RESUMO
PURPOSE: A mutation in the ß(1) subunit of the voltage-gated sodium (Na(V)) channel, ß(1) (C121W), causes genetic epilepsy with febrile seizures plus (GEFS+), a pediatric syndrome in which febrile seizures are the predominant phenotype. Previous studies of molecular mechanisms underlying neuronal hyperexcitability caused by this mutation were conducted at room temperature. The prevalence of seizures during febrile states in patients with GEFS+, however, suggests that the phenotypic consequence of ß(1) (C121W) may be exacerbated by elevated temperature. We investigated the putative mechanism underlying seizure generation by the ß(1) (C121W) mutation with elevated temperature. METHODS: Whole-cell voltage clamp experiments were performed at 22 and 34°C using Chinese Hamster Ovary (CHO) cells expressing the α subunit of neuronal Na(V) channel isoform, Na(V) 1.2. Voltage-dependent properties were recorded from CHO cells expressing either Na(V) 1.2 alone, Na(V) 1.2 plus wild-type (WT) ß(1) subunit, or Na(V) 1.2 plus ß(1) (C121W). KEY FINDINGS: Our results suggest WT ß(1) is protective against increased channel excitability induced by elevated temperature; protection is lost in the absence of WT ß(1) or with expression of ß(1) (C121W). At 34°C, Na(V) 1.2 + ß(1) (C121W) channel excitability increased compared to NaV1.2 + WT ß(1) by the following mechanisms: decreased use-dependent inactivation, increased persistent current and window current, and delayed onset of, and accelerated recovery from, fast inactivation. SIGNIFICANCE: Temperature-dependent changes found in our study are consistent with increased neuronal excitability of GEFS+ patients harboring C121W. These results suggest a novel seizure-causing mechanism for ß(1) (C121W): increased channel excitability at elevated temperature.
Assuntos
Química Encefálica/genética , Epilepsia/genética , Predisposição Genética para Doença/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Convulsões Febris/genética , Canais de Sódio/genética , Animais , Temperatura Corporal/genética , Células CHO , Cricetinae , Cricetulus , Citoproteção/genética , Resistência à Doença/genética , Epilepsia/metabolismo , Epilepsia/fisiopatologia , Temperatura Alta/efeitos adversos , Potenciais da Membrana/genética , Canal de Sódio Disparado por Voltagem NAV1.2 , Técnicas de Patch-Clamp , Subunidades Proteicas/genética , Ratos , Convulsões Febris/metabolismo , Convulsões Febris/fisiopatologiaRESUMO
The effects on slow inactivation (SI) of charge substitutions, neutralizations, and reversals were studied for the negatively charged residues D1309 and EE1314,15 surrounding the IFM motif in the DIII-DIV cytoplasmic linker - the putative fast inactivation particle - of human skeletal muscle voltage-gated sodium channel (hNa(V)1.4). Changing aspartate (D) at position 1309 to glutamate (E) (substitution) did not strongly affect SI, whereas charge neutralization to glutamine (Q) and charge reversal to arginine (R) right-shifted the midpoint of the steady-state SI curve. Charge neutralization (D-->Q) at position 1309 also reduced the apparent valence associated with SI. Glutamates (E) at positions 1314 and 1315 were similarly mutated. Charge reversal (EE-->RR) right-shifted the steady-state SI curve and both reversal and substitution (EE-->DD) reduced its apparent valence. Charge neutralization (EE-->QQ) and reversal decreased the maximum probability of SI. These mutations also had differential effects on the rate of SI onset and recovery. These results suggest that charged residues in the DIII-DIV linker may interact with structures that control SI.