Identification and validation of the epilepsy associated KCNQ2 complexes in the brain

摘要/项目摘要 最近的人类遗传学研究表明，电压门控的功能变体的丧失或获得， 钾通道KCNQ 2引起新生儿癫痫性脑病。目前假定 KCNQ 2通道主要与大脑中的KCNQ 3通道相关;然而，一些研究表明， KCNQ 2通道也可能与其他跨膜蛋白相互作用。 为了了解KCNQ 2在大脑中的作用， KCNQ 2通道功能障碍可能导致癫痫，并设计更好的治疗方法。到 为了解决这个问题，我们已经开发了一种新的表位标记的小鼠系，使我们能够分析 使用质谱法从海马和新皮质中亲和纯化KCNQ 2复合物。 事实上，我们的初步数据表明，KCNQ 2通道可以与KCNQ 3以及 KCNQ 5频道。这一发现违背了目前的教条，即KCNQ 2只与 神经系统中的KCNQ 3通道因此，在本建议中，我们计划研究 脑中的天然KCNQ 2复合物通道由KCNQ 3和KCNQ 5通道组成。本 最后，我们将：（i）确定大脑中KCNQ 2/5或KCNQ 2/3/5复合物的存在，以及（ii） 探测含有KCNQ 2/5的KCNQ通道的生物物理特性。拟议的研究将 为我们更广泛地理解KCNQ 2、KCNQ 3和KCNQ 5如何 通道控制神经元兴奋性，并为预防和治疗疾病奠定基础 与神经元KCNQ功能障碍有关。

Abstract/Project Summary Recent human genetic studies have shown that loss or gain of function variants of the voltage-gated potassium channel KCNQ2 causes neonatal epileptic encephalopathy. It is currently assumed that KCNQ2 channels primarily associate with KCNQ3 channels in the brain; however, several studies have suggested that KCNQ2 channels may also interact with additional transmembrane proteins. Identifying the KCNQ2 membrane complex in the brain is necessary in order to understand how KCNQ2 channels dysfunction could lead to epilepsy and to also design better therapeutics. To address this question, we have developed a new epitope tagged mouse line allowing us to analyze KCNQ2 affinity purified complexes from the hippocampus and neocortex using mass spectrometry. Indeed, our preliminary data show that KCNQ2 channels could associate with KCNQ3 as well as KCNQ5 channels. This finding goes against the current dogma that KCNQ2 only associates with KCNQ3 channels in the nervous system. Therefore, in this proposal we plan to examine whether native KCNQ2 complex channels in the brain are comprised of KCNQ3 and KCNQ5 channels. To this end, we will: (i) establish the presence of KCNQ2/5 or KCNQ2/3/5 complexes in the brain and (ii) probe the biophysical properties of KCNQ2/5-containing KCNQ channels. The proposed research will make a significant contribution to our broader understanding of how KCNQ2, KCNQ3, and KCNQ5 channels control neuronal excitability, and build a foundation for preventing and treating disorders associated with neuronal KCNQ dysfunction.