HCN channel trafficking in epilepsy

描述（由申请人提供）：颞叶癫痫（TLE）是内科和外科治疗难治性癫痫发作的常见原因。TLE中癫痫发作倾向的增加可能是由异常的神经元兴奋性引起的。神经元兴奋性的重要控制器是超极化激活电流Ih，其由超极化激活的环核苷酸门控（HCN）通道介导。HCN通道由四个孔形成亚基（HCN 1 -4）的同源或异源组装体组成，所述孔形成亚基在海马神经元中与辅助亚基、含三肽重复序列（TPR）的Rab 8b相互作用蛋白（TRIP 8b）缔合。HCN通道在海马锥体神经元树突中明显富集，但这些通道在TLE的啮齿动物模型中偏离树突质膜，导致异常的神经元兴奋性。我们认为阻断或逆转这一通道的错误定位和由此产生的过度兴奋可以减少或消除TLE的复发性癫痫发作。因此，尽管现有的治疗方法通过直接降低神经元兴奋性来统一靶向癫痫症状，但我们的工作旨在阐明导致癫痫的上游分子变化，从而可以建立预防或逆转癫痫发生的新治疗靶点。离子通道的定位和功能通常由亚基蛋白在特定位点的磷酸化控制。沿着这些路线，我们已经表明，在海马锥体神经元树突的正常HCN通道运输需要TRIP 8 b和N-甲基-D-天冬氨酸受体（NMDAR）和钙调蛋白依赖性蛋白激酶II（CaMK II）活性的激活。我们建议进一步表征HCN通道亚基磷酸化在控制正常和癫痫海马中HCN通道定位和功能中的作用。我们假设1）HCN通道亚基磷酸化调节HCN通道在神经元中的定位，2）癫痫发生导致HCN通道亚基磷酸化的变化，从而导致TLE中的异常通道定位和功能，3）操纵HCN通道亚基磷酸化可以防止HCN通道错误定位并减少颞叶癫痫（TLE）中的复发性癫痫发作。为了解决这些假设，我们建议使用生理学，细胞生物学和生物化学技术来解决以下具体目标：1）鉴定正常和癫痫海马中HCN通道亚单位磷酸化的位点，2）确定HCN通道亚单位磷酸化是否调节通道定位，3）确定阻断HCN通道磷酸化的癫痫相关变化是否防止TLE中的通道错误定位和复发性癫痫发作。

DESCRIPTION (provided by applicant): Temporal lobe epilepsy (TLE) is a common cause of seizures refractory to medical and surgical treatment. Increased seizure propensity in TLE is likely caused by abnormal neuronal excitability. An important controller of neuronal excitability i the hyperpolarization-activated current, Ih, which is mediated by the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel. HCN channels are comprised of homo- or heteromeric assemblies of four pore-forming subunits (HCN1-4), which in hippocampal neurons associate with an auxiliary subunit, tetratricopeptide repeat (TPR)-containing Rab8b interacting protein (TRIP8b). HCN channels are markedly enriched in hippocampal pyramidal neuron dendrites, but these channels are mislocalized away from the dendritic plasma membrane in a rodent model of TLE, leading to abnormal neuronal excitability. We reason that blocking or reversing this channel mislocalization and the resultant hyperexcitability could reduce or eliminate recurrent seizures in TLE. Thus, whereas existing treatments uniformly target the symptoms of epilepsy by directly reducing neuronal excitability, our work stands to elucidate the upstream molecular changes leading to epilepsy that could establish novel therapeutic targets for preventing or reversing epileptogenesis. Ion channel localization and function is often controlled by phosphorylation of subunit proteins at specific sites. Along these lines, we have shown that normal HCN channel trafficking in hippocampal pyramidal neuron dendrites requires TRIP8b and activation of N-methyl-D-aspartate receptors (NMDAR) and calmodulin-dependent protein kinase II (CaMKII) activity. We propose to further characterize the role of HCN channel subunit phosphorylation in controlling HCN channel localization and function in normal and epileptic hippocampus. We hypothesize that 1) HCN channel localization in neurons is regulated by HCN channel subunit phosphorylation, 2) epileptogenesis leads to changes in HCN channel subunit phosphorylation that cause aberrant channel localization and function in TLE, and 3) manipulating HCN channel subunit phosphorylation can prevent HCN channel mislocalization and reduce recurrent seizures in temporal lobe epilepsy (TLE). To address these hypotheses, we propose to use physiological, cell biological and biochemical techniques to address the following specific aims: 1) to identify sites of HCN channel subunit phosphorylation in the normal and epileptic hippocampus, 2) to determine whether HCN channel subunit phosphorylation regulates channel localization, 3) to determine if blocking epilepsy-associated changes in HCN channel phosphorylation prevents channel mislocalization and recurrent seizures in TLE.