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. 2001 Oct 1;21(19):7481-90.
doi: 10.1523/JNEUROSCI.21-19-07481.2001.

Functional effects of two voltage-gated sodium channel mutations that cause generalized epilepsy with febrile seizures plus type 2

J Spampanato  1 , A Escayg, M H Meisler, A L Goldin
Affiliations

Functional effects of two voltage-gated sodium channel mutations that cause generalized epilepsy with febrile seizures plus type 2

J Spampanato et al. J Neurosci. .

Abstract

Two mutations that cause generalized epilepsy with febrile seizures plus (GEFS+) have been identified previously in the SCN1A gene encoding the alpha subunit of the Na(v)1.1 voltage-gated sodium channel (Escayg et al., 2000). Both mutations change conserved residues in putative voltage-sensing S4 segments, T875M in domain II and R1648H in domain IV. Each mutation was cloned into the orthologous rat channel rNa(v)1.1, and the properties of the mutant channels were determined in the absence and presence of the beta1 subunit in Xenopus oocytes. Neither mutation significantly altered the voltage dependence of either activation or inactivation in the presence of the beta1 subunit. The most prominent effect of the T875M mutation was to enhance slow inactivation in the presence of beta1, with small effects on the kinetics of recovery from inactivation and use-dependent activity of the channel in both the presence and absence of the beta1 subunit. The most prominent effects of the R1648H mutation were to accelerate recovery from inactivation and decrease the use dependence of channel activity with and without the beta1 subunit. The DIV mutation would cause a phenotype of sodium channel hyperexcitability, whereas the DII mutation would cause a phenotype of sodium channel hypoexcitability, suggesting that either an increase or decrease in sodium channel activity can result in seizures.

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Figures

Fig. 1.
Fig. 1.
Sample sodium channel currents from wild-type rNav1.1 and GEFS+ 2 mutant channels. Currents were recorded for the wild-type rNav1.1, DII (T875M), and DIV (R1648H) mutants expressed as α subunits alone and as α + β1 subunits. Mutant and wild-type channels were expressed in Xenopusoocytes, and currents were recorded at 20°C by using the cut-open oocyte 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: 2 msec, 200 nA.
Fig. 2.
Fig. 2.
Voltage dependence of activation and steady-state inactivation for wild-type rNav1.1 and GEFS+ 2 mutant channels. The voltage dependencies of activation (circles) and inactivation (diamonds) were determined for the wild-type rNav1.1 (white symbols), DII (gray symbols), and DIV (black symbols) mutants expressed as α subunits alone (A) and as α + 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. Conductance values were calculated by dividing the peak current amplitude by the driving force at each potential and normalizing to the maximum conductance, as described in Materials and Methods. The values shown are averages; the error bars indicate SD. The data were fit with a two-state Boltzmann equation, and the parameters of the fits are shown in Table 1. The voltage dependence of inactivation was determined by using a two-step protocol in which a conditioning pulse was applied from a holding potential of −100 mV, consisting of 100 msec 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; the error bars indicate SD. The data were fit with a two-state Boltzmann equation, and the parameters of the fits are shown in Table 1.
Fig. 3.
Fig. 3.
Kinetics of fast inactivation of wild-type rNav1.1 and GEFS+ 2 mutant channels. Sodium currents were recorded from oocytes expressing wild-type rNav1.1 (white symbols andbars), DII (gray symbols andbars), and DIV (black symbols andbars) channels, as described in the legend to Figure 1. Current traces were fit with either a single or a double exponential equation, as described in Materials and Methods, and time constants for the fast (τfast, circles) and slow (τslow, triangles) components of fast inactivation are plotted on a logarithmic scale in thetoppanels for α subunits alone (A) and α + β1 subunits (B). The bottom panels indicate the fraction of current inactivating with τfast. In all cases the sum of the components is one. The values shown are averages; the error bars indicate SD. Sample sizes were rNav1.1 α (4), DII α (5), DIV α (4), rNav1.1 α + β1 (4), DII α + β1 (5), DIV α + β1 (7).
Fig. 4.
Fig. 4.
Recovery from inactivation for wild-type rNav1.1 and GEFS+ 2 mutant channels. Recovery from inactivation was determined by using three separate two-pulse protocols for wild-type rNav1.1 (white symbols), DII (gray symbols), and DIV (black symbols) channels. Each protocol was performed with a holding potential of −100 mV and consisted of a conditioning depolarization to −5 mV for 50 msec (which inactivated >95% of the channels), a decreasing recovery time interval at −100 mV, and a test depolarization to −5 mV. The three protocols differed only in the maximum length of recovery time and the time interval by which that recovery period decreased: 25 msec maximum and 1 msec decrements in the early protocol, 200 msec maximum and 5 msec decrements in the intermediate protocol, and 3000 msec maximum and 100 msec decrements in the late protocol. 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 α subunits alone (A) and α + β1 subunits (B). The values shown are averages; the error bars indicate SD.
Fig. 5.
Fig. 5.
Frequency dependence of wild-type rNav1.1 and GEFS+ 2 mutant channels. Use dependence was analyzed at 10, 20, and 39 Hz for wild-type rNav1.1 (white symbols), DII (gray symbols), and DIV (black symbols) channels. Currents were elicited at each frequency by using 17.5 msec depolarizations to −10 mV from a holding potential of −100 mV. Each protocol was performed until an equilibrium current had been reached: 2 sec at 10 Hz, 2.5 sec at 20 Hz, and 2.56 sec at 39 Hz. Peak current amplitudes were normalized to the initial peak current amplitude and plotted against pulse number for α subunits alone (A,circles) and α + β1 subunits (B,diamonds). The values shown are averages; the error bars indicate SD. Sample sizes were rNav1.1 α (5), DII α (5), DIV α (5), rNav1.1 α + β1 (3), DII α + β1 (5), DIV α + β1 (5).
Fig. 6.
Fig. 6.
Slow-gated properties of wild-type rNav1.1 and GEFS+ 2 mutant channels. The slow-gated properties were determined for the wild-type rNav1.1 (white circles), DII (gray circles), and DIV (black circles) mutants expressed as α + β1 subunits. The voltage dependence of slow inactivation (A) was analyzed by using a two-step protocol consisting of 60 sec depolarizations from a holding potential of −120 mV to a range of potentials between −120 and −10 mV, followed by a hyperpolarization to −120 mV for 20 msec to allow for recovery from fast inactivation and a test pulse to −5 mV. The data were fit with a two-state Boltzmann equation, as described in Materials and Methods, and the parameters of the fits are shown in Table 2. The recovery from slow inactivation (B) was analyzed by using two separate two-pulse protocols consisting of a 60 sec depolarization to −5 mV from a holding potential of −120 mV, followed by a variable recovery time at −120 mV, a hyperpolarization to −120 mV to allow for recovery from fast inactivation, and a test depolarization to −5 mV. The data were fit with a double exponential equation, as described in Materials and Methods, and the parameters of the fits are shown in Table 2. The rate of entry into the slow-inactivated state (C, D) was analyzed by using a two-step protocol consisting of a variable length conditioning pulse at either −45 or −10 mV from a holding potential of −120 mV, followed by a hyperpolarization to −120 mV to allow for recovery from fast inactivation and a test depolarization to −5 mV. The data were fit with a double exponential decay, as described in Materials and Methods, and the parameters of the fits are shown in Table 2. For each graph the values shown are averages; the error bars indicate SD.
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