RESUMO
We identified nine patients from four unrelated families harboring three biallelic variants in SCN1B (NM_001037.5: c.136C>T; p.[Arg46Cys], c.178C>T; p.[Arg60Cys], and c.472G>A; p.[Val158Met]). All subjects presented with early infantile epileptic encephalopathy 52 (EIEE52), a rare, severe developmental and epileptic encephalopathy featuring infantile onset refractory seizures followed by developmental stagnation or regression. Because SCN1B influences neuronal excitability through modulation of voltage-gated sodium (NaV ) channel function, we examined the effects of human SCN1BR46C (ß1R46C ), SCN1BR60C (ß1R60C ), and SCN1BV158M (ß1V158M ) on the three predominant brain NaV channel subtypes NaV 1.1 (SCN1A), NaV 1.2 (SCN2A), and NaV 1.6 (SCN8A). We observed a shift toward more depolarizing potentials of conductance-voltage relationships (NaV 1.2/ß1R46C , NaV 1.2/ß1R60C , NaV 1.6/ß1R46C , NaV 1.6/ß1R60C , and NaV 1.6/ß1V158M ) and channel availability (NaV 1.1/ß1R46C , NaV 1.1/ß1V158M , NaV 1.2/ß1R46C , NaV 1.2/ß1R60C , and NaV 1.6/ß1V158M ), and detected a slower recovery from fast inactivation for NaV 1.1/ß1V158M . Combined with modeling data indicating perturbation-induced structural changes in ß1, these results suggest that the SCN1B variants reported here can disrupt normal NaV channel function in the brain, which may contribute to EIEE52.
Assuntos
Espasmos Infantis/genética , Subunidade beta-1 do Canal de Sódio Disparado por Voltagem/genética , Canais de Sódio Disparados por Voltagem/genética , Canais de Sódio Disparados por Voltagem/metabolismo , Criança , Pré-Escolar , Mapeamento Cromossômico , DNA/genética , Epilepsia Resistente a Medicamentos/etiologia , Eletroencefalografia , Exoma , Feminino , Variação Genética , Humanos , Lactente , Masculino , Modelos Moleculares , Mutação de Sentido Incorreto/genética , Linhagem , Convulsões/etiologiaRESUMO
Nav1.1 is an important pharmacological target as this voltage-gated sodium channel is involved in neurological and cardiac syndromes. Channel activators are actively sought to try to compensate for haploinsufficiency in several of these pathologies. Herein we used a natural source of new peptide compounds active on ion channels and screened for drugs capable to inhibit channel inactivation as a way to compensate for decreased channel function. We discovered that JzTx-34 is highly active on Nav1.1 and subsequently performed a full structure-activity relationship investigation to identify its pharmacophore. These experiments will help interpret the mechanism of action of this and formerly identified peptides as well as the future identification of new peptides. We also reveal structural determinants that make natural ICK peptides active against Nav1.1 challenging to synthesize. Altogether, the knowledge gained by this study will help facilitate the discovery and development of new compounds active on this critical ion channel target.
Assuntos
Peptídeos , Canais de Sódio Disparados por Voltagem , Humanos , Peptídeos/farmacologia , Peptídeos/química , Relação Estrutura-AtividadeRESUMO
Background: Voltage-gated sodium (NaV) channels help regulate electrical activity of the plasma membrane. Mutations in associated subunits can result in pathological outcomes. Here we examined the interaction of NaV channels with cardiac arrhythmia-linked mutations in SCN2B and SCN4B, two genes that encode auxiliary ß-subunits. Materials and Methods: To investigate changes in SCN2B R137H and SCN4B I80T function, we combined three-dimensional X-ray crystallography with electrophysiological measurements on NaV1.5, the dominant subtype in the heart. Results: SCN4B I80T alters channel activity, whereas SCN2B R137H does not have an apparent effect. Structurally, the SCN4B I80T perturbation alters hydrophobic packing of the subunit with major structural changes and causes a thermal destabilization of the folding. In contrast, SCN2B R137H leads to structural changes but overall protein stability is unaffected. Conclusion: SCN4B I80T data suggest a functionally important region in the interaction between NaV1.5 and ß4 that, when disrupted, could lead to channel dysfunction. A lack of apparent functional effects of SCN2B R137H on NaV1.5 suggests an alternative working mechanism, possibly through other NaV channel subtypes present in heart tissue. Indeed, mapping the structural variations of SCN2B R137H onto neuronal NaV channel structures suggests altered interaction patterns.