Nonspecific, Reversible Inhibition of Voltage-Gated Calcium Channels by CaMKII Inhibitor CK59
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ORIGINAL RESEARCH
Nonspecific, Reversible Inhibition of Voltage-Gated Calcium Channels by CaMKII Inhibitor CK59 Andrew S. Karls • Michelle Mynlieff
Received: 6 March 2013 / Accepted: 24 April 2013 / Published online: 9 May 2013 Ó Springer Science+Business Media New York 2013
Abstract 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–calmodulindependent 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 (AntAIP-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 lM, 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 voltagegated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.
A. S. Karls M. Mynlieff (&) Department of Biological Sciences, Marquette University, P.O. Box 1881, Milwaukee, WI 53201, USA e-mail: [email protected] A. S. Karls e-mail: [email protected]
Keywords Calcium–calmodulin-dependent kinase II (CaMKII) Calcium channel Kinase inhibitor Nonspecific inhibition
Introduction Calcium–calmodulin-dependent kinase II (CaMKII) is a crucial signaling molecule involved in many of the most fundamental behaviors of neurons, such as gene expression and synaptic plasticity due to long-term potentiation. It follows that the distribution of this protein is widespread throughout the nervous system, and makes up between 1 and 2 % of total protein content in neurons. Given both its importance and its abundance, CaMKII-mediated processes are currently under intense investigation. One of the most common ways to study the activity of CaMKII is to inhibit it with pharmacological agents and then investigate changes in the neuron when CaMKII is no longer active. Unfortunately, a common problem with kinase inhibitors, including CaMKII inhibitors, is their lack of specificity. Therefore, new, more specific inhibitors are always in demand. This demand extends to a clinical setting, where more specific CaMKII inhibitors may help avoid potentially harmful side effects. Over 30 different iso
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