P2X7 receptor cross-talk regulates ATP-induced pannexin 1 internalization

Abstract

In the nervous system, extracellular ATP levels transiently increase in physiological and pathophysiological circumstances, effecting key signalling pathways in plasticity and inflammation through purinergic receptors. Pannexin 1 (Panx1) forms ion- and metabolite-permeable channels that mediate ATP release and are particularly enriched in the nervous system. Our recent study demonstrated that elevation of extracellular ATP triggers Panx1 internalization in a concentration- and time-dependent manner. Notably, this effect was sensitive to inhibition of ionotropic P2X7 purinergic receptors (P2X7Rs). Here, we report our novel findings from the detailed investigation of the mechanism underlying P2X7R-Panx1 cross-talk in ATP-stimulated internalization. We demonstrate that extracellular ATP triggers and is required for the clustering of P2X7Rs and Panx1 on Neuro2a cells through an extracellular physical interaction with the Panx1 first extracellular loop (EL1). Importantly, disruption of P2X7R-Panx1 clustering by mutation of tryptophan 74 within the Panx1 EL1 inhibits Panx1 internalization. Notably, P2X7R-Panx1 clustering and internalization are independent of P2X7R-associated intracellular signalling pathways (Ca influx and Src activation). Further analysis revealed that cholesterol is required for ATP-stimulated P2X7R-Panx1 clustering at the cell periphery. Taken together, our data suggest that extracellular ATP induces and is required for Panx1 EL1-mediated, cholesterol-dependent P2X7R-Panx1 clustering and endocytosis. These findings have important implications for understanding the role of Panx1 in the nervous system and provide important new insights into Panx1-P2X7R cross-talk.

Pathophysiological Role of Purines and Pyrimidines in Neurodevelopment: Unveiling New Pharmacological Approaches to Congenital Brain Diseases

“…The discovery of the first patient bearing a homozygous Pannexin 1 (Panx1) loss-of-function gene variant associated with multisystem dysfunction and intellectual disability has been recently published (Shao et al, 2016 ). Panx1 is a membrane channel allowing the passage of ions and small molecules, among which ATP is one of the most common (Boyce and Swayne, 2017 ). In the CNS, Panx1 is highly expressed by developing and mature neurons, and contributes to brain development, maturation, and to synaptic plasticity (Wicki-Stordeur et al, 2016 ).…”

Influence of the three‐dimensional culture of human bone marrow mesenchymal stromal cells within a macroporous polysaccharides scaffold on Pannexin 1 and Pannexin 3

“…Moreover, very recently (Boyce & Swayne, ) new results have provided striking new details on the mechanisms underlying ATP‐induced Panx1 endocytosis. This includes the discovery of an ATP‐dependent extracellular interaction between the Panx1 and P2X7R.…”

Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease

“…Our recent work has shown that elevation of extracellular ATP triggers clustering of Panx1 with P2X7Rs and subsequent internalization ( Boyce et al, 2015 ; Boyce and Swayne, 2017 ). In this hypothesis paper, we have outlined two scenarios where we predict this down-regulation of Panx1 surface expression could play an important role.…”

“…P2X7Rs are also present in NPCs and NPC model cell lines, where they play key roles in maintenance of stemness, proliferation, differentiation and programmed cell death (reviewed in Burnstock and Ulrich, 2011 ; Cavaliere et al, 2015 ). In N2a cells, a model of neuronal differentiation that we used to study Panx1 trafficking in ( Boyce et al, 2015 ; Boyce and Swayne, 2017 ), P2X7Rs are the primary functional P2XR subtype ( Gomez-Villafuertes et al, 2009 ). P2X7Rs are expressed in the embryonic ( Tsao et al, 2013 ) and postnatal ( Messemer et al, 2013 ) ventricular zone, as well as the early postnatal subgranular zone ( Tsao et al, 2013 ), an NPC niche within the hippocampus.…”

“…Adding further complexity to P2X7R-Panx1 crosstalk, we recently demonstrated that elevation of extracellular ATP leads to Panx1 internalization ( Boyce et al, 2015 ; Boyce and Swayne, 2017 ), thereby reducing Panx1 surface expression. ATP-induced internalization required activation of P2X7Rs ( Boyce et al, 2015 ) as well as their physical interaction with the Panx1 first extracellular loop ( Boyce and Swayne, 2017 ). This was the first report to identify the interaction site for the P2X7R within the Panx1 sequence.…”