P2X4 receptor channels form large noncytolytic pores in resting and activated microglia.
P2X4 ATP-gated cation channels have been shown to contribute to the microglial component of central sensitization, making their functional regulation a key element in chronic pain pathologies. Here we show that prolonged activation of native P2X4 receptor channels by ATP induces opening of a pore permeable to NMDG(+) and large fluorescent dyes in BV-2 microglial cells and primary murine microglia. This intrinsic pore formation mechanism is potentiated by LPS treatment, known to upregulate P2X4 expression in microglial cells and to mimic the microglial activation observed in neuropathic pain states. Sustained activation of the P2X7 channel subtype, also expressed in microglia, induces a pore formation that requires pannexin hemichannels and leads to plasma membrane blebbing and cytotoxicity. In contrast, P2X4 pore formation is unaffected by the pannexin blocker carbenoxolone, does not induce cytoskeletal rearrangements and does not lead to cell death. Furthermore, we show that P2X4 pore dilation is modulated by phosphoinositides (PIP(n) ) levels as it is inhibited by wortmannin, a blocker of PIP(n) synthesis, suggesting possible regulation by phospholipase C-coupled pathways. Nonlethal P2X4 pore dilation could play a role in neuropathic pain by allowing the flux of large organic molecules in microglia. Different outcomes of P2X4 and P2X7 membrane permeabilization point to subtype-specific microglial responses to ATP in normal and pathological neuro-immune crosstalks.
Department of Neurology & Neurosurgery, Montreal Neurological Institute, Alan Edwards Center for Research on Pain, McGill University, Montréal, Québec, Canada. philippe.seguela@mcgill.ca
Louis-Philippe
LP
Bernier
Ariel R
AR
Ase
Eric
E
Boué-Grabot
Philippe
P
Séguéla
D000818:Animals; D000831:Animals, Newborn; D002463:Cell Membrane Permeability / Q000502:physiology*; D003601:Cytotoxicity Tests, Immunologic; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D005971:Glutamates / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000378:metabolism*; D058848:Receptors, Purinergic P2X4 / Q000737:chemistry / Q000378:metabolism*; D016192:Resting Phase, Cell Cycle / Q000502:physiology*; D014982:Xenopus laevis
D005456:Fluorescent Dyes; D005971:Glutamates; D058848:Receptors, Purinergic P2X4; C014574:gamma-glutamylmethylamide