Abstracts

Rationale: Previous studies in our laboratory used two common inbred strains of mice, B6 (relatively seizure resistant) and D2 (relatively seizure susceptible), to map the location of genes which influence the robust strain difference in seizure threshold. A locus of large effect was mapped to distal Chr.1 and then confirmed and fine-mapped with congenic strains. A critical interval of about 6 Mb was defined as containing the causative gene(s) and this interval was reduced to less than 200 Kb with an in vivo BAC transgenesis strategy. Of the 5 tandem genes on the BAC construct, Kcnj10 is considered the most likely seizure susceptibility candidate not only by virtue of its biological function as the gene which encodes a potassium buffering protein in glial cells, but also because of a non-synonymous single nucleotide polymorphism (SNP) producing a strain variation in the protein C-terminus at amino acid position 262 between B6 (Threonine) and D2 (Serine). Methods: We performed electrophysiological measurements on cortical astrocytes in culture and hippocampal astrocytes in slice preparations using tissue from B6 and D2 parental mice as well as from Kcnj10-BAC transgenic mice. To confirm an effect of the T262S Kcnj10 variant in vivo, we created a knock-in mouse strain in which a SNP leading to the D2-derived Ser262 variant was engineered into B6-derived embryonic stem cells and we analyzed the strain for seizure susceptibility. Results: Electrophysiological studies in vitro reveal that potassium currents elicited from B6 astroctyes are substantially larger than those elicited from D2 astrocytes. Results also show that application of barium inhibits currents in B6 astrocytes but has a reduced effect on currents measured in D2 astrocytes. Potassium buffering capacity of D2 cortical astrocytes is also impaired relative to B6. Results of seizure testing document that homozygous Ser262 knock-in mice have a significantly lower threshold for induction of electroshock seizures compared to wild type Thr262 mice. Heterozygous Thr/Ser mice have an intermediate seizure phenotype. Conclusions: Overall, results are consistent with a role for Kcnj10 Ser262 in enhancing seizure susceptibility. Studies documenting that the biophysical properties of Kcnj10-containing channels are similar for the two subunit isoforms suggest that enhanced seizure susceptibility in vivo is related to a spatial or temporal alteration of protein expression. Studies are currently in progress to elucidate this mechanism.