Phosphorylation of TRIP8b Modulates Binding to HCN Channels and is Dysregulated in Temporal Lobe EpilepsyPublic
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Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are non-selective cation channels expressed in the brain and heart where they exert control over many diverse physiological properties. In the hippocampus, HCN channels are enriched in a dendritic gradient within CA1 pyramidal cells where they reduce dendritic integration and dampen neuronal excitability. HCN channels are highly regulated through interactions with an auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b). TRIP8b is a cytosolic protein that directly binds to HCN channels at two distinct locations to facilitate dendritic channel enrichment and influence channel gating. Loss of this dendritic enrichment and HCN channel function in the hippocampus is associated with increased excitability and enhanced seizure susceptibility. In animal models of temporal lobe epilepsy (TLE), a reduction in Ih and channel expression in the distal dendrites of CA1 pyramidal cells is well established. Prior work from the Chetkovich laboratory has demonstrated that the loss of HCN channel expression in chronic epilepsy is also associated with a reduction in the interaction between TRIP8b and HCN channels. This thesis investigates the possibility that post-translational modifications in TRIP8b mediate the interaction with HCN channels and that these modifications in TRIP8b are regulated during epileptogenesis. In Chapter 2, we use mass spectrometry to identify phosphorylation sites on HCN1, HCN2, and TRIP8b in mouse brain. The locations of the phosphorylation sites provide information on which functions of the proteins may be altered with changes in phosphorylation, as well as which kinase may regulate phosphorylation levels. Through these experiments we were able to identify a phosphorylation site on residue S237 of TRIP8b located in a domain that regulates binding, trafficking, and gating of HCN channels. In Chapter 3, I characterize the function and location of phosphorylated TRIP8b using a newly generated phosphospecific antibody. I use several in vitro assays to determine that the phosphorylation of residue S237 is necessary for TRIP8b binding to HCN channels and for the TRIP8b-mediated hyperpolarization of channel gating. Using immunohistochemistry, I determined that phosphorylated TRIP8b is located in the distal dendrites of the hippocampus and neocortex, and is a feature of one distinct isoform of the protein. These data advance our understanding of the relationship between HCN channels and TRIP8b and highlight the significance of kinase and phosphatase pathways in regulating channel binding and function. In Chapter 4, we turn our attention to TLE to analyze how TRIP8b phosphorylation may change over the course of epileptogenesis. Using the well-established kainic acid (KA) model of TLE in rats, I determine that S237 phosphorylation is significantly reduced during the acute, latent, and chronic stages of epileptogenesis. This reduction may be due to reduced kinase activity of the enzyme CaMKIIa as observed in this study, or to enhanced phosphatase activity as reported previously. These results implicate TRIP8b dephosphorylation in the channel expression and trafficking deficits observed in TLE. Finally, I also test the impact of the commonly prescribed anti-epileptic drug phenobarbital (PB) on TRIP8b phosphorylation. The data indicate that PB treatment enhances S237 phosphorylation in both epileptic and non-epileptic animals. This observation raises the possibility that TRIP8b phosphorylation and its effect on regulating HCN channel function may be part of the mechanism by which PB reduces spontaneous seizures. Overall, this project establishes that TRIP8b phosphorylation at S237 is a significant modulator of the relationship between TRIP8b and HCN channels. Furthermore, these data highlight the possibility that restoring TRIP8b phosphorylation through altering kinase or phosphatase pathways may enhance HCN channel function in TLE and reduce hippocampal excitability.
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