The interplay between α7 nicotinic acetylcholine receptors, pannexin‐1 channels and P2X7 receptors elicit exocytosis in chromaffin cells

Abstract

Pannexin‐1 (Panx1) forms plasma membrane channels that allow the exchange of small molecules between the intracellular and extracellular compartments, and are involved in diverse physiological and pathological responses in the nervous system. However, the signaling mechanisms that induce their opening still remain elusive. Here, we propose a new mechanism for Panx1 channel activation through a functional crosstalk with the highly Ca2+ permeable α7 nicotinic acetylcholine receptor (nAChR). Consistent with this hypothesis, we found that activation of α7 nAChRs induces Panx1‐mediated dye uptake and ATP release in the neuroblastoma cell line SH‐SY5Y‐α7. Using membrane permeant Ca2+ chelators, total internal reflection fluorescence microscopy in SH‐SY5Y‐α7 cells expressing a membrane‐tethered GCAMP3, and Src kinase inhibitors, we further demonstrated that Panx1 channel opening depends on Ca2+ signals localized in submembrane areas, as well as on Src kinases. In turn, Panx1 channels amplify cytosolic Ca2+ signals induced by the activation of α7 nAChRs, by a mechanism that seems to involve ATP release and P2X7 receptor activation, as hydrolysis of extracellular ATP with apyrase or blockage of P2X7 receptors with oxidized ATP significantly reduces the α7 nAChR‐Ca2+ signal. The physiological relevance of this crosstalk was also demonstrated in neuroendocrine chromaffin cells, wherein Panx1 channels and P2X7 receptors contribute to the exocytotic release of catecholamines triggered by α7 nAChRs, as measured by amperometry. Together these findings point to a functional coupling between α7 nAChRs, Panx1 channels and P2X7 receptors with physiological relevance in neurosecretion.

A physiologic rise in cytoplasmic calcium ion signal increases pannexin1 channel activity via a C-terminus phosphorylation by CaMKII

“…In order to detect submembrane localized Ca 2+ signals, HeLa cells grown on coverslips were transfected with a genetically encoded Ca 2+ indicator protein pN-Lck-GCAMP3 plasmid (a generous gift from Baljit Khakh, Los Angeles, CA, Addgene plasmid no. 26974) as previously described (47). Cells were imaged 24 to 48 h posttransfection by TIRF microscopy using an inverted microscope (Eclipse Ti-E; Nikon) implemented with a 100× APO TIRF objective (numerical aperture 1.49; Nikon) and a Perfect Focus Unit TI-ND6-PFS (Nikon).…”

Recent developments concerning the investigation of exocytosis with amperometry

“…With the same cell model, the central role of extracellular and intracellular sphingosine-1-phosphate, a complex signaling lipid, is demonstrated for the first time by using CFA and especially by analyzing the PSF properties (that reflects the fusion pore stability; see Figure 2) [9]. CFA also contributes to important findings related to the interconnected role between pannexin-1 channels and some purinergic and nicotinic acetylcholine receptors to trigger exocytosis [10]. In the same way, the effects of some signaling pathways from a receptor tyrosine kinaselike EPHB6 on catecholamines secretion are undoubtedly proven with CFA at chromaffin cells with important consequences on the relationship between effect on catecholamine secretion and blood pressure regulation [11].…”

Oxidative Stress, Inflammation and Connexin Hemichannels in Muscular Dystrophies

“…Panx1 channels localize in the cell membrane and play a relevant role in the release of ATP from different cell types [ 170 ]. Different stimuli capable of activating Panx1 channels have been reported, including voltage [ 173 ], membrane stretching [ 174 , 175 ], intracellular Ca 2+ augmentation [ 175 , 176 ], and its C-terminal domain proteolysis [ 177 ]. However, it was recently shown that Panx1 is not directly sensitive to stretching instead, stretch stimulates other channels that allow the entry of Ca 2+ promoting the activation of the Ca 2+ /calmodulin-dependent protein kinase II (CaMK ll), which phosphorylates Panx1, causing a conformational change of the channel that allows the passage of ATP through its “lateral tunnels” [ 175 ].…”