Abstract
Temporal lobe epilepsy (TLE) is a prevalent neurological disorder with many patients experiencing poor seizure control with existing anti-epileptic drugs. Thus, novel insights into the mechanisms of epileptogenesis and identification of new drug targets can be transformative. Changes in ion channel function have been shown to play a role in generating the aberrant neuronal activity observed in TLE. Previous work demonstrates that hyperpolarization-activated cyclic nucleotide-gated (HCN) channels regulate neuronal excitability and are mislocalized within CA1 pyramidal cells in a rodent model of TLE. The subcellular distribution of HCN channels is regulated by an auxiliary subunit, tetratricopeptide repeat-containing Rab8b-interacting protein (TRIP8b), and disruption of this interaction correlates with channel mislocalization. However, the molecular mechanisms responsible for HCN channel dysregulation in TLE are unclear. Here we investigated whether changes in TRIP8b phosphorylation are sufficient to alter HCN channel function. We identified a phosphorylation site at residue Ser237 of TRIP8b that enhances binding to HCN channels and influences channel gating by altering the affinity of TRIP8b for the HCN cytoplasmic domain. Using a phosphospecific antibody, we demonstrate that TRIP8b phosphorylated at Ser237 is enriched in CA1 distal dendrites and that phosphorylation is reduced in the kainic acid model of TLE. Overall, our findings indicate that the TRIP8b-HCN interaction can be modulated by changes in phosphorylation and suggest that loss of TRIP8b phosphorylation may affect HCN channel properties during epileptogenesis. These results highlight the potential of drugs targeting posttranslational modifications to restore TRIP8b phosphorylation to reduce excitability in TLE.
Highlights
Temporal lobe epilepsy (TLE) is a prevalent neurological disorder with many patients experiencing poor seizure control with existing anti-epileptic drugs
In this study, we have shown that tetratricopeptide repeat– containing Rab8b-interacting protein (TRIP8b) phosphorylation regulates hyperpolarization-activated cyclic nucleotide-gated (HCN) channel binding and function
Residue Ser237 is located within the nano domain, where it is predicted to influence the effect of TRIP8b on channel gating and trafficking [21, 22, 26, 29, 54]
Summary
A previous global phosphoproteome analysis identified several possible phosphorylation sites on TRIP8b, including on residue Ser237 [53]. Enhanced the pSer237 signal compared with the nonphosphorylated protein without ATP These data demonstrate that both CaMKII␣ and PKA can phosphorylate the Ser237 residue of TRIP8b in vitro. These data show that phosphorylated TRIP8b is enriched in the CA1 region (relative pSer237 level to total TRIP8b, 1.0) compared with the levels found in the dentate gyrus (0.2 Ϯ 0.1), thalamus (0.3 Ϯ 0.1), neocortex (0.5 Ϯ 0.1), and cerebellum (0.05 Ϯ 0.04) (p Ͻ 0.05, n ϭ 3) Taken together, these results demonstrate that TRIP8b isoform 1a-4 is phosphorylated at Ser237 and highly enriched in the distal dendrites of CA1 pyramidal cells. WT TRIP8b was basally phosphorylated in HEK293T cells, whereas both phosphoablative constructs show reduced signal from the ␣-pSer237 antibody (Fig. 5B) These constructs were transfected into HEK293T cells along with HCN1, HCN1 protein was immunoprecipitated from the lysates, and the level of TRIP8b bound to the channel was quantified (Fig. 5, C and D). These data establish that phosphorylated TRIP8b at Ser237 is reduced throughout the time course of epileptogenesis and that the change is localized to the epileptogenic zone
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