to determine the effects of staurosporine (ST), a potent protein kinase C (PKC) inhibitor, on L-type Ca(2+) channel (LTCC) activity in rabbit atrial myocytes. Bath application of ST (300 nM) caused a significant reduction in peak I-V relationship of LTCC (from -16.8+/-2.55 to -3.74+/-1.22pApF(-1) at 0 mV). The level of L-type Ca(2+) current (I(Ca,L)) inhibition produced by ST was independent of the voltage at which the effect was measured. ST inhibited the I(Ca,L) in a dose-dependent manner with a K(d) value of 61.98+/-6.802 nM. ST shifted the activation curve to more positive potentials, but did not have any significant effect on the voltage dependence of the inactivation curve. Other PKC inhibitors, GF 109203X (1 microM) and chelerythrine (3 microM), and PKA inhibitor, PKA-IP (5 microM), did not show any inhibitory effect on I(Ca,L). Additional application of ST in the presence of isoproterenol (1 microM), a selective beta-adrenoreceptor agonist, reduced peak I(Ca,L) (78.2%) approximately to the same level with single application of ST (77.8%). In conclusion, our results indicate that ST directly blocks the LTCC in a PKC or PKA-independent manner on LTCC and it should be taken into consideration when ST is used in functional studies of ion channel modulation by protein phosphorylation.
采用全细胞膜片钳记录技术，以确定强效蛋白激酶C（PKC）抑制剂星形孢菌素（ST）对兔心房肌细胞L型钙通道（LTCC）活性的影响。浴槽中加入ST（300 nM）可使LTCC的I-V关系峰值显著降低（在0 mV时，从-16.8±2.55降至-3.74±1.22 pApF-1）。ST产生的L型钙电流（I(Ca,L)）抑制水平与测量该效应时的电压无关。ST以剂量依赖性方式抑制I(Ca,L)，K(d)值为61.98±6.802 nM。ST使激活曲线向更正的电位移动，但对失活曲线的电压依赖性没有显著影响。其他PKC抑制剂，GF 109203X（1 μM）和白屈菜红碱（3 μM），以及PKA抑制剂PKA-IP（5 μM），对I(Ca,L)均未显示出任何抑制作用。在选择性β-肾上腺素能受体激动剂异丙肾上腺素（1 μM）存在的情况下额外加入ST，使I(Ca,L)峰值降低（78.2%），与单独应用ST时（77.8%）大致相同。总之，我们的结果表明，ST以不依赖PKC或PKA的方式直接阻断LTCC，在利用ST进行蛋白磷酸化对离子通道调节的功能研究时应予以考虑。
