A Ca_v3.2 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

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.

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).

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].