P2X7 Receptor–Pannexin 1 Hemichannel Association: Effect of Extracellular Calcium on Membrane Permeabilization

Abstract

Activation of P2X(7) receptor (P2X(7)R) and pannexin have been implicated in membrane permeabilization associated with ischemic cell death and many other inflammatory processes. P2X(7)R has a unique property of forming large pore upon repeated or prolonged application of agonist like ATP or 2', 3'-(4-benzoyl) benzoyl ATP. It has been proposed that pannexin 1 (panx1) hemichannel associates with P2X(7)R to form large pore, though the actual mechanism is not yet understood. Calcium concentration in extracellular milieu drops in many patho-physiological conditions, e.g. ischemia, when P2X(7)R/pannexin is also known to be activated. Therefore, we hypothesize that extracellular calcium ([Ca(2+)](o)) plays an important role in the coupling of P2X(7)R-panx1 and subsequent membrane permeabilization. In this study we show that membrane permeability of the P2X(7)R and panx1 expressing N2A cell increases in ([Ca(2+)](o))-free solution. In [Ca(2+)](o)-free solution, fluorescent dye calcein trapped cells exhibited time-dependent dye leakage resulting in about 50% decrease of fluorescence intensity in 30 min. Control cells in 2 mM [Ca(2+)](o) did not show such leakage. Like N2A cells, mixed culture of neuron and glia, derived from hippocampal progenitor cells showed similar dye leakage. Dye leakage was blocked either by pannexin-specific blocker, carbenoxolone or P2X(7)R antagonists, Brilliant Blue G, and oxidized ATP. Furthermore P2X(7)R and panx1 were co-immunoprecipitated. The amount of P2X(7)R protein pulled-down with panx1, increased by twofold when cells were incubated 30 min in [Ca(2+)](o)-free buffer. Taken together, the results of this study demonstrate the activation and association of P2X(7)R-panx1, triggered by the removal of [Ca(2+)](o).

Contribution of Pannexin 1 and Connexin 43 Hemichannels to Extracellular Calcium–Dependent Transport Dynamics in Human Blood-Brain Barrier Endothelial Cells

“…1). This is consistent with the reported characteristics of Px1 (Poornima et al, 2012) and Cx43 (De Bock et al, 2011). Considering that the extracellular Ca 21 concentration in the brain interstitial fluid (ISF) falls during ischemic stroke (Silver and Erecinska, 1990), it seems reasonable to consider that decrease of extracellular Ca 21 concentration plays a key role as a trigger of the opening of Px1 and Cx43 and the consequent increase of membrane permeability in brain capillary endothelial cells.…”

“…Considering that the extracellular Ca 21 concentration in the brain interstitial fluid (ISF) falls during ischemic stroke (Silver and Erecinska, 1990), it seems reasonable to consider that decrease of extracellular Ca 21 concentration plays a key role as a trigger of the opening of Px1 and Cx43 and the consequent increase of membrane permeability in brain capillary endothelial cells. Poornima et al (2012) have proposed a model in which Px1 associates with P 2 X 7 receptor (P 2 X 7 R) to form a large calcein dye-permeable pore upon Px1-mediated ATP release under conditions of extracellular Ca 21 deprivation. Since hCMEC/ D3 cells express several types of P 2 X receptors, including P 2 X 7 R, at the transcriptional level (Bintig et al, 2012), it is probable that the association of Px1 and P 2 X 7 R plays a role in promoting formation of a fluorescent dye-permeable pore in hCMEC/D3 cells.…”

Dispensable ATP permeability of Pannexin 1 channels in a heterologous system and in mammalian taste cells

“…Ample evidence suggests that Panx1 interacts with a1D-adrenergic receptors (Billaud, et al, 2011) and with several components of the multiprotein inflammasome complex, including the P2X7 receptor, caspase-1, caspase-11, and some other proteins (Silverman et al, 2008). In N2A cells, the association of P2X7 and Panx1 and activation of this complex require low extracellular Ca 2+ (Poornima et al, 2012). The K + channel subunit Kvb3 was identified as a potential interacting partner of Panx1 (Bunse et al, 2005).…”

Activation, Permeability, and Inhibition of Astrocytic and Neuronal Large Pore (Hemi)channels

“…A range of large pore (hemi)channels appears to open to the extracellular space in response to a variety of stimuli, such as removal of divalent cations from the extracellular solution, addition of receptor agonist, and/or at positive membrane potentials (4, 6, 8 -10). Channel opening is commonly measured with various fluorescent dyes of molecular sizes ranging from ϳ300 to ϳ700 Da (9,11,12). Proportionality between dye uptake and permeability to biologically relevant molecules has, on occasion, been implied.…”