title: 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).
authors.affiliation: Department of Biotechnology, IIT Madras, Chennai, 600036, India.
authors.forename: V
authors.initials: V
authors.lastname: Poornima
authors.forename: M
authors.initials: M
authors.lastname: Madhupriya
authors.forename: S
authors.initials: S
authors.lastname: Kootar
authors.forename: G
authors.initials: G
authors.lastname: Sujatha
authors.forename: Arvind
authors.initials: A
authors.lastname: Kumar
authors.forename: Amal Kanti
authors.initials: AK
authors.lastname: Bera
mesh_terms: D000818:Animals; D002118:Calcium / Q000502:physiology*; D002229:Carbenoxolone / Q000494:pharmacology; D045744:Cell Line, Tumor; D002463:Cell Membrane Permeability / Q000187:drug effects / Q000235:genetics; D017630:Connexins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D045604:Extracellular Fluid / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D051379:Mice; D009419:Nerve Tissue Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D058953:Neural Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009447:Neuroblastoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D009457:Neuroglia / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D058486:Receptors, Purinergic P2X7 / Q000235:genetics / Q000378:metabolism*
chemical_list: D017630:Connexins; D009419:Nerve Tissue Proteins; D058486:Receptors, Purinergic P2X7; C479936:pannexin 1, rat; D002229:Carbenoxolone; D002118:Calcium