A missense mutation of the Na+ channel alpha II subunit gene Na(v)1.2 in a patient with febrile and afebrile seizures causes channel dysfunction

Abstract

Generalized epilepsy with febrile seizures plus (GEFS+), a clinical subset of febrile seizures (FS), is characterized by frequent episodes beyond 6 years of age (FS+) and various types of subsequent epilepsy. Mutations in beta1 and alpha(I)-subunit genes of voltage-gated Na(+) channels have been associated with GEFS+1 and 2, respectively. Here, we report a mutation resulting in an amino acid exchange (R188W) [corrected] in the gene encoding the alpha-subunit of neuronal voltage-gated Na(+) channel type II (Na(v)1.2) in a patient with FS associated with afebrile seizures. The mutation R188W [corrected] occurring on Arg(187), a highly conserved residue among voltage-gated Na(+) channels, was not found in 224 alleles of unaffected individuals. Whole-cell patch clamp recordings on human embryonic kidney (HEK) cells expressing a rat wild-type (rNa(v)1.2) and the corresponding mutant channels showed that the mutant channel inactivated more slowly than wild-type whereas the Na(+) channel conductance was not affected. Prolonged residence in the open state of the R188W [corrected] mutant channel may augment Na(+) influx and thereby underlie the neuronal hyperexcitability that induces seizure activity. Even though a small pedigree could not show clear cosegregation with the disease phenotype, these findings strongly suggest the involvement of Na(v)1.2 in a human disease and propose the R188W [corrected] mutation as the genetic defect responsible for febrile seizures associated with afebrile seizures.

Figure 1

Missense mutations identified in a Japanese family with febrile seizures associated with afebrile seizures. (A) Pedigree of Japanese family K1 with FS and afebrile seizures (proband) and FS only (parents). c.562C → T (R187W) mutations appeared in proband P1 and his father. Circles = females, squares = males, arrow = proband, filled square = FS associated with afebrile seizures, half-filled square and circle = FS. (B) Electropherogram of the mutation in the gene for Na_v1.2 identified in family K1. The nucleotide sequence of the relevant region of exon 4 of the proband is shown. Arrow indicates nucleotide 562, where a heterozygous C-to-T transition resulted in an amino acid substitution, R187W.

Figure 2

Diagram of Na_v1.2 and amino acid sequence alignments of Na⁺ channel α-subunit family members. (A) Locations of missense mutations identified in this study on Na_v1.2. Filled square indicates the mutation proposed to be responsible for the disease phenotype. The amino acid exchanges indicated by open circles are ones assumed to be benign variants. (B–D) Partial amino acid sequences of Na_v1.2 and other α-subunit family members (GenBank accession nos.: M94055, M22253, Y00766, M81758, M77235, AB027567, X82835, AF117907, AF188679, D37977, L19979, M22252, and M32078). Dark shaded background indicates identical amino acids, light shaded background indicates conserved amino acids, and white background depicts nonconserved amino acids. Arrows above the sequences indicate the positions of the missense mutations, R19K (B), R187W (C), and R524Q (D). Arg187 is highly conserved among the α-subunits of voltage-gated Na⁺ channels (C).

Figure 3

Differences in the kinetics between WT and mutant sodium channels. (A) Sodium currents of WT and mutant Na⁺ channels. Currents were induced by a step depolarization to 0 mV from a holding potential of −120 mV. (B) Enlargement of the area within dashed line in A. Peak amplitudes were 5.2 nA for WT, 3.9 nA for R524Q, 4.4 nA for R187W, and 4.1 nA for R19K. The peak currents were normalized. (C) Time constant of the decay during a step depolarization. Current decay was fitted by a single exponential function. The decay time constant was significantly slower in R187W channels than in WT, R19K, or R524Q channels. Each point is given as mean ± SEM (n = 7). Statistical test was done by ANOVA. **, the statistical difference at 1% level.

Figure 4

Effects of missense mutations on conductance-voltage relationships and steady-state inactivation. (A) Peak conductance-voltage relationships for WT (open circle), R524Q (filled circle), R187W (open diamond), and R19K (filled diamond) Na⁺ channels expressed in HEK cells. No significant differences were detected between WT and mutant channels. Number of experiments was four, and all data were pooled. Curves are fitted to the data by using the equations given in Materials and Methods. (B) Steady-state voltage dependence of inactivation for WT (open circle), R524Q (filled circle), R187W (open diamond), and R19K (filled diamond) Na⁺ channels. Two different protocols were used. The prepulses were applied from −155 to −15 mV for WT, R524Q, and R19K mutant channels, and from −140 to −10 mV for R187W mutant channel with 10-mV increments. The half-inactivation potential and slope factors were estimated to be −76.9 mV and 6.7 for WT, −76.4 mV and 6.8 for R524Q, −88.6 mV and 8.7 for R187W, and −77.2 mV and 6.9 for R19K. All data were pooled and fitted as in A. (C) Voltage dependence of the inactivation time constant for WT and the R187W mutant channels. Note the clear difference in maximal rate of inactivation at the depolarized membrane potentials between WT and the R187W mutant.