Probenecid Disrupts a Novel Pannexin 1-Collapsin Response Mediator Protein 2 Interaction and Increases Microtubule Stability.

Neurite formation relies on finely-tuned control of the cytoskeleton. Here we identified a novel protein-protein interaction between the ion and metabolite channel protein Pannexin 1 (Panx1) and collapsin response mediator protein 2 (Crmp2), a positive regulator of microtubule polymerization and stabilization. Panx1 and Crmp2 co-precipitated from both Neuro-2a (N2a) cells and mouse ventricular zone (VZ) tissue. In vitro binding assays between purified proteins revealed the interaction occurs directly between the Panx1 C-terminus (Panx1 CT) and Crmp2. Because Crmp2 is a well-established microtubule-stabilizing protein, and we previously observed a marked increase in neurite formation following treatment with the Panx1 blocker, probenecid, in N2a cells and VZ neural precursor cells (NPCs), we investigated the impact of probenecid on the Panx1-Crmp2 interaction. Probenecid treatment significantly disrupted the Panx1-Crmp2 interaction by both immunoprecipitation (IP) and proximity ligation analysis, without altering overall Crmp2 protein expression levels. In the presence of probenecid, Crmp2 was concentrated at the distal ends of growing neurites. Moreover, probenecid treatment increased tubulin polymerization and microtubule stability in N2a cells. These results reveal that probenecid disrupts a novel interaction between Panx1 and the microtubule stabilizer, Crmp2, and also increases microtubule stability.

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

Pannexin 1 (Panx1) channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs). Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

The emerging Pannexin 1 signalome: a new nexus revealed?

Pannexins (Panxs) are a family of single-membrane, large-pore ion, and metabolite permeable channels. Of the three Panx proteins, Panx1 has been most extensively studied, and has recently emerged as an exciting, clinically relevant target in many physiological and pathophysiological settings. This channel is widely expressed across various cell and tissue types; however its links to precise signaling pathways are largely unknown. Here we review the current literature surrounding presently identified Panx1-protein interactions, a critical first step to unraveling the Panx1 signalome. First we elucidate the reported associations of Panx1 with other ion channels, receptors, and channel signaling complexes. Further, we highlight recently identified Panx1-cytoskeleton interactions. Finally, we discuss the implications of these protein-protein interactions for Panx1 function in various cell and tissue types, and identify key outstanding questions arising from this work.