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. 2005 Dec 1;569(Pt 2):433-45.
doi: 10.1113/jphysiol.2005.094326. Epub 2005 Oct 6.

Sodium channel dysfunction in intractable childhood epilepsy with generalized tonic-clonic seizures

Thomas H Rhodes  1 , Carlos G Vanoye, Iori Ohmori, Ikuo Ogiwara, Kazuhiro Yamakawa, Alfred L George Jr
Affiliations

Sodium channel dysfunction in intractable childhood epilepsy with generalized tonic-clonic seizures

Thomas H Rhodes et al. J Physiol. .

Abstract

Mutations in SCN1A, the gene encoding the brain voltage-gated sodium channel alpha(1) subunit (Na(V)1.1), are associated with genetic forms of epilepsy, including generalized epilepsy with febrile seizures plus (GEFS+ type 2), severe myoclonic epilepsy of infancy (SMEI) and related conditions. Several missense SCN1A mutations have been identified in probands affected by the syndrome of intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC), which bears similarity to SMEI. To test whether ICEGTC arises from molecular mechanisms similar to those involved in SMEI, we characterized eight ICEGTC missense mutations by whole-cell patch clamp recording of recombinant human SCN1A heterologously expressed in cultured mammalian cells. Two mutations (G979R and T1709I) were non-functional. The remaining alleles (T808S, V983A, N1011I, V1611F, P1632S and F1808L) exhibited measurable sodium current, but had heterogeneous biophysical phenotypes. Mutant channels exhibited lower (V983A, N1011I and F1808L), greater (T808S) or similar (V1611F and P1632S) peak sodium current densities compared with wild-type (WT) SCN1A. Three mutations (V1611F, P1632S and F1808L) displayed hyperpolarized conductance-voltage relationships, while V983A exhibited a strong depolarizing shift in the voltage dependence of activation. All mutants except T808S had hyperpolarized shifts in the voltage dependence of steady-state channel availability. Three mutants (V1611F, P1632S and F1808L) exhibited persistent sodium current ranging from approximately 1-3% of peak current amplitude that was significantly greater than WT-SCN1A. Several mutants had impaired slow inactivation, with V983A showing the most prominent effect. Finally, all of the functional alleles exhibited reduced use-dependent channel inhibition. In summary, SCN1A mutations associated with ICEGTC result in a wide spectrum of biophysical defects, including mild-to-moderate gating impairments, shifted voltage dependence and reduced use dependence. The constellation of biophysical abnormalities for some mutants is distinct from those previously observed for GEFS+ and SMEI, suggesting possible, but complex, genotype-phenotype correlations.

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Figures

Figure 1
Figure 1. Location of SCN1A mutations
Schematic diagram illustrating the transmembrane topology of a voltage-gated sodium channel and location of ICEGTC mutations characterized in this study. Filled symbols represent non-functional mutations and open symbols represent the location of mutations with preserved activity.
Figure 2
Figure 2. Representative WT and mutant whole-cell SCN1A sodium currents
Whole-cell currents recorded from tsA201 cells transiently expressing the indicated alleles during voltage steps to various potentials between −80 and +60 mV in 10 mV intervals from a holding potential of −120 mV (see pulse protocol illustrated in Fig. 3B). Vertical and horizontal scale bars represent 0.5 nA and 2 ms, respectively. The lower right panel illustrates superimposed averaged whole-cell sodium currents evoked by depolarization to 0 mV from a holding potential of −120 mV and normalized to peak current amplitude. Values in parentheses indicate the number of independent cells used to obtain each averaged current trace.
Figure 3
Figure 3. Inactivation and activation properties of ICEGTC-associated mutants
A, current–voltage relationships of whole-cell currents from transiently transfected tsA201 cells. Currents were elicited by test pulses to various potentials (B, inset) and normalized to cell capacitance (WT-SCN1A, n = 14; T808S, n = 12; P1632S, n = 10; V1611F, n = 12; F1808L, n = 11; N1011I, n = 6; V983A, n = 9). T808S current density is significantly larger than WT between −50 and +60 mV (P < 0.05). P1632S current density is significantly larger than WT between −40 and −20 mV (P < 0.05). F1808L and N1011I current density is significantly smaller than WT between −20 and +60 mV (P < 0.05). V983A current density is significantly smaller than WT between −30 and 0 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 step 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 ms pulse and then stepped 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 fast and slow recovery time constants. Fitted values from these experiments are provided in Table 1.
Figure 4
Figure 4. Altered voltage dependence of activation and inactivation
A, plots of steady-state inactivation and conduction–voltage curves for WT-SCN1A (▪) and P1632S (○). B, similar plots for WT-SCN1A (▪) and V983A (⊳). Data are the same as shown in Fig. 3B and C, with fit parameters given in Table 1.
Figure 5
Figure 5. Voltage dependence of fast inactivation time constants and fractional amplitudes
A, inactivation time constants for WT and mutant SCN1A currents plotted against test voltage. The decay phase of the transient sodium current was fitted with a two-exponential function as described in the Methods. Fast time constants (τ1) were significantly smaller for P1632S (−10 to −30 mV, P < 0.005; and 0 mV, P < 0.05), V983A (−10 and +30 mV, P < 0.05), F1808L (−30 mV, P < 0.05), and V1611F (−30 mV, P < 0.05). Significantly larger τ1 values were observed for T808S (+20 mV, P < 0.05) and V1611F (+30 mV, P < 0.05). B, fractional amplitudes of the slower component (τ2) of fast inactivation plotted against voltage (values significantly different from WT are indicated as *P < 0.05 and **P < 0.005).
Figure 6
Figure 6. ICEGTC mutants exhibit persistent sodium current
A, sodium current was measured at the end of a 200 ms depolarization to −10 mV from a holding potential of −120 mV. TTX-sensitive currents were obtained by digital subtraction of sodium currents recorded before and after TTX addition. Sodium currents were normalized to peak amplitude. The inset shows an expanded vertical scaled to emphasize the relative proportion of persistent current. B, persistent sodium current expressed as the percentage of peak current amplitude for WT-SCN1A (n = 9) and the six ICEGTC mutants (n = 4–6). Values significantly different from WT are indicated as *P < 0.05 and **P < 0.005.
Figure 7
Figure 7. Slow inactivation properties of WT and mutant SCN1A channels
A, onset of slow inactivation. Cells were stepped to −10 mV for 0.001–100 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. Cells were allowed to recover from fast inactivation at −120 mV for 50 ms before a test pulse to −10 mV. C, recovery from slow inactivation. Cells were conditioned at −10 mV for 30 s, allowed to recover at −120 mV for 0.1–100 s, and immediately tested at −10 mV. Because the recovery period always exceeded 100 ms, effects of fast inactivation were considered negligible. Data were fitted to a two-exponential (A and C) or a Boltzmann function (B), as described in the Methods. Fitted values from all experiments are provided in Table 2.
Figure 8
Figure 8. Use dependence of WT and mutant SCN1A channels
A, frequency dependence of WT-SCN1A and ICEGTC mutants. Cells were stimulated with depolarizing pulse trains (100 pulses, 5 ms, 0 mV) from a holding potential of −120 mV at the indicated frequencies. A recovery interval (15 s, −120 mV) followed each pulse train experiment. Currents were normalized to the value recorded after the first pulse in each frequency train for the corresponding allele and plotted on a log scale. Data are means ± s.e.m. from 3 or 4 cells. Significant differences (P < 0.05) from WT-SCN1A were observed for V983A (40–80 Hz), V1611F (60–80 Hz) and F1808L (50–80 Hz). B, residual normalized sodium current after the 60th pulse during a 60 Hz pulse train (n = 5–7 cells). Significant differences from WT-SCN1A are indicated as *P < 0.05 and **P < 0.005.
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