Mechanisms of activation of lysosomal two-pore channel TPC2, a potential novel target for the treatment of lysosomal storage disorders and neurodegenerative diseases

内溶酶体离子通道，特别是双孔通道（TPCs），由于其病理生理相关性（与几种疾病有关）和结构角度（新兴的低温电镜结构），目前引起了人们的极大兴趣。但是围绕TPCs基本特性的不确定性，包括它们是如何被激活的以及它们的离子渗透性，再加上有限的药理学工具集，正在减缓进展。本提案将详细解决这些差距。我们将1)验证新的化学工具来研究TPCs，特别是在生理和病理生理背景下的TPC2，我们将2)通过为更清楚地了解TPC2如何通过不同的激活信号传导不同的离子来响应潜在的不同生理后果奠定基础，从而促进TPC2药物的发现和设计。通过结合Ca2+成像、分子生物学和电生理学，我们应用了我们发现的TPC2的亲脂性激动剂，以证明TPC2的离子选择性是激动剂依赖的，而不是固定的。这对TPC2的生理和病理生理作用和内源性激活机制以及药物发现和药物设计具有重要意义。为了将TPC2作为治疗lsd或神经退行性疾病的靶点，深入了解这些不同的激活机制和内源性刺激途径至关重要，本工作旨在弥补缺失的知识空白。

Endolysosomal ion channels, in particular the two-pore channels (TPCs) are currently garnering much interest due to its pathophysiological relevance (given links to several diseases) and from a structural standpoint (with emerging cryoEM structures). But uncertainty surrounding fundamental properties of TPCs including how they are activated and their ion permeability, coupled with a limited pharmacological tool set is slowing progress. This proposal will address these gaps in detail. We will 1) validate new chemical tools to study TPCs, in particular TPC2 in a physiological and pathophysiological context and we will 2) facilitate TPC2 drug discovery and design by laying the foundations for a clearer understanding of how TPC2 can conduct different ions in response to different activating cues with potentially differential physiological consequences. By using a combination of Ca2+ imaging, molecular biology and electrophysiology, we apply the first-in-kind lipophilic agonists of TPC2 that we have discovered to demonstrate that the ion selectivity of TPC2 is agonist-dependent and not fixed. This has very important implications for the physiological and pathophysiological roles and endogenous activation mechanisms of TPC2 and consequently for drug discovery and drug design. In order to establish TPC2 as a target to treat, e.g. LSDs or neurodegenerative diseases, insights into these different activation mechanisms and endogenous stimulation pathways are crucial and this work aims to deliver the missing knowledge gaps.