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-Mg(2+) medium (low-Mg(2+) neurons) results in induction of continuous seizure activity. However, the underlying mechanism of the contribution of low Mg(2+) 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(2+) neurons, but the channel activity was more sensitive to Ca(2+)/CaM regulation in low-Mg(2+) than normal neurons. The increased sensitivity of VGSCs in low-Mg(2+) 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(2+) sites are disabled, was used, indicating that functional Ca(2+)-binding sites from either lobe of CaM are required for modulation of VGSCs in low-Mg(2+) 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- Mg(2+) than normal neurons, as shown by immunofluorescence. Our main finding is that low-Mg(2+) treatment increases sensitivity of VGSCs to Ca(2+)/CaM-mediated regulation. Our data reveal that CaM, as a core regulating factor, connects the functional roles of the three main intracellular ions, Na(+), Ca(2+), and Mg(2+), by modulating VGSCs and provides a possible explanation for the seizure discharge observed in low-Mg(2+) neurons.

The role of potassium channels and calcium in the relaxation mechanism of magnesium sulfate on the isolated rat uterus

“…It is known that Mg 2+ inhibits the ryanodine receptor (RyR) Ca 2+ -release channels by competing with Ca 2+ at the cytosolic activation sites of the channel in the mM range [31][32][33] and influences the fidelity of coupling between L-type Ca 2+ channels and RyRs [34]. On the other hand, cytosolic levels of H + , Ca 2+ , adenine nucleotides and Mg 2+ during fatigue influence the gating properties of the SR Ca 2+ channel [35], and the functional roles of the three main intracellular ions, Na + , Ca 2+ and Mg 2+ , are modulated by calmodulin connected voltage-gated Na + channels [36].…”

The Effect of Ca2+, Lobe-Specificity, and CaMKII on CaM Binding to NaV1.1

“…A previous study had shown that CaM overexpression in HEK1.1 cells increases the peak current of Na V 1.1 in a calcium-dependent manner [ 12 ]. Our previous study has also demonstrated that neuronal VGSC activity is modulated by CaM in a concentration-dependent manner in normal and low Mg 2+ condition [ 13 ]. Thus, we propose the following hypothetical model ( Figure 7 C) for the modulation of Ca 2+ /CaM/CaMKII on Na V 1.1 based on our present study and other studies [ 12 , 13 ]: At low [Ca 2+ ]—a nonphosphorylated state of Ca 2+ concentration—C-lobe is the predominant domain for apoCaM binding to IQ domain, and the binding of apoCaM to Na V 1.1 IQ is not affected by CaMKII.…”

“…Our previous study has also demonstrated that neuronal VGSC activity is modulated by CaM in a concentration-dependent manner in normal and low Mg 2+ condition [ 13 ]. Thus, we propose the following hypothetical model ( Figure 7 C) for the modulation of Ca 2+ /CaM/CaMKII on Na V 1.1 based on our present study and other studies [ 12 , 13 ]: At low [Ca 2+ ]—a nonphosphorylated state of Ca 2+ concentration—C-lobe is the predominant domain for apoCaM binding to IQ domain, and the binding of apoCaM to Na V 1.1 IQ is not affected by CaMKII. At high [Ca 2+ ] but at a nonphosphorylated state of IQ domain, N-lobe becomes the predominant domain since some of the CaM binds with Ca 2+ .…”

“…Voltage-gated sodium channels (VGSCs) are basic signaling molecules in excitable cells and are molecular targets for local anesthetic agents and antiepileptic agents [ 1 , 2 ]. So far, ten isoform members have been identified—Na V 1.1-Na V 1.9 and NaX—forming the VGSCs superfamily [ 3 , 4 ] in which Na V 1.1 is widely expressed in cell bodies and axon initial segments of neurons in the brains [ 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 ]. A sequence within C-terminal of Na V 1.1 contains a classical calmodulin (CaM)-binding IQ (isoleucine and glutamine) domain [ 14 , 15 , 16 , 17 ], which is involved in Ca 2+ signal transduction and alters the activity in response to changes in free Ca 2+ concentration ([Ca 2+ ]).…”

EGAR, A Food Protein-Derived Tetrapeptide, Reduces Seizure Activity in Pentylenetetrazole-Induced Epilepsy Models Through α-Amino-3-Hydroxy-5-Methyl-4-Isoxazole Propionate Receptors

“…While NMDAR antagonists inhibit these forms of synaptic plasticity, AMPAR antagonists do not impair synaptic plasticity and do not inhibit memory formation or retrieval. Hippocampal hyperexcitability induced by magnesium reduction or removal is a well-suited in vitro model of epileptiform seizure activity to elucidate the cellular mechanisms that underlie epileptogenesis and the spontaneous recurrent epileptiform discharge activity associated with epilepsy [60]. The observed increase in spiking after the low magnesium treatment was significantly reduced after the addition of EGAR, indicating a rescuing effect of the agent.…”