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. 2004 Jul 27;101(30):11147-52.
doi: 10.1073/pnas.0402482101. Epub 2004 Jul 19.

Noninactivating voltage-gated sodium channels in severe myoclonic epilepsy of infancy

Thomas H Rhodes  1 , Christoph Lossin, Carlos G Vanoye, Dao W Wang, Alfred L George Jr
Affiliations

Noninactivating voltage-gated sodium channels in severe myoclonic epilepsy of infancy

Thomas H Rhodes et al. Proc Natl Acad Sci U S A. .

Abstract

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel alpha(1) subunit (Na(V)1.1), are associated with at least two forms of epilepsy, generalized epilepsy with febrile seizures plus and severe myoclonic epilepsy of infancy (SMEI). We examined the functional properties of five SMEI mutations by using whole-cell patch-clamp analysis of heterologously expressed recombinant human SCN1A. Two mutations (F902C and G1674R) rendered SCN1A channels nonfunctional, and a third allele (G1749E) exhibited minimal functional alterations. However, two mutations within or near the S4 segment of the fourth repeat domain (R1648C and F1661S) conferred significant impairments in fast inactivation, including persistent, noninactivating channel activity resembling the pattern of channel dysfunction observed for alleles associated with generalized epilepsy with febrile seizures plus. Our data provide evidence for a range of SCN1A functional abnormalities in SMEI, including gain-of-function defects that were not anticipated in this disorder. Our results further indicate that a complex relationship exists between phenotype and aberrant sodium channel function in these inherited epilepsies.

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Figures

Fig. 1.
Fig. 1.
Functional properties of WT and mutant SCN1A sodium channels. Representative whole-cell currents recorded from tsA201 cells expressing WT (a) or mutant SCN1A channels R1648C (b), F1661S (c), and G1749E (d). Cells were stepped to various potentials between –80 and +40 mV in 10-mV increments from a holding potential of –120 mV (see Fig. 3b for pulse protocol). All experiments were performed in the whole-cell patch-clamp configuration at room temperature 24–72 h after transfection.
Fig. 2.
Fig. 2.
Voltage dependence of fast inactivation for WT and mutant channels. (a) Representative normalized whole-cell sodium currents from cells stepped to a potential of –10 mV from a holding potential of –120 mV. Peak current amplitudes were normalized. (b) Inactivation time constants of WT and mutant SCN1A currents. The decay phase of voltage-sensitive inward currents was fitted with a two-exponential function, It/Imax = A1·exp(–t/τ1) + A2·exp(–t/τ2) + C, where An and τn refer to fractional amplitude and time constant, respectively. (c) Fractional amplitudes of the slower component (designated as τ2) of fast inactivation plotted against voltage. Values significantly different from WT are indicated: *, P < 0.005; †, P < 0.05.
Fig. 3.
Fig. 3.
Inactivation and activation properties of SMEI-associated SCN1A mutants. (a) Current–voltage relationships of whole-cell currents. Currents were elicited by test pulses to various potentials (see b Inset) and normalized to cell capacitance (WT, n = 17; R1648C, n = 7; F1661S, n = 8; G1749E, n = 8). F1661S current density is significantly smaller than WT between –30 and +50 mV (P < 0.05). G1749E current density is significantly smaller than WT between –20 and +30 mV (P < 0.05). (b) Voltage dependence of activation. The voltage dependence of channel activation was estimated by measuring peak sodium current during a variable test potential from a holding potential of –120 mV. The current at each membrane potential was divided by the electrochemical driving force for sodium ions and normalized to the maximum sodium conductance. (c) Voltage dependence of inactivation. The two-pulse protocol illustrated by Inset was used to examine channel availability after conditioning at various potentials. Currents were normalized to the peak current amplitude. (d) Recovery from fast inactivation. Channels were inactivated by a 100-ms pulse then stepped to –120 mV for various durations. Currents were normalized to the peak current amplitude measured during the inactivation pulse and fitted to a two-exponential function, It/Imax = A1·[1 – exp(–t/τ1) + A2·[1 – exp(–t/τ2), generating fast and slow recovery time constants. Fit parameters for all experiments are provided in Table 1.
Fig. 4.
Fig. 4.
Noninactivating sodium currents. Sodium current was elicited by a 200-ms depolarization from –120 to 10 mV. TTX-sensitive currents were obtained by digital subtraction of sodium currents recorded before and after TTX addition. Peak sodium currents were normalized. Inset shows an expanded y axis scaled to emphasize the relative proportion of noninactivating current.
Fig. 5.
Fig. 5.
Slow inactivation properties of WT and mutant SCN1A channels. (a) Onset of slow inactivation. Cells were depolarized to –10 mV for durations ranging from 1 ms to 100 s, allowed to recover from fast inactivation at –120 mV for 50 ms, and subjected to a –10-mV test pulse. (b) Steady-state slow inactivation after a 30-s depolarization to potentials between –140 and –10 mV. (c) Recovery from slow inactivation. Cells were conditioned at –10 mV for 30 s, allowed to recover from inactivation at –120 mV for 0.1–100 s, and immediately tested at –10 mV. Because the intermediate recovery period always exceeded 100 ms, effects of fast inactivation were negligible. All data were fitted to a two-exponential (see Fig. 3 legend) or a Boltzmann function (I/Imax = {1 + exp[(V – V1/2)/k]}–1), where V denotes the stepping potential, V1/2 denotes the stepping potential where half-maximal slow inactivation is achieved, and k is the slope factor of the fitting curve. Fit parameters for all experiments are provided in Table 2.
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