OBJECTIVE A slowly persistent Na + current (INaP) has been found in neurons and the abnormal change of in INaP has been found in epilepsy.Calmodulin (CaM) serving as a Ca2 + sensor is an important regulator of ion channels.Ca2 + bindings to the carboxy-terminal (C-lobe) and amino-terminal (N-lobe) of CaM have been widely suggested to induce sodium channel regnlations.However, the detailed mechanisms of how CaM modulates INaP in Mg2 +-free induced epileptiform discharge model of cultured rat hippocampal neurons are not yet determined.METHODS 1.Molecular biology CaM poptide was purified using glutathione-Sepharose 4B.The point mutations for CaMa1234 (E31A + E67A + S101F + E140A) were introduced into the Ca2+ binding sites in CaM by the site-directed mutagenesis kit.2.Cell culture The postnatal hippocampal tissue were taken from 1-day-old Wistar rats and were used for primary culture.3.Patch clamp technique Neurons after 10 d culture were treated with Mg2+-free extracellular fluid for 3 h, then the discharge activity was recorded in the current-clamp mode.Single-channel currents were recorded in the cell-attached mode and "insideout" mode.The depolarization was from-100 mV to-60 mV at a holding potential of-100 mV for 200 ms.RESULTS 1.Neurons showed " continuous tonic high frequency burst discharges" and the " wedge shaped depolarization" after Mg2+-free treatment, showing that Mg2+-free induced epileptiform discharge model was successfully established.2.The slope conductance was not changed (22.85 ± 1.85 ps) in Mg2+ free treated model compared with the control neurons (22.69 ± 2.69 ps).The mean amplitude of single-channel current at-60 mV was-1.81 ±0.03 pA in normal cells and-1.86 ±0.20 pA in model cells, respectively, and the differences between two group was not significant (P >0.05).However, NPo of model cells was larger than that of control ceils (0.066 ± 0.020 in model vs 0.049 ± 0.008 in control, P < 0.05).3.Application of 0.7, 1.4 and 2.1 mol· L-1 CaM at free [Ca2 +] of 100 nmol· L-1 in normal cells induced NPo in a concentration-dependent manner (41.5 ± 6.3% , 76.6 ± 9.6% and 101.5 ± 18.2%) compared with control condition, but NPo in model cells was 122.6 ± 16.5% even at 0.7 mol ·L-1 CaM.Application of CaM1234 in normal cells and model cells induced NP. in a concentration-independent manner.CONCLUSION Modulation of CaM on sodium channel activity is more sensitive in Mg2 +-free model, which might be related to the underlying mechanism of spontaneous epileptiform discharge in Mg2+-free neuron model.