Abstracts

Rationale: Alterations in hyperpolarization-activated, cyclic nucleotide-gated (HCN) channel expression and function have been reported following status epilepticus (SE) in the pilocarpine-induced epileptic rat model (Jung et al., 2010, 2011; Williams et al., 2015). We have shown in these epileptic rats that the altered HCN properties in hippocampal principal neurons (such as changes in surface expression and current (Ih) amplitude) are regulated by phosphorylation. However, the actual phosphorylation sites (phosphosites) on HCN channel proteins that are altered in epilepsy have not been identified. In our current study, our objective was to identify these phosphosites on HCN1 and HCN2 channels through tandem mass spectrometry (MS). Methods: For rat samples, we homogenized CA1 hippocampal tissues from male P90-101 epileptic rats (5-6 weeks post SE, n = 7) and age-matched naﶥ controls (n = 8). For human samples, we homogenized neocortical tissue from 6 human patients with refractory epilepsy undergoing resection of the epileptogenic zone. HCN1 enrichment was achieved by immunoprecipitating with a monoclonal a-HCN1 antibody. After in-gel digestion by trypsin, samples were submitted to the University of Washington Proteomics Resource facility (UWPR) for tandem mass spectrometry. Results: Mass spectrometry analyses of the rat samples revealed 6 phosphorylation sites on HCN1 in both our experimental and control samples, located in the cytoplasmic side of the protein at both the N- and C-terminal regions. No statistically significant differences were observed in the frequency of phosphosite identification in epileptic versus naﶥ samples. For HCN2 channels, we observed 10 phosphorylation sites. Interestingly, we found a statistically significant reduction in the frequency of phosphorylation at one phosphosite in the C-terminus in epileptic tissue. Analyses of the human neocortical epileptogenic zone tissue revealed: on HCN1 channels, five phosphosites, three of which were homologous to rat phosphosites, one which was novel, and one with no corresponding homolog on the rat sequence; on HCN2 channels, nine phosphosites, eight of which corresponded to rat homologs, and one which was novel. Conclusions: In summary, we have identified novel phosphosites on both HCN1 and HCN2 channels, and provide the first evidence of differential phosphorylation of HCN2 channels in an animal model of epilepsy. Also, we show for the first time that phosphorylation of human neocortical HCN channels occurs at predominantly the same phosphosites as in rat hippocampal HCN channels. Further quantitative exploration of HCN channel phosphorylation may lead to better understanding of the downregulation of HCN channel expression and biophysical properties that occurs during epileptogenesis and in chronic epilepsy, and that contributes to neuronal hyperexcitability. Funding: NIH R01 NS050229