Mechanism of P2X7 receptor-dependent enhancement of neuromuscular transmission in pannexin 1 knockout mice

Abstract

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 wildtype (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 CaMKIIdependent 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.

Interaction between Calcium Chelators and the Activity of P2X7 Receptors in Mouse Motor Synapses

“…Next, we tested the contribution of P2X7 receptors, which might provide specific Ca 2+ influx to nerve terminals, to the regulation of ACh secretion in NMJs. Like we demonstrated recently [10], the activation of P2X7 receptors by their exogenous agonist BzATP (30 µM) at the NMJs of WT mice did not change either spontaneous or evoked ACh release during rhythmic synaptic activity ( Figure 3A,B).…”

“…Further analysis of the specific activity of P2X7 receptors was performed in the NMJs of Panx1 −/− mice. Recently, we found that in the motor synapses of Panx1 −/− mice but not in those of WT mice, BzATP induces the potentiation of evoked ACh release, which can be prevented by the inhibition of CaMKII or L-type VDCCs [10]. Here, we reproduced the potentiating effect of BzATP in the NMJs of Panx1 −/− mice (Figure 4 A-C).…”

“…We found that the application of BzATP in the presence of EGTA did not affect the MEPP amplitude, but the ability of BzATP to provoke a significant increase in the quantal content of EPPs was fully prevented by EGTA; the mean value of this parameter for the first EPP in the train was 25.10 ± 2.21 (n = 15, p > 0.05), which was indistinguishable from the control ( Figure 4D, E). Thus, the loading of EGTA-AM into motor nerve terminals produced the same effect as that observed upon the blockade of L-type VDCCs: the BzATP-induced potentiation of EPPs was prevented completely in motor synapses [10]. We found that when BAPTA-AM was used instead of EGTA-AM, the incubation of Panx1 −/− NMJs with 50 µM BAPTA-AM did not alter any of the parameters of MEPPs but decreased both the amplitude of EPPs and their quantal content, as in WT mice.…”

“…Here, we reproduced the potentiating effect of BzATP in the NMJs of Panx1 −/− mice (Figure 4 A-C). We speculated that this effect of BzATP is triggered by the activation of P2X7 receptors followed by the CaMKII-mediated disinhibition of L-type VDCCs, leading to the potentiation of Ca 2+ -dependent ACh release [10]. The role of P2X7 receptor-mediated Ca 2+ influx as a form of Ca 2+ entry (in comparison with Ca 2+ influx through L-type VDCCs) in enhancing ACh secretion in Panx1 −/− motor synapses remains unknown.…”

“…Recently, we showed that the activation of P2X7 receptors by BzATP (30 µM) did not alter evoked end-plate potential (EPP) parameters in wild-type (WT) mice. At the neuromuscular junctions (NMJs) of pannexin-1 knockout (Panx1 −/− ) mice, lacking the pannexin-1-dependent tonic activation of inhibitory presynaptic purinergic receptors, the same activation of presynaptic P2X7 receptors may be accompanied by signaling pathway activation, which includes the activation of calcium-calmodulin-dependent protein kinase type II (CaMKII), the disinhibition of L-type VDCCs and the potentiation of evoked ACh release [10]. However, which of the two modes of activated Ca 2+ entry, directly via the channels of P2X7 receptors itself or via L-type VDCCs, provides Ca 2+ -dependent facilitation of ACh release and the extent of this facilitation remain unclear.…”