Pannexin-1 channels as facilitators of anandamide membrane transport

离子和小信号分子通过促进的运输(通道和转运体)在质膜上的调节运动是细胞生理学的基本基础。例如，对钙离子内流的密切调控可以决定正常和有害信号之间的差异。几十年来，我的研究项目一直在研究便利运输的机制，我们取得了几项开创性的发现。作为一名NSERC资助的博士后，我对ATP进入高尔基复合体是由经典转运蛋白介导的概念提出了质疑。相反，我展示了它是一种三磷酸腺苷可渗透的阴离子通道。自从我开始自己的实验室(2008年)以来，我们的研究计划发现谷氨酸的亲离子受体N-甲基-D-天冬氨酸受体(NMDAR)除了具有典型的亲离子功能外，还具有代谢性信号转导作用。因此，我们继续质疑通道只是通道，转运体只是转运体，离子亲和性受体和代谢性受体之间有明显区别的概念。这项发现拨款提案的重点是了解pAnnexin-1(Panx1)通道的一种新的促进运输作用的生物物理学和神经生理学。*Panx1是一种非选择性离子和代谢产物通道，属于缝隙连接超家族。重要的是，它不形成细胞内缝隙连接，而是在质膜上作为真正的通道发挥作用，连接细胞内和细胞外空间。Panx1最好的特征是参与并受嘌呤能信号通路调节的ATP通透性通道。我们之前的NSERC资助侧重于了解Panx1在嘌呤能信号和免疫反应中的作用。在这一筹资期间，我们有几个关键的见解，这些见解决定了本提案的形成。例如，调节Panx1通道开放的机制特征不佳。我们对可穿透通道的离子和分子类型的了解范围有限，这限制了对Panx1可能不同的生理作用的表征。在这方面，我们取得了一个重要的发现，Panx1可以促进信号脂质N-花生四烯基乙醇胺(andamine；AEA)的运输，这是内源性大麻系统中的一个关键配体。*这项发现拨款将检验Panx1促进AEA运输以调节大脑中的内源性大麻素信号这一普遍假设。*我们的方法是独特和创新的，为高素质人才的培训和发展提供了令人兴奋的机会。这项工作将极大地增加我们对Panx1的不同生物学作用的理解，通过识别一种新的和独特的anandamide转运机制，并确定这种机制如何调节神经元功能和电路输出。

The regulated movement of ions and small signaling molecules across the plasma membrane occurring through facilitated transport (channels and transporters) is a fundamental basis of cellular physiology. The close regulation of Ca2+ influx, for example, can determine the difference between normal and detrimental signaling. For several decades, my research program has been investigating mechanisms of facilitated transport and we having made several seminal discoveries. As an NSERC funded postdoc, I challenged the concept that ATP transport into the Golgi complex was mediated by a classical transporter. Rather I showed it was an ATP permeable anion channel. Since starting my own lab (in 2008), our research program has discovered that the ionotropic receptor for glutamate, the N-Methyl-D-Aspartate receptor (NMDAR) has a metabotropic signaling role in addition to its canonical ionotropic function. Thus, we continue to question the concept that channels are only channels, transporters are only transporters and that there is a clear distinction between ionotropic and metabotropic receptors. This Discovery grant proposal is focused on understanding the biophysics and neurophysiology of a novel facilitated transport role of the pannexin-1 (Panx1) channel. *** Panx1 is a non-selective ion and metabolite channel belonging to the gap junction superfamily. Importantly, it does not form intracellular gap junctions, rather it functions in plasma membranes as a bona fide channel, joining the intra- and extracellular spaces. Panx1 is best characterized as an ATP permeable channel that contributes to, and is regulated by, purinergic signaling pathways. Our previous NSERC grant focused on understanding the roles of Panx1 in purinergic signaling and immune responses. During this funding period we had several critical insights that have shaped the present proposal. For example, the mechanisms that regulate the opening of Panx1 channels are poorly characterized. Our understanding of the scope of the types of ions and molecules that can permeate the channels is limited, which restricts characterization of the likely diverse physiological roles of Panx1. In this regard, we made the important discovery that Panx1 can facilitate the transport of the signaling lipid, N-arachidonoylethanolamine (andandamide; AEA), a critical ligand in the endocannabinoid system. ******This Discovery grant will test the general hypothesis that Panx1 facilitates the transport of AEA to regulate endocannabinoid signaling in the brain. ******Our approach is unique and innovative, providing exciting opportunities for the training and development of highly qualified personnel. The work here will dramatically increase our understanding of the diverse biological roles of Panx1 through identifying a new and unique transport mechanism for anandamide and determining how this modulates neuronal function and circuit output.