Nonspecific, reversible inhibition of voltage-gated calcium channels by CaMKII inhibitor CK59

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-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.

Fig. 1

CaMKII inhibitor CK59 but not Ant-AIP-II significantly attenuates the amount of high KCl-induced increase in intracellular calcium when measured with Fura-2 ratiometric imaging. a Example of 340/380 ratio obtained with high KCl solution alone or high KCl solution in the presence of 50 μM CK59. Each line shown represents an individual cell (N = 6). b The same conditions as in A, but using 50 nM Ant-AIP-II (N = 6). c Average change in intracellular calcium as determined by the 340/380 ratios with KCl alone (solid bars), KCl with CK59 (crossed hash bar, N = 128), or KCl with Ant-AIP-II (diagonal hashed bar, N = 255). *Paired t test, p < 0.001)

Fig. 2

Various doses (0.5, 5, 20, 50, and 250 μM; N = 122, 51, 166, 326, and 122, respectively) of CK59 were applied to cells during high KCl-induced depolarization. The percent inhibition of high KCl-induced intracellular calcium by CK59 was calculated as $100\%-(100\times \frac{(\mathit{Fura}\mathit{Ratio}\mathit{in}\mathit{CK}59+\mathit{KCl})}{(\mathit{Fura}\mathit{Ratio}\mathit{in}\mathit{KCl})}).$ Data were fit with a 3-parameter sigmoidal curve (r ² = 0.983). This allowed calculation of the IC₅₀ value for CK59, 52 μM

Fig. 3

Calcium currents elicited by a 300 ms depolarizing pulse to +10 mV from a holding potential of −80 mV demonstrates the nonspecific effects of CK59 on calcium channel types. a CK59 application reduces current in normal recording solution. Voltage-gated calcium currents were recorded before (black trace), during (gray trace), and after (light gray trace) application of CK59. b Control calcium currents were measured before and after CK59 application (filled circles) and fit with a linear regression line and 95 % confidence interval (dashed line). Calcium currents elicited in the presence of CK59 (gray diamonds) fell outside of the confidence interval for the control currents. The percent of current blocked by CK59 was calculated by comparing the current in the presence of CK59 to the regression line for that specific time point. c 20 μM nimodipine was bath applied and voltage-gated calcium currents were recorded before (black trace), during (gray trace), and after (light gray trace) application of CK59 (50 μM). d 2 μM ω-conotoxin MVIIC was bath applied and voltage-gated calcium currents were recorded before (black trace), during (gray trace), and after (light gray trace) application of CK59 (50 μM). The traces illustrated in a, c, and d were recorded in different neurons. e Average decrease in voltage-gated calcium current seen with CK59 application (50 μM) in control bathing solution (N = 15), with 20 μM nimodipine (N = 7), and with 2 μM ω-conotoxin MVIIC (N = 5) in the bath. *Unpaired t test, p < 0.001

Fig. 4

CaMKII inhibitor CK59 attenuates the amount of high KCl-induced increase in intracellular calcium in the presence of the L-type channel blocker, nimodipine when measured with Fura-2 ratiometric imaging. Nimodipine (20 μM) was perfused during the entire measurement period. The 340/380 ratio was measured in response to high KCl solution before and during CK59 (50 μM) application and representative cells are shown (N = 7). Each line represents an individual cell