Abstracts

Rationale: Previous studies in our laboratory used two common inbred strains of mice, B6 (relatively seizure resistant) and D2 (relatively seizure susceptible) to identify kcnj10 as a putative seizure susceptibility gene. This gene encodes the inwardly rectifying potassium channel Kir4.1 that is differentially expressed in glial cells and regulates both potassium and glutamate buffering in the brain. Impairment of these functions results in hyperexcitability of neurons. Kcnj10 contains 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) mice. The purpose of the present study was to confirm that genetic variations in kcnj10 confer a seizure phenotype (regardless of other mutations/variations in D2) by determining seizure susceptibility and astrocytic Kir4.1 channel function in a knock-in mouse strain in which a SNP leading to the D2-derived Ser262 variant was engineered into B6-derived embryonic stem cells.Methods: Using wild-type and knock-in male mice (8-12 weeks of age), we performed behavioral studies to assay seizure susceptibility and electrophysiological measurements on hippocampal astrocytes in slice preparations to determine functionality of Kir4.1 channels. We also utilized quantitative immunohistochemistry to assess overall levels of Kir4.1 protein in hippocampus of D2 and B6 mice. Animals were used in accordance with IACUC approved protocols at each institution.Results: Results of seizure testing document that homozygous Ser262 knock-in mice have a significantly lower threshold for maximal electroshock seizures compared to wild type Thr262 mice. Heterozygous Thr/Ser mice do not differ from wild type suggesting that the Ser allele is recessive. Although previous biophysical studies demonstrated no difference in ion channel conductance between these two channel isoforms, our whole cell electrophysiological recordings from astrocytes in hippocampal slices reveal that Kir channel function is impaired in astrocytes from homozygous Ser262 knock-in mice. This is reflected by the significantly smaller effects of the Kir channel blocker barium on membrane potentials, on potassium-induced inward currents and on the I/V curves of astrocytes recorded from knock-in mice as compared to those from wild type mice. In complementary immunohistochemical studies, we found that the overall level of total Kir4.1 immunoreactivity was not reduced in the hippocampal region of D2 mice relative to the B6 mice. Taken together with the electrophysiological data this suggests that while transcription/translation of kcnj10 was not affected by the Ser262 variation, trafficking of the channel to the astrocytic membrane may be impaired.Conclusions: In summary, our results are consistent with a role for Kcnj10 Ser262 in enhancing seizure susceptibility due to decreased functional Kir4.1 channels in the astrocytic membrane. Reduced plasma membrane expression of Kir4.1 in astrocytes of Ser262 mice would lead to decreased potassium and glutamate buffering which, in turn, would lead to seizure susceptibility.