P2X7 receptor-Pannexin1 complex: pharmacology and signaling.
Pannexin 1 (Panx1), an ortholog to invertebrate innexin gap junctions, has recently been proposed to be the pore induced by P2X(7) receptor (P2X(7)R) activation. We explored the pharmacological action of compounds known to block gap junctions on Panx1 channels activated by the P2X(7)R and the mechanisms involved in the interaction between these two proteins. Whole cell recordings revealed distinct P2X(7)R and Panx1 currents in response to agonists. Activation of Panx1 currents following P2X(7)R stimulation or by membrane depolarization was blocked by Panx1 small-interfering RNA (siRNA) and with mefloquine > carbenoxolone > flufenamic acid. Incubation of cells with KN-62, a P2X(7)R antagonist, prevented current activation by 2'(3')-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP). Membrane permeabilization to dye induced by BzATP was also prevented by Panx1 siRNA and by carbenoxolone and mefloquine. Membrane permeant (TAT-P2X(7)) peptides, provided evidence that the Src homology 3 death domain of the COOH-terminus of the P2X(7)R is involved in the initial steps of the signal transduction events leading to Panx1 activation and that a Src tyrosine kinase is likely involved in this process. Competition assays indicated that 20 microM TAT-P2X(7) peptide caused 50% reduction in Src binding to the P2X(7)R complex. Src tyrosine phosphorylation following BzATP stimulation was reduced by KN-62, TAT-P2X(7) peptide, and by the Src tyrosine inhibitor PP2 and these compounds prevented both large-conductance Panx1 currents and membrane permeabilization. These results together with the lack Panx1 tyrosine phosphorylation in response to P2X(7)R stimulation indicate the involvement of an additional molecule in the tyrosine kinase signal transduction pathway mediating Panx1 activation through the P2X(7)R.
Iglesias
R
R
The Dominick P. Purpura Department of Neuroscience, Albert Einstein College of Medicine, Bronx, New York, USA.
Locovei
S
S
Roque
A
A
Alberto
A P
AP
Dahl
G
G
Spray
D C
DC
Scemes
E
E
D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine / Q000031:analogs & derivatives / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002229:Carbenoxolone / Q000494:pharmacology; D002460:Cell Line; D002463:Cell Membrane Permeability / Q000187:drug effects; D017630:Connexins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005439:Flufenamic Acid / Q000494:pharmacology; D017629:Gap Junctions / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D006801:Humans; D008264:Macrophages / Q000378:metabolism; D015767:Mefloquine / Q000494:pharmacology; D008564:Membrane Potentials; D051379:Mice; D009419:Nerve Tissue Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D009865:Oocytes; D010766:Phosphorylation; D034622:RNA Interference; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D058486:Receptors, Purinergic P2X7; D015398:Signal Transduction / Q000187:drug effects; D014981:Xenopus; D019061:src-Family Kinases / Q000378:metabolism
D017630:Connexins; D009419:Nerve Tissue Proteins; C546468:P2RX7 protein, human; C546469:P2rx7 protein, mouse; C492764:PANX1 protein, human; C503631:Panx1 protein, mouse; D034741:RNA, Small Interfering; D018048:Receptors, Purinergic P2; D058486:Receptors, Purinergic P2X7; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D005439:Flufenamic Acid; C063302:KN 62; D019307:1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; D000255:Adenosine Triphosphate; D019061:src-Family Kinases; D002229:Carbenoxolone; D015767:Mefloquine