A Cav3.2 T-type calcium channel point mutation has splice-variant-specific effects on function and segregates with seizure expression in a polygenic rat model of absence epilepsy

Abstract

Low-voltage-activated, or T-type, calcium (Ca(2+)) channels are believed to play an essential role in the generation of absence seizures in the idiopathic generalized epilepsies (IGEs). We describe a homozygous, missense, single nucleotide (G to C) mutation in the Ca(v)3.2 T-type Ca(2+) channel gene (Cacna1h) in the genetic absence epilepsy rats from Strasbourg (GAERS) model of IGE. The GAERS Ca(v)3.2 mutation (gcm) produces an arginine to proline (R1584P) substitution in exon 24 of Cacna1h, encoding a portion of the III-IV linker region in Ca(v)3.2. gcm segregates codominantly with the number of seizures and time in seizure activity in progeny of an F1 intercross. We have further identified two major thalamic Cacna1h splice variants, either with or without exon 25. gcm introduced into the splice variants acts "epistatically," requiring the presence of exon 25 to produce significantly faster recovery from channel inactivation and greater charge transference during high-frequency bursts. This gain-of-function mutation, the first reported in the GAERS polygenic animal model, has a novel mechanism of action, being dependent on exonic splicing for its functional consequences to be expressed.

Figure 1.

Representative EEG traces from m/m (a, d), +/m (b, e), and m/m (c, f) animals over a 10 s period (a–c) and a 5 min period (d–f). +/+ animals are null for the R1584P mutation (gcm), +/m animals carry one copy of the mutation, and m/m animals are homozygous for the gcm mutation.

Figure 2.

The gcm mutation positively correlates with the epileptic phenotype in double-crossed (F2) GAERS versus NEC rats. a, Percentage of recording time spent in seizure activity. Animals homozygous for the mutation spend more time in seizure activity than animals null for the gcm (p < 0.05, Mann–Whitney one-tailed test). b, Number of seizures. Animals homozygous for the gcm experience more seizures than animals null for the mutation (p < 0.05, Mann–Whitney one-tailed test). c, The interval between the seizures was significantly shorter for animals homozygous for the mutation compared with animals null for the mutation (p < 0.05, Mann–Whitney one-tailed test). d, The length of individual seizures did not significantly differ between the genotypes (p > 0.05, Mann–Whitney one-tailed test). e, The cycle frequency of the spike-and-wave discharges (hertz) did not significantly differ between the genotypes (p > 0.05, Mann–Whitney one-tailed test). +/+ animals are null for the gcm, +/m animals have one copy of the gcm, and m/m animals are homozygous for the gcm. Data are expressed as mean ± SEM. *p < 0.05.

Figure 3.

Differential expression of Ca_v3.2 splice variants in NEC and GAERS animals. Exon 25 of the rat Cacna1h gene is alternatively spliced to produce Ca_v3.2 (+25) and Cav3.2 (−25) isoforms. The Ca_v3.2 (−25) variant channels have a lysine residue at position 1598. This lysine residue is replaced by the 7 aa sequence (STFPNPE) in the Ca_v3.2 (+25) variant. The R1584P mutation (gcm) site is located 13 aa upstream of the beginning of exon 25 region (underlined arginine residue).

Figure 4.

The gcm accelerates rate of recovery from inactivation in the Ca_v3.2 (+25) splice variant. a, b, The conductance (filled symbols) of Ca_v3.2 (+25) (a) and Ca_v3.2 (−25) (b) and steady-state inactivation (open symbols) of Ca_v3.2 (+25) (a) and Ca_v3.2 (−25) (b) were not significantly altered by the gcm. Insets (a, b) show overlaid gcm and wild-type macroscopic currents during a 150 ms depolarizing pulse from a holding potential of −110 to −20 mV. Activation and inactivation kinetics of Ca_v3.2 (+25) (a, inset) and Ca_v3.2 (−25) (b, inset) splice variant currents are not affected by the gcm. Ca_v3.2 conductance was calculated from currents recorded during a series of depolarizing steps from a holding potential of −110 mV to various membrane potentials and normalized to maximum conductance. Steady-state inactivation was calculated from Ca_v3.2 currents recorded during a test pulse to −30 mV directly after a 2 s inactivating prepulse of varying membrane potentials and normalized to peak current. c, d, The effect of the gcm on fractional recovery (determined by the ratio of the peak current at the test pulse to the peak current at the prepulse and fitted to a double exponential) is shown for Ca_v3.2 (+25) (c) and Ca_v3.2 (−25) (d). Ca_v3.2 currents were recorded during test voltage pulses from a holding potential of −110 to −30 mV after an inactivating prepulse, with an increasing interpulse interval. e, f, Representative traces obtained at test pulses after 160, 320, 640, and 1280 ms interpulse intervals are shown for Ca_v3.2 (+25) (e) and Ca_v3.2 (−25) (f) currents. Normalized Ca_v3.2 (+25) currents from 80 to 2560 ms interpulse intervals were significantly increased in the gcm [80 ms: wild type, 0.25 ± 0.02; gcm, 0.31 ± 0.02 (p < 0.05); 160 ms: wild type, 0.35 ± 0.02; gcm, 0.45 ± 0.02 (p < 0.01); 320 ms: wild type, 0.52 ± 0.03; gcm, 0.67 ± 0.03 (p < 0.005); 640 ms: wild type, 0.70 ± 0.04; gcm, 0.92 ± 0.04 (p < 0.005); 1280 ms: wild type, 0.94 ± 0.05; gcm, 1.12 ± 0.05 (p < 0.05); 2560: wild type, 1.04 ± 0.04; gcm, 1.16 ± 0.04 (p < 0.05); wild type, n = 11; gcm, n = 12].

Figure 5.

The gcm increases the charge transference of Ca_v3.2 (+25) during high-frequency burst depolarizing trains. a–c, Representative traces of Ca_v3.2 (+25) wild-type (a) and Ca_v3.2 (+25) gcm (b) currents recorded during high-frequency depolarizing train pulses (125 Hz for 80 ms) from −70 to −20 mV occurring in bursts (5 Hz for 1 s) (c). Charge transference of Ca_v3.2 during each burst was divided by the peak current on first pulse of the first burst to account for variations in current magnitude. d, In Ca_v3.2 (+25), the gcm significantly increased the charge transference factor in all subsequent bursts after one 125 Hz burst. e, In Ca_v3.2 (−25), the gcm had no significant effect on the charge transference factor. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, significant difference between charge transference factors (ANOVA).