Modulation of heteromeric P2X1/5 receptors by phosphoinositides in astrocytes depends on the P2X1 subunit.
Purinergic signaling is critical for neuron-glia communication. Glial cells participate in synaptic transmission and express metabotropic P2Y as well as ionotropic P2X ATP receptors. In astrocytes, endogenous ATP-evoked currents with kinetics and pharmacology characteristic of the heteromeric P2X1/5 receptor channel have recently been reported. We investigated the interaction of major phosphoinositides with heteromeric P2X1/5 channels. Using patch-clamp electrophysiology on enhanced green fluorescent protein-expressing astrocytes acutely isolated from cortical slices of transgenic mice, we report a strong modulation of P2X1/5-like currents by phosphoinositides. Wortmannin-induced depletion of phosphoinositides decreases the amplitude of both the fast and sustained component of the P2X1/5-like currents although recovery and kinetics remain intact. In transfected human embryonic kidney cells, we provide evidence that depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] levels significantly decreases P2X1/5 currents while intracellular application of PI(4,5)P(2) completely rescued P2X1/5 currents, ruling out the involvement of phosphatidylinositol 3,4,5-trisphosphate. In contrast to P2X1, homomeric P2X5 current responses were found insensitive to phosphoinositides, and the C-terminus of P2X5 subunit lacked binding to phospholipids in an overlay assay. Our results suggest that the contribution of calcium-permeable heteromeric P2X1/5 receptor channels to the excitability of astrocytes is modulated by PI(4,5)P(2) through the P2X1 lipid-binding domain.
Ase
Ariel R
AR
Montreal Neurological Institute, Department of Neurology & Neurosurgery, McGill University, Montreal, Quebec, Canada.
Bernier
Louis-Philippe
LP
Blais
Dominique
D
Pankratov
Yuriy
Y
Séguéla
Philippe
P
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes; D001921:Brain / Q000166:cytology; D002461:Cell Line, Transformed; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation / Q000379:methods; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005904:Glial Fibrillary Acidic Protein / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D008563:Membrane Lipids / Q000378:metabolism; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D018408:Patch-Clamp Techniques / Q000379:methods; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000494:pharmacology; D010716:Phosphatidylinositols / Q000378:metabolism*; D051381:Rats; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058469:Receptors, Purinergic P2X; D058485:Receptors, Purinergic P2X5; D014162:Transfection / Q000379:methods; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D005904:Glial Fibrillary Acidic Protein; D008563:Membrane Lipids; C546465:P2RX5 protein, human; C546466:P2rx5 protein, mouse; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D058485:Receptors, Purinergic P2X5; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000255:Adenosine Triphosphate; C002630:alpha,beta-methyleneadenosine 5'-triphosphate; D000077191:Wortmannin