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以治愈的癫痫的常见原因。TLE癫痫发作倾向的增加可能是由神经元兴奋性异常引起的。神经元兴奋性的一个重要控制因素是超极化激活电流Ih，它是由超极化激活的环核苷酸门控(HCN)通道介导的。HCN通道由四个致孔亚基(HCN1-4)组成，在海马神经元中，HCN1-4与一个辅助亚基--含有四肽重复序列(TPR)的Rab8b相互作用蛋白(TRIP8b)相连。在TLE动物模型中，HCN通道在海马区锥体神经元树突中显著丰富，但这些通道错位地远离树突质膜，导致神经元兴奋性异常。我们认为阻断或逆转这种通道错误定位以及由此产生的过度兴奋性可以减少或消除TLE中反复发作的癫痫。因此，尽管现有的治疗方法都是通过直接降低神经元的兴奋性来统一针对癫痫的症状，但我们的工作旨在阐明导致癫痫的上游分子变化，从而为预防或逆转癫痫的发生建立新的治疗靶点。离子通道的定位和功能通常由特定位置的亚基蛋白的磷酸化控制。沿着这些路线，我们已经证明了正常的HCN通道在海马锥体神经元树突中的运输需要TRIP8b和激活N-甲基-D-天冬氨酸受体(NMDAR)和钙调蛋白依赖的蛋白激酶II(CaMKII)的活性。我们建议进一步研究HCN通道亚单位磷酸化在控制正常和癫痫海马区HCN通道定位和功能中的作用。我们假设1)HCN通道亚基在神经元中的定位受HCN通道亚单位磷酸化的调节，2)癫痫的发生导致HCN通道亚单位磷酸化的改变，从而导致TLE的通道定位和功能异常，3)操纵HCN通道亚单位磷酸化可以防止HCN通道错误定位，减少颞叶癫痫(TLE)的反复发作。为了解决这些假说，我们建议使用生理学、细胞生物学和生化技术来解决以下具体目标：1)确定正常和癫痫海马区HCN通道亚单位磷酸化的位置，2)确定HCN通道亚单位磷酸化是否调节通道定位，3)确定阻断癫痫相关的HCN通道磷酸化改变是否可以防止通道错误定位和TLE反复发作。

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.