Low-Mg2+ treatment increases sensitivity of voltage-gated Na+ channels to Ca2+/calmodulin-mediated modulation in cultured hippocampal neurons

Abstract

Culture of hippocampal neurons in low-Mg2+ medium (low-Mg2+ neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg2+ 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-Mg2+ neurons, but the channel activity was more sensitive to Ca2+/CaM regulation in low-Mg2+ than normal neurons. The increased sensitivity of VGSCs in low-Mg2+ 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 Ca2+ sites are disabled, was used, indicating that functional Ca2+-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg2+ 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 low- Mg2+ than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg2+ treatment increases sensitivity of VGSCs to Ca2+/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na+, Ca2+, and Mg2+, by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg2+ neurons.