Department of Biological Sciences, Marquette University, Milwaukee, WI 53201, USA.
Cell Mol Neurobiol. 2013 Jul;33(5):723-9. doi: 10.1007/s10571-013-9941-8. Epub 2013 May 9.
Investigation of kinase-related processes often uses pharmacological inhibition to reveal pathways in which kinases are involved. However, one concern about using such kinase inhibitors is their potential lack of specificity. Here, we report that the calcium-calmodulin-dependent kinase II (CaMKII) inhibitor CK59 inhibited multiple voltage-gated calcium channels, including the L-type channel during depolarization in a dose-dependent manner. The use of another CaMKII inhibitor, cell-permeable autocamtide-2 related inhibitory peptide II (Ant-AIP-II), failed to similarly decrease calcium current or entry in hippocampal cultures, as shown by ratiometric calcium imaging and whole-cell patch clamp electrophysiology. Notably, inhibition due to CK59 was reversible; washout of the drug brought calcium levels back to control values upon depolarization. Furthermore, the IC50 for CK59 was approximately 50 μM, which is only fivefold larger than the reported IC50 values for CaMKII inhibition. Similar nonspecific actions of other CaMKII inhibitors KN93 and KN62 have previously been reported. In the case of all three kinase inhibitors, the IC50 for calcium current inhibition falls near that of CaMKII inhibition. Our findings demonstrate that CK59 attenuates activity of voltage-gated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.
钙调蛋白依赖性激酶 II（CaMKII）抑制剂 CK59 可剂量依赖性地抑制多种电压门控钙通道，包括去极化时的 L 型通道。我们的研究表明，当使用另一种 CaMKII 抑制剂——细胞通透型自磷酸化肽 II（Ant-AIP-II）时，钙电流或进入海马培养物的情况并未得到类似的抑制，这通过比率钙成像和全细胞膜片钳电生理学得到了证明。值得注意的是，CK59 的抑制作用是可逆的；药物洗脱后，在去极化时钙水平恢复到对照值。此外，CK59 的 IC50 约为 50 μM，仅比报道的 CaMKII 抑制的 IC50 值大五倍。先前曾报道过其他 CaMKII 抑制剂 KN93 和 KN62 的类似非特异性作用。在所有三种激酶抑制剂的情况下，钙电流抑制的 IC50 接近 CaMKII 抑制的 IC50。我们的研究结果表明 CK59 可减弱电压门控钙通道的活性，因此在依赖药理学抑制来研究激酶依赖性现象时，需要更加谨慎。
