The neuromodulatory inward current (IMI) 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 IMIloses 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 IMIis mediated by intracellular signaling pathways activated by extracellular calcium. We demonstrate that calmodulin inhibitors and a ryanodine antagonist can reduce IMIvoltage dependence in normal Ca2+, but that, in conditions of low Ca2+, calmodulin activators do not restore IMIvoltage dependence. Further, we show evidence that CaMKII alters IMIvoltage dependence. These results suggest that calmodulin is necessary but not sufficient for IMIvoltage dependence. We therefore hypothesize that the Ca2+/calmodulin requirement for IMIvoltage dependence is due to an active sensing of extracellular calcium by a GPCR family calcium-sensing receptor (CaSR) and that the reduction in IMIvoltage 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 IMIvoltage dependence, and a CaSR antagonist reduced IMIvoltage dependence. Additionally, myosin light chain kinase, which is known to act downstream of the CaSR, seems to play a role in regulating IMIvoltage dependence. Finally, a Gß?-subunit inhibitor also affects IMIvoltage dependence, in support of the hypothesis that this process is regulated by a G-protein-coupled CaSR.
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