Epilepsy-associated dysfunction in the voltage-gated neuronal sodium channel SCN1A

Abstract

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel alpha1 subunit (NaV1.1), are associated with at least two forms of epilepsy, generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI). We examined the functional properties of four GEFS+ alleles and one SMEI allele using whole-cell patch-clamp analysis of heterologously expressed recombinant human SCN1A. One previously reported GEFS+ mutation (I1656M) and an additional novel allele (R1657C), both affecting residues in a voltage-sensing S4 segment, exhibited a similar depolarizing shift in the voltage dependence of activation. Additionally, R1657C showed a 50% reduction in current density and accelerated recovery from slow inactivation. Unlike three other GEFS+ alleles that we recently characterized, neither R1657C nor I1656M gave rise to a persistent, noninactivating current. In contrast, two other GEFS+ mutations (A1685V and V1353L) and L986F, an SMEI-associated allele, exhibited complete loss of function. In conclusion, our data provide evidence for a wide spectrum of sodium channel dysfunction in familial epilepsy and demonstrate that both GEFS+ and SMEI can be associated with nonfunctional SCN1A alleles.

Figure 1.

WT and mutant whole-cell currents from tsA201 cells. a-c, Cells were stepped to various potentials between -80 and +60 mV in 10 mV intervals from a holding potential of -120 mV (see Fig. 2b). All experiments were performed in the whole-cell patch-clamp configuration at room temperature 24-72 hr after transfection of the indicated DNA. Calibration: 1 nA vs. 2 msec. d, Inactivation time constants of WT and mutant SCN1A currents. The decay phase of voltage-sensitive inward currents was fitted with a two-exponential function, as described in Materials and Methods, generating τ_fast (open symbols) and τ_slow (closed symbols). No significant difference between WT and mutant currents was found.

Figure 2.

Biophysical characterization of GEFS+-associated mutants. a, Current-voltage relationships of whole-cell currents from transiently transfected cells. Currents were elicited by test pulses to various potentials (b, inset) and normalized to cell capacitance. WT, n = 17; I1656M, n = 8; R1657C, n = 17. R1657C current density is significantly smaller than WT between -30 and +40 mV (p < 0.005). b, Voltage dependence of activation of whole-cell currents. 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 outlined in the 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 msec pulse and stepped back to -120 mV for increasingly long periods. Currents were normalized to the peak current amplitude measured during the inactivation pulse and fitted to a two-exponential function generating a fast and a slower recovery time constant. Fit parameters for all experiments shown are provided in Table 1.

Figure 3.

Slow inactivation properties of I1656M and R1657C. a, Onset of slow inactivation. Cells were stepped to -10 mV for 0.001-100 sec, allowed to recover from fast inactivation at -120 mV for 50 msec, and subjected to a -10 mV test pulse. b, Steady-state slow inactivation after a 30 sec depolarization to potentials between -140 and -10 mV. Cells were allowed to recover from fast inactivation at -120 mV for 50 msec before the actual test pulse to -10 mV. c, Recovery from slow inactivation. Cells were conditioned at -10 mV for 30 sec, allowed to recover at -120 mV for 0.1-100 sec, and immediately tested at -10 mV. Because the intermediate recovery period always exceeded 100 msec, effects of fast inactivation were deemed to be negligible. All data were fitted to a two-exponential or Boltzmann function, as described in Materials and Methods. Fit parameters for all experiments shown are provided in Table 2.