Abstracts

Rationale: Recent evidence implicates the Cav3.2 T-type Ca²⁺ channel in the pathogenesis of genetic absence epilepsy, although whether functional abnormalities in this channel play a causative role is unknown. Linking an absence phenotype to a mutation in this channel would provide a priori case for a causative role. We have previously reported that GAERS (a genetic rat model of absence epilepsy) carry a single nucleotide missense mutation in the highly conserved III-IV linker region of the Cav3.2 T-type Ca²⁺ gene (R1584P) (Powell et. al, AES abstract 2007 A.12). In addition, we demonstrated that the R1584P mutation segregated co-dominantly with the number of seizures and time spent in seizure activity in F₂ progenies derived from second generation crosses of GAERS and non-epileptic NEC rats. This study examines the mechanistic role that Cav3.2 gene alterations contribute to the GAERS epileptic phenotype. Methods: Splice variant screening: Exon scanning was performed on full-length rat thalamic Cav3.2 cDNA libraries to identify sites of alternative splicing. The proportion of each splice variant was then measured using quantitative PCR. Electrophysiology: The R1584P mutation was introduced into the Cav3.2 splice variants using site directed mutagenesis. Cav3.2 splice variant channel function ± R1584P mutation was assessed electrophysiologically in vitro using whole-cell patch clamp technique in HEK293 cells. Results: Two splice variants of the rat Cav3.2 gene (Cacna1h) were identified in the thalamus, differing with respect to the presence [Cav3.2 (+25)] or absence [Cav3.2 (-25)] of exon 25. The location of the R1584P mutation in exon 24 is 13 amino acids upstream from the site of exonic splicing. In adult Wistar rat (n=8) thalamus, Cav3.2 (+25) represents 34.3% and Cav3.2 (-25) represents 48.6% of the total pool of Cav3.2 channels. These splice variants were also similarly distributed in the somatosensory cortex - a critical thalamocortical region believed to be the focus for the origin of seizures in GAERS [Cav3.2 (+25) = 36.9% and Cav3.2 (-25) = 48.6% (n=8)]. Cav3.2 (+25) and Cav3.2 (-25) are thus the two major splice variants commonly expressed in the rat thalamus and somatosensory cortex. Functionally, it was found that the R1584P mutation accelerates the rate of recovery from inactivation specifically in the full-length Cav3.2 (+25) splice variant (p<0.05), resulting in significantly larger Ca²⁺ currents (p<0.05) and potentially leading to an increase in neuronal excitability as well as promoting epileptogenesis. Conversely, these mutation-mediated effects were not observed in the Cav3.2 (-25) splice variant. Conclusions: These findings provide a gain-of-function mechanism to explain the pro-epileptic effect of the R1584P mutation, being completely dependent on exonic splicing for its functional consequences to be expressed. Overall, our data further highlight the dysfunction of Cav3.2 channels as a critical determinant of the pathophysiological mechanisms underlying absence seizures.