Voltage Dependence of a Neuromodulator-Activated Ionic Current123

The neuromodulatory inward current (I(MI)) generated by crab Cancer borealis stomatogastric ganglion neurons is an inward current whose voltage dependence has been shown to be crucial in the activation of oscillatory activity of the pyloric network of this system. It has been previously shown that I(MI) loses its voltage dependence in conditions of low extracellular calcium, but that this effect appears to be regulated by intracellular calmodulin. Voltage dependence is only rarely regulated by intracellular signaling mechanisms. Here we address the hypothesis that the voltage dependence of I(MI) is mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce I(MI) voltage dependence in normal Ca(2+), but that, in conditions of low Ca(2+), calmodulin activators do not restore I(MI) voltage dependence. Further, we show evidence that CaMKII alters I(MI) voltage dependence. These results suggest that calmodulin is necessary but not sufficient for I(MI) voltage dependence. We therefore hypothesize that the Ca(2+)/calmodulin requirement for I(MI) voltage dependence is due to an active sensing of extracellular calcium by a GPCR family calcium-sensing receptor (CaSR) and that the reduction in I(MI) voltage dependence by a calmodulin inhibitor is due to CaSR endocytosis. Supporting this, preincubation with an endocytosis inhibitor prevented W7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride)-induced loss of I(MI) voltage dependence, and a CaSR antagonist reduced I(MI) voltage dependence. Additionally, myosin light chain kinase, which is known to act downstream of the CaSR, seems to play a role in regulating I(MI) voltage dependence. Finally, a Gβγ-subunit inhibitor also affects I(MI) voltage dependence, in support of the hypothesis that this process is regulated by a G-protein-coupled CaSR.