FUNCTIONAL COUPLING BETWEEN VOLTAGE-GATED CA2 CHANNELS AND TRESK K+ CHANNELS

电压门控 CA2 通道和 Tresk K 通道之间的功能耦合

• 批准号: 8920176
• 负责人: YUQING CAO
• 金额: $ 22.88万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-15 至 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8920176
• 关键词: Action Potentials; Afferent Neurons; Binding; Binding Sites; Calcineurin; Caliber; Calmodulin; Cells; Chelating Agents; Classic Migraine; Co-Immunoprecipitations; Coupled; Coupling; Dependence; Disease; Dominant-Negative Mutation; Egtazic Acid; Exhibits; Family; Foundations; Frameshift Mutation; Frequencies; Future; Genes; Health; Human; Ion Channel; Isolectin; Knock-out; Knockout Mice; Lead; Light; Link; Mediating; Migraine; Modeling; Mus; Mutate; Mutation; Neurons; Nociception; Outcome; Outcome Study; Pathway interactions; Physiological; Plants; Potassium Channel; Proteins; Research; Signal Pathway; Source; Spinal Cord; Spinal Ganglia; Stimulus; Structure of trigeminal ganglion; System; Testing; Transmembrane Domain; Trigeminal Pain; Trigeminal System; Work; channel blockers; genetic pedigree; insight; interdisciplinary approach; loss of function; loss of function mutation; mutant; neuronal excitability; protein K; protein complex; research study; response; voltage

DESCRIPTION (provided by applicant): TWIK-related spinal cord K+ (TRESK) channel belongs to the two-pore-domain K+ (K2P) channel family and is the only K2P channel that exhibits Ca2+-dependent activation. Previous studies have shown that TRESK is a major background K+ channel in primary afferent neurons and controls neuronal excitability in both normal and disease settings. Despite the recent progress in the studies of TRESK and other K2P channels, it is still not clear whether endogenous TRESK channels are activated by intracellular Ca2+ and if so, what is the source of the Ca2+ and to what degree this contributes to the overall background K+ currents and neuronal excitability. Recently, a frameshift mutation in the KCNK18 gene encoding human TRESK subunit has been associated with migraine with aura in a large pedigree. The mutation results in the truncation of the TRESK protein. We have expressed the mutant TRESK subunit in cultured trigeminal ganglion (TG) neurons and found that it has a dominant-negative effect on the endogenous TRESK currents. Furthermore, neurons expressing mutant TRESK subunits exhibited a lower current threshold to elicit action potential as well as a higher spike frequency in response to supra-threshold stimuli, indicating that the mutation resulted in hyper-excitability of TG neurons. Interestingly, we have shown previously that a migraine-associated P/Q-type voltage-gated Ca2+ channel mutation also results in hyper-excitability of TG neurons, raising the possibility that P/Q and TRESK channels may regulate TG neuron excitability through a common pathway. The research objective of this proposal is to test the hypothesis that P/Q-type Ca2+ channels and TRESK K+ channels are functionally coupled in a subpopulation of TG nociceptive neurons to control neuronal excitability. Specifically, we hypothesize that Ca2+ influx through P/Q-type channels activates TRESK channels via the Ca2+/calmodulin-calcineurin signaling pathway, thereby regulating the excitability of TG neurons. First, we will investigate whether the endogenous TRESK channel activity is necessary for P/Q-type channels to regulate TG neuron excitability. Secondly, we will test whether P/Q-mediated Ca2+ influx is necessary for the endogenous TRESK channels to control the excitability of TG neurons. In addition, we will use HEK cells co-expressing P/Q and TRESK channels as a platform to explore the mechanisms underlying the P/Q-TRESK coupling. We will investigate whether the rise in local Ca2+ near P/Q channels is sufficient to enhance TRESK activity and whether the Ca2+ preferentially binds to the calmodulin pre-associated with P/Q-type channels. We will also test whether the P/Q-TRESK coupling requires the activation of calcineurin anchored to the TRESK channels. Lastly, we will investigate whether P/Q and TRESK channels interact with each other within a protein complex. The outcome of this study will lead to better understanding of the mechanisms underlying the P/Q-TRESK as well as its functional significance. Ultimately this will shed light on the common mechanisms through which P/Q and TRESK channel mutations cause migraine headache.

描述（由申请人提供）：TWIK相关的脊髓K+（TRESK）通道属于双孔域K+（K2P）通道家族，并且是唯一表现出Ca2+依赖性激活的K2P通道。先前的研究表明，TRESK 是初级传入神经元中的主要背景 K+ 通道，并控制正常和疾病环境中的神经元兴奋性。尽管 TRESK 和其他 K2P 通道的研究最近取得了进展，但仍不清楚内源性 TRESK 通道是否被细胞内 Ca2+ 激活，如果是，Ca2+ 的来源是什么以及它对整体背景 K+ 电流和神经元兴奋性的贡献程度如何。 最近，编码人类 TRESK 亚基的 KCNK18 基因中的移码突变已在一个大谱系中与先兆偏头痛相关。该突变导致 TRESK 蛋白截短。我们在培养的三叉神经节（TG）神经元中表达了突变的 TRESK 亚基，并发现它对内源性 TRESK 电流具有显性负效应。此外，表达突变TRESK亚基的神经元表现出较低的引发动作电位的电流阈值以及响应超阈值刺激的较高尖峰频率，表明该突变导致TG神经元过度兴奋。有趣的是，我们之前已经证明，偏头痛相关的 P/Q 型电压门控 Ca2+ 通道突变也会导致 TG 神经元过度兴奋，这增加了 P/Q 和 TRESK 通道可能通过共同途径调节 TG 神经元兴奋性的可能性。 本提案的研究目的是检验以下假设：P/Q 型 Ca2+ 通道和 TRESK K+ 通道在 TG 伤害性神经元亚群中功能耦合以控制神经元兴奋性。具体来说，我们假设 Ca2+ 通过 P/Q 型通道流入，通过 Ca2+/钙调蛋白-钙调磷酸酶信号通路激活 TRESK 通道，从而调节 TG 神经元的兴奋性。首先，我们将研究内源性 TRESK 通道活性是否是 P/Q 型通道调节 TG 神经元兴奋性所必需的。其次，我们将测试 P/Q 介导的 Ca2+ 内流是否是内源性 TRESK 通道控制 TG 神经元兴奋性所必需的。此外，我们将使用共表达 P/Q 和 TRESK 通道的 HEK 细胞作为平台来探索 P/Q-TRESK 耦合的机制。我们将研究 P/Q 通道附近局部 Ca2+ 的增加是否足以增强 TRESK 活性，以及​​ Ca2+ 是否优先与与 P/Q 型通道预关联的钙调蛋白结合。我们还将测试 P/Q-TRESK 偶联是否需要激活锚定于 TRESK 通道的钙调神经磷酸酶。最后，我们将研究 P/Q 和 TRESK 通道是否在蛋白质复合物中相互作用。这项研究的结果将有助于更好地理解 P/Q-TRESK 的机制及其功能意义。最终，这将揭示 P/Q 和 TRESK 通道突变导致偏头痛的常见机制。