Inhibition of P2X4 function by P2Y6 UDP receptors in microglia
Louis-Philippe Bernier1, Ariel R Ase, Éric Boué-Grabot
1Department of Neurology and Neurosurgery, Montreal Neurological Institute, Alan Edwards Center for Research on Pain, McGill University, Montréal, Québec, H3A 2B4, Canada.
Abstract:
ATP-gated P2X4 receptor channels expressed in spinal microglia actively participate in central sensitization, making their functional regulation a key process in chronic pain pathologies. P2Y6 metabotropic Gq -coupled receptors, also expressed in microglia, are involved in the initial response to nerve injury, triggering phagocytosis upon activation by UDP. It has been reported recently that expression of both P2X4 and P2Y6 is upregulated in activated microglia following nerve injury. We show here, in resting as well as LPS-activated primary microglia, that P2Y6 decreases P2X4-mediated calcium entry and inhibits the dilation of P2X4 channels into a large-conductance pore measured with a YO-PRO-1 uptake assay. Furthermore, P2Y6 activation modulates the ATP-dependent migration of microglia, a process likely involved in their shift from migratory to phagocytic phenotype. Reconstituting the P2X4-P2Y6 interaction in recombinant systems shows that P2Y6 activation decreases P2X4 current amplitude, activation and desensitization rates, and reduces P2X4 channel permeability to the large cation NMDG(+) . Phospholipase C-mediated hydrolysis of the phosphoinositide PI(4,5)P2 , a necessary cofactor for P2X4 channel function, underlies this inhibitory crosstalk. As extracellular levels of both ATP and UDP are increased in the spinal cord following nerve injury, the control of P2X4 activity by P2Y6 might play a critical role in regulating neuropathic pain-inducing microglial responses.
Insights
The P2Y6 receptor inhibits the P2X4 channel in microglia, reducing calcium entry and channel dilation. This interaction may regulate microglial responses involved in neuropathic pain.
Area of Science:
- Neuroscience
- Immunology
- Pharmacology
Background:
- Spinal microglia express ATP-gated P2X4 receptors involved in central sensitization and chronic pain.
- P2Y6 receptors, activated by UDP, are also in microglia and mediate initial responses to nerve injury.
- Both P2X4 and P2Y6 receptor expression increase in microglia after nerve injury.
Purpose of the Study:
- To investigate the functional interaction between P2Y6 and P2X4 receptors in primary microglia.
- To elucidate the molecular mechanisms underlying P2Y6 modulation of P2X4 channel activity.
- To assess the role of this crosstalk in microglial function and neuropathic pain.
Main Methods:
- Primary microglia cultures (resting and LPS-activated)
- Calcium imaging assays
- YO-PRO-1 uptake assay for channel dilation
- Recombinant system studies of P2X4-P2Y6 interaction
- Patch-clamp electrophysiology
- Phospholipase C activity assays
Main Results:
- P2Y6 activation significantly decreased P2X4-mediated calcium influx and channel dilation.
- P2Y6 modulated ATP-dependent microglial migration.
- In recombinant systems, P2Y6 activation reduced P2X4 current, altered gating kinetics, and decreased channel permeability.
- This inhibition was mediated by Phospholipase C-dependent hydrolysis of PI(4,5)P2.
Conclusions:
- P2Y6 receptor activation inhibits P2X4 channel function in microglia through PI(4,5)P2 hydrolysis.
- This P2Y6-P2X4 crosstalk influences microglial migration and phenotype.
- Targeting this interaction could offer a novel strategy for managing neuropathic pain.
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