CALL FOR PAPERS Proteomic and Metabolomic Approaches to Cell Physiology and Pathophysiology Low-Mg treatment increases sensitivity of voltage-gated Na channels to Ca /calmodulin-mediated modulation in cultured hippocampal neurons

Guo F, Zhou PD, Gao QH, Gong J, Feng R, Xu XX, Liu SY, Hu HY, Zhao MM, Adam HC, Cai JQ, Hao LY. Low-Mg treatment increases sensitivity of voltage-gated Na channels to Ca /calmodulin-mediated modulation in cultured hippocampal neurons. Am J Physiol Cell Physiol 308: C594 –C605, 2015. First published February 4, 2015; doi:10.1152/ajpcell.00174.2014.— Culture of hippocampal neurons in low-Mg medium (low-Mg neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg to hyperexcitability of neurons has not been clarified. Our data, obtained using the patch-clamp technique, show that voltage-gated Na channel (VGSC) activity, which is associated with a persistent, noninactivating Na current (INa,P), was modulated by calmodulin (CaM) in a concentration-dependent manner in normal and low-Mg neurons, but the channel activity was more sensitive to Ca /CaM regulation in low-Mg than normal neurons. The increased sensitivity of VGSCs in low-Mg neurons was partially retained when CaM12 and CaM34, CaM mutants with disabled binding sites in the N or C lobe, were used but was diminished when CaM1234, a CaM mutant in which all four Ca sites are disabled, was used, indicating that functional Ca binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg neurons. Furthermore, the number of neurons exhibiting colocalization of CaM with the VGSC subtypes NaV1.1, NaV1.2, and NaV1.3 was significantly higher in lowMg than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg treatment increases sensitivity of VGSCs to Ca /CaMmediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na , Ca , and Mg , by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg neurons.