Mechanism of P2X7 receptor-dependent enhancement of neuromuscular transmission in pannexin 1 knockout mice
P2X7 receptors are present in presynaptic membranes of motor synapses, but their regulatory role in modulation of neurotransmitter release remains poorly understood. P2X7 receptors may interact with pannexin 1 channels to form a purinergic signaling unit. The potential mechanism of P2X7 receptor-dependent modulation of acetylcholine (ACh) release was investigated by recording miniature endplate potentials (MEPPs) and evoked endplate potentials (EPPs) in neuromuscular junctions of wild-type (WT) and pannexin 1 knockout (Panx1−/−) mice. Modulation of P2X7 receptors with the selective inhibitor A740003 or the selective agonist BzATP did not alter the parameters of either spontaneous or evoked ACh release in WT mice. In Panx1−/− mice, BzATP-induced activation of P2X7 receptors resulted in a uniformly increased quantal content of EPPs during a short stimulation train. This effect was accompanied by an increase in the size of the readily releasable pool, while the release probability did not change. Inhibition of calmodulin by W-7 or of calcium/calmodulin-dependent kinase II (CaMKII) by KN-93 completely prevented the potentiating effect of BzATP on the EPP quantal content. The blockade of L-type calcium channels also prevented BzATP action on evoked synaptic activity. Thus, the activation of presynaptic P2X7 receptors in mice lacking pannexin 1 resulted in enhanced evoked ACh release. Such enhanced release was provoked by triggering the calmodulin- and CaMKII-dependent signaling pathway, followed by activation of presynaptic L-type calcium channels. We suggest that in WT mice, this pathway is downregulated due to pannexin 1-dependent tonic activation of inhibitory presynaptic purinergic receptors, which overcomes P2X7-mediated effects.
KeywordsP2X7 receptors Neuromuscular junction Pannexin 1 CaMKII L-type calcium channels
A.S. Miteva, A.E. Gaydukov, and O.P. Balezina conceptualized ideas, designed the study, and wrote the manuscript. A.S. Miteva and A.E. Gaydukov performed the experiments and analyzed the data; V.I. Shestopalov assisted in the discussion of the results and crafting the manuscript.
This study was supported by the Russian Foundation for Basic Research grant 18-34-00189 and the NIH grants EY R01-021517 and P30 EY014801.
Compliance with ethical standards
Conflicts of interest
Anna S. Miteva declares that she has no conflict of interest.
Alexander E. Gaydukov declares that he has no conflict of interest.
Valery I. Shestopalov declares that he has no conflict of interest.
Olga P. Balezina declares that she has no conflict of interest.
All experimental procedures in this study were approved by the Bioethics Committee of Moscow State University.
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