Abstracts

Rationale: Previously, we described a loss of surface membrane expression of hyperpolarization-activated, cyclic nucleotide-gated type 1 (HCN1) channels following status epilepticus (SE), contributing to chronic loss of I_h (the current mediated by HCN channels) in a rat model of epilepsy (Jung et al., J. Neurosci., 2010, 2011). We also showed that HCN1 channel surface membrane expression was modulated by phosphorylation (Williams et al., J. Physiol. 2015). We therefore hypothesized that changes in phosphorylation at individual HCN1 phosphosites occur in both the acute and chronic stages of epileptogenesis, the development of epilepsy after a brain insult. Methods: We collected CA1 hippocampal tissue from male Sprague Dawley rats one hour after the onset of pilocarpine-induced SE (1 hr post-SE), and from their age-matched naïve controls (n=12 each), as well as from chronically epileptic rats (6-8 weeks post-SE, n=8). We also collected tissue from human refractory epilepsy patients undergoing resection at both temporal and extratemporal sites (n=4). After HCN1 enrichment by immunoprecipitation and trypsin in-gel digestion, the samples were analyzed by mass spectrometry. Results: Six HCN1 phosphosites were detected in 100% of rat samples. Of these phosphosites evaluated at 1 hr post-SE, a statistically significant phosphorylation change was seen only at S891: a 50% decrease compared to control (1 hr post- SE phosphorylation level 11.5 ± 2.99%; control 22.8 ± 3.18%; p=0.016). Loss of phosphorylation at S891 was maintained in tissue from chronically epileptic rats, with a 58% decrease in epilepsy (chronic: 9.64 ± 4.06%; p=0.020). Also seen in chronic epilepsy was a 49% increase in phosphorylation at S791 (chronic 33.1 ± 3.51%; control 22.1 ± 2.52%; p=0.018). No change was seen at other phosphosites. Human HCN1 phosphosites were detected at the identical residues for which homologous phosphosites exist in rat. Conclusions: These data represent a novel survey of HCN1 phosphorylation sites. Human HCN1 phosphosite identification almost exactly mirrored that in rat, demonstrating conservation of upstream phosphorylation signaling across species. During epileptogenesis, persistent dephosphorylation occurs at S891, a residue located 20 amino acids from the carboxyl end of the HCN1 protein, and a potential region of interactions with accessory proteins. This dephosphorylation of HCN1 channels near the carboxyl terminal may mediate their loss of surface expression during epileptogenesis. Future experiments will seek to validate the role of phosphorylation changes at S891 and S791 in the loss of function of HCN1 channels seen in epilepsy. Funding: Funding from NIH R01 NS050229 (NPP).