CaMKII Regulation of Calcium Homeostasis and Neuronal Activity

Abstract

Calcium/calmodulin-dependent kinase II (CaMKII) is a multifunctional serine/threonine protein kinase that regulates multiple ion channels and receptors that are essential for neuronal activity and plasticity. To explore the role of CaMKII in regulating neuronal calcium homeostasis, we applied pharmacological inhibitors of CaMKII (10 micromolar tat-CN21, tat-AIP, and myr-AIP) to cultured cortical and hippocampal neurons and measured changes in intracellular calcium levels using Fluo-4AM and Fura-2FF. CaMKII inhibition induced a rapid increase in intracellular calcium levels. Dysregulated calcium signaling was not limited to neurons, as CaMKII inhibitors also initiated calcium waves in cultured astrocytes that preceeded neuronal effects. Inhibitors that disrupt neuronal activity, VGSCs, and AMPA-Rs, block this calcium dysregulation in neurons. Although L-type calcium channel blockers had no effect, eliminating extracellular calcium, inhibiting NMDA-Rs or N-type calcium channels did prevent calcium dysregulation in neurons, implicating aberrant glutamate release and/or post-synaptic activity in this calcium dysregulation. Increased glutamate levels (2-4 micromolar) were observed in the media following CaMKII inhibition. Enzymatic buffering of glutamate in the media prevented the aberrant calcium influx. Finally, to directly test whether CaMKII inhibition increases neuronal activity, voltage-clamp electrophysiology was utilized to measure action potential generation to a depolarizing ramp current. Unlike inactive controls, CaMKII inhibitors induced a three-fold increase in the number of action potentials when applied in the patch pipette. In total, a loss of CaMKII activity results in dysregulation of intracellular calcium homeostasis and a subsequent dysregulation of glutamate signaling and neuronal excitability. Thus, CaMKII not only responds to neuronal activity via changes in intracellular calcium levels, it also appears to serve as a master regulator of neuronal excitability through regulation of calcium homeostasis.