An epilepsy mutation in the sodium channel SCN1A that decreases channel excitability

Abstract

Mutations in three voltage-gated sodium channel genes, SCN1A, SCN2A, and SCN1B, and two GABAA receptor subunit genes, GABRG2 and GABRD, have been identified in families with generalized epilepsy with febrile seizures plus (GEFS+). A novel mutation, R859C, in the Nav1.1 sodium channel was identified in a four-generation, 33-member Caucasian family with a clinical presentation consistent with GEFS+. The mutation neutralizes a positively charged arginine in the domain 2 S4 voltage sensor of the Nav1.1 channel alpha subunit. This residue is conserved in mammalian sodium channels as well as in sodium channels from lower organisms. When the mutation was placed in the rat Nav1.1 channel and expressed in Xenopus oocytes, the mutant channel displayed a positive shift in the voltage dependence of sodium channel activation, slower recovery from slow inactivation, and lower levels of current compared with the wild-type channel. Computational analysis suggests that neurons expressing the mutant channel have higher thresholds for firing a single action potential and for firing multiple action potentials, along with decreased repetitive firing. Therefore, this mutation should lead to decreased neuronal excitability, in contrast to most previous GEFS+ sodium channel mutations, which have changes predicted to increase neuronal firing.

Figure 1.

The R859C mutation cosegregates with disease in a large GEFS+ family. DNA was obtained from eight family members in three generations. Shading indicates diagnosis. +/m denotes individuals with mutation. +/+ denotes individuals without mutation. The arrow indicates the proband. FS, Febrile seizures.

Figure 2.

Location and conservation of R859C. A, R859C results in the substitution of a positively charged arginine residue in the S4 segment of domain 2. Published GEFS+ mutations (filled circles) are distributed throughout SCN1A. B, R859 is invariant in mammalian sodium channel genes and in sodium channels from lower organisms.

Figure 3.

Currents through wild-type and R859C channels. Currents were recorded through the wild-type rNa_v1.1 and R859C channels expressed as α subunits alone and as α plus β1 subunits. Mutant and wild-type channels were expressed in Xenopus oocytes, and currents were recorded at 20°C using the two-electrode voltage clamp as described in Materials and Methods. Membrane depolarizations from a holding potential of −100 mV to a range of potentials from −50 to +50 mV in 10 mV increments are shown. Calibration: 1 ms, 1 μA.

Figure 4.

Voltage dependence and kinetics for wild-type and R859C channels. The voltage dependences of activation (circles) and inactivation (triangles) were determined for the wild-type rNa_v1.1 (filled symbols) and R859C (open symbols) channels expressed as α subunits alone (A) and as α plus β1 subunits (B). Sodium currents were recorded from a holding potential of −100 mV by depolarizations to a range of potentials from −95 to +50 mV in 5 mV increments. Normalized conductances were calculated by dividing the conductance at each potential by the maximum conductance, as described in Materials and Methods. The values shown are averages, and the error bars are SDs. The data were fit with a two-state Boltzmann equation, and the parameters of the fits are shown in Table 2. The voltage dependence of inactivation was determined using a two-step protocol in which a conditioning pulse was applied from a holding potential of −100 mV, consisting of 500 ms depolarizations to a range of potentials from −100 to +15 mV in 5 mV increments, followed by a test pulse to −5 mV. The peak current amplitude during each test pulse was normalized to the current amplitude of the first test pulse and plotted as a function of the conditioning pulse potential. The values shown are averages, and the error bars are SDs. The data were fit with a two-state Boltzmann equation, and the parameters of the fits are shown in the Table 2. The kinetics of inactivation was determined for wild-type rNa_v1.1 (filled symbols and bars) and R859C (open symbols and bars) channels by fitting the current traces with a double exponential equation as described in Materials and Methods. The time constants for the fast (τ_fast; triangles) and slow (τ_slow; circles) components of fast inactivation are plotted on a logarithmic scale in the top panels for α subunits alone (C) and α plus β1 subunits (D). The bottom panels indicate the fraction of current inactivating with τ_fast. In all cases, the sum of the components is one. The values shown are averages, and the error bars indicate SDs. Sample sizes were rNa_v1.1 α, 5; R859C α, 5; rNa_v1.1 α plus β1, 5; R859C α plus β1, 5.

Figure 5.

Recovery from inactivation and use dependence for wild-type and R859C mutant channels. Recovery from inactivation was determined using three separate, two-pulse protocols for wild-type rNa_v1.1 (filled symbols) and R859C (open symbols) channels. Each protocol was performed from a holding potential of −100 mV and consisted of a conditioning depolarization to −5 mV for 50 ms (which inactivated >95% of the channels), a decreasing recovery time interval at −100 mV, and a test depolarization to −5 mV. Fractional recovery was calculated by dividing the maximum current amplitude of the test pulse by the maximum current amplitude of the corresponding conditioning pulse. Fractional recovery is plotted on a log scale as a function of time for subunits alone (A) and α plus β1 subunits (B). The values shown are averages, and the error bars are SDs. Use dependence was analyzed at 30 Hz for wild-type rNa_v1.1 (filled symbols) and R859C (open symbols) channels. Currents were elicited using 17.5 ms depolarizations to −5 mV from a holding potential of −100 mV. Each protocol was performed until an equilibrium current had been reached (1.32 s). Peak current amplitudes were normalized to the initial peak current amplitude and plotted against pulse number for a subunits alone (C) and α plus β1 subunits (D). The values shown are averages, and the error bars are SDs. Sample sizes were rNa_v1.1 α, 5; R859C α, 5; rNa_v1.1 α plus β1, 5; R859C α plus β1, 5.

Figure 6.

Slow-gated properties of wild-type and R859C channels. The slow-gated properties were determined for the wild-type rNa_v1.1 (filled circles) and R859C (open circles) channels expressed as α plus β1 subunits. A, The voltage dependence of slow inactivation was analyzed using a two-step protocol in which a conditioning pulse was applied from a holding potential of −120 mV to potentials ranging from −115 to 0 mV in 5 mV increments for a period of 90 s. The conditioning pulse was immediately followed by a hyperpolarization to −120 mV for 20 ms to allow for recovery from fast inactivation and a subsequent test pulse to −5 mV for 17.5 ms to assess slow inactivation. The data were fit with a two-state Boltzmann equation as described in Materials and Methods, and the parameters of the fits and sample sizes are shown in Table 3. B, The rate of entry into the slow inactivated state was analyzed using a two-step protocol with a variable length conditioning pulse followed by a test pulse. The conditioning potential of −10 mV was applied from a holding potential of −120 mV for times ranging from 0 to 29 s. The conditioning pulse was immediately followed by a hyperpolarization to −120 mV for 20 ms to allow for recovery from fast inactivation and a subsequent test pulse to −5 mV. The peak current amplitudes during the subsequent test pulses were normalized to the peak current amplitude during the first test pulse and plotted against the period of the conditioning pulse. The data were fit with a double exponential decay as described in Materials and Methods, and the parameters of the fits and the sample sizes are shown in Table 3. C, Recovery from slow inactivation was analyzed using a two-pulse protocol beginning with a conditioning depolarization from a holding potential of −120 mV to −5 mV for 60 s, which inactivated >95% of the channels. This was followed by a decreasing recovery time interval at −120 mV for recovery times between 100 s and 0 s. The conditioning pulse was immediately followed by a hyperpolarization to −120 mV for 20 ms to allow for recovery from fast inactivation and a subsequent test pulse to −5 mV. Fractional recovery was calculated by dividing the maximum current amplitude during the test pulse by the average maximum current amplitude during three single-step depolarizations to −5 mV recorded before each recovery protocol and plotted against the length of the recovery interval. The data were fit with a double exponential equation as described in Materials and Methods, and the parameters of the fits and the sample sizes are shown in Table 3. For each graph, the values shown are averages, and the error bars are SDs.

Figure 7.

Expression of mixed populations of wild-type and R859C channels. A, Mutant and wild-type channels were expressed in Xenopus oocytes by injection of 20 pg of RNA for either 24 h (α plus β1) or 48 h (α), after which sodium current amplitudes were measured during a depolarization to −5 mV from a holding potential of −100 mV. Sample sizes were wild-type α, 5; R859C α, 5; wild-type α plus β1, 5; R859C α plus β1, 5. B, C, The voltage dependence of activation was determined as described in the legend to Figure 4 for wild-type rNa_v1.1 (filled circles), R859C (open circles), and wild-type rNa_v1.1 plus R859C (open squares) in the absence (B) and presence (C) of the β1 subunit. The values shown are averages, and the error bars are SDs. The data were fit with a two-state Boltzmann equation, and the parameters of the fits and sample sizes are shown in Table 2. D, Recovery from slow inactivation was determined as described in the legend to Figure 6 for the wild-type rNa_v1.1 plus β1 (filled circles), R859C plus β1 (open circles), and wild-type rNa_v1.1 plus R859C plus β1 (open squares). The data were fit with a double exponential equation as described in Materials and Methods, and the parameters of the fits and sample sizes are shown in Table 3. The values shown are averages, and the error bars are SDs.

Figure 8.

Computer simulation of neuronal firing. Action potential thresholds for model neurons with wild-type (A) or R859C (B) sodium channels are shown. Model neurons were injected with increasing amounts of current starting at 130 pA for a duration of 200 ms. The intensity of the stimulus was increased by 10 pA until a single action potential was observed. Steps shown correspond to 10 pA increments. C, Model neurons with R859C mutant sodium channels fire fewer action potentials for a given stimulus compared with those with wild-type sodium channels. A current injection of 120 pA was applied to model neurons for a duration of 200 ms, and the number of action potentials was counted and plotted versus the stimulus intensity. This was repeated for increasing current injections in increments of 10 pA. D, Model neurons were assigned a mixed population ranging from 0 to 100% mutant (100 to 0% wild-type) sodium channels, and the number of action potentials elicited during injection of 150 pA was determined and plotted versus the percentage of mutant channels.