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

描述(申请人提供)：Twik相关脊髓K+(Tresk)通道属于双孔区域K+(K2P)通道家族，是唯一表现出钙依赖激活的K2P通道。先前的研究表明，Tresk是初级传入神经元的主要背景K+通道，控制着正常和疾病环境下神经元的兴奋性。尽管最近对Tresk和其他K2P通道的研究取得了进展，但仍不清楚内源性Tresk通道是否由细胞内钙激活，如果是，钙的来源是什么，以及这在多大程度上对整体背景K+电流和神经元的兴奋性有贡献。最近，编码人类Tresk亚单位的KCNK18基因的移码突变与偏头痛先兆相关，在一个大的家系中。这种突变会导致Tresk蛋白的截断。我们在培养的三叉神经节(TG)神经元中表达了突变的Tresk亚单位，发现它对内源性Tresk电流具有显性-负性效应。此外，表达突变Tresk亚单位的神经元表现出较低的诱发动作电位的电流阈值和较高的尖峰频率，表明该突变导致了TG神经元的高兴奋性。有趣的是，我们以前已经证明偏头痛相关的P/Q型电压门控型钙通道突变也会导致TG神经元的高兴奋性，这增加了P/Q和Tresk通道可能通过共同的途径调节TG神经元兴奋性的可能性。本研究的目的是验证P/Q型钙通道和Tresk K+通道在TG伤害性感受神经元亚群中功能偶联的假设，以控制神经元的兴奋性。具体地说，我们假设通过P/Q型通道的钙离子内流通过钙/钙调素-钙调神经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通道活动是否对P/Q型通道调节TG神经元的兴奋性是必要的。其次，我们将测试内源性Tresk通道是否需要P/Q介导的钙内流来控制TG神经元的兴奋性。此外，我们将以共表达P/Q和Tresk通道的HEK细胞为平台，探索P/Q-Tresk偶联的机制。我们将研究P/Q通道附近局部Ca~(2+)的升高是否足以增强Tresk的活性，以及Ca~(2+)是否优先与P/Q-型通道预先关联的钙调蛋白结合。我们还将测试P/Q-Tresk偶联是否需要锚定在Tresk通道上的钙调神经磷酸酶的激活。最后，我们将研究P/Q通道和Tresk通道是否在蛋白质复合体中相互作用。这项研究的结果将有助于更好地理解P/Q-Tresk的潜在机制及其功能意义。最终，这将阐明P/Q和Tresk通道突变导致偏头痛的常见机制。

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.