Abstracts

Rationale: We recently reported a missense mutation (gcm) in exon 24 of the T-type calcium channel Cacna1h gene in Genetic Absence Epilepsy Rats from Strasbourg (GAERS) which segregated co-dominantly with seizure expression in F₂ progenies derived from GAERS and non-epileptic control (NEC) rats. We further identified two major Cacna1h splice variants, either with or without exon 25 in rat brain tissue. The gcm specifically resulted in a gain of function effect (faster recovery from channel inactivation and greater charge transference during high-frequency burst firing) in a splice variant-specific manner, requiring the presence of exon 25 to manifest its effects. The current study aims to extend these novel findings by examining the effect of this gcm mutation on the relative expression of the Ca_v3.2 splice variants. Methods: RNA samples from the somatosensory cortex and thalamus of age-matched GAERS and their NEC counterparts, as well as the F₂ progeny of GAERS×NEC crosses, were isolated and reversed transcribed into cDNA. Quantitative real time-PCR was used to measure the relative expression of mRNA for Ca_v3.2 splice variants. The relationship between mRNA levels, genotype and seizure activity in the F₂ animals was assessed by non-parametric Spearman’s correlations. Results: There were no significant differences in the relative copy number of Ca_v3.2 mRNA [calculated from the sum of Ca_v3.2 (+25) and Ca_v3.2 (-25) mRNA levels] between NEC and GAERS animals (p>0.05, n=3-7 for both strains) at either pre-epileptic (6 weeks) or epileptic (>18 weeks) ages. However, the ratio of Ca_v3.2 (+25)/Ca_v3.2 (-25) splice variants was ~1.6-fold greater in the thalamocortical structures of both pre-epileptic and epileptic GAERS compared to NEC animals (p<0.05, n=3-7 for both strains). In the somatosensory cortex and thalamus of F₂ animals, the Ca_v3.2 (+25)/Ca_v3.2 (-25) expression ratio was significantly increased (~3.8-fold) in individuals homozygous for the gcm (n=10-12) compared to those null for the gcm (n=8, p<0.001). This increase in Ca_v3.2 (+25)/Ca_v3.2 (-25) ratio was due to a significant down-regulation (~74%) of Ca_v3.2 (-25) splice variant mRNA (p<0.01). In addition, there was a significant positive correlation between Ca_v3.2 (+25)/Ca_v3.2 (-25) expression ratio in the somatosensory cortex and the number of seizures in F₂ animals (R=0.46, p=0.04). Conclusions: The presence of the gcm mutation is associated with a concomitant change in the expression ratios of the splice variants, with a greater proportion of the variant in which the mutation has a gain-of-function effect; Ca_v3.2 (+25). Given the gcm is located 13 amino acids upstream from the exon 25 splicing site, we are currently investigating the underlying relationship between Cacna1h splicing events and the mechanisms of the pro-epileptic effect of the mutation within the thalamocortical circuitry.