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

The ability of P2X7 receptors to potentiate rhythmically evoked acetylcholine (ACh) release through Ca(2+) entry via P2X7 receptors and via L-type voltage-dependent Ca(2+) channels (VDCCs) was compared by loading Ca(2+) chelators into motor nerve terminals. Neuromuscular preparations of the diaphragms of wild-type (WT) mice and pannexin-1 knockout (Panx1(−/−)) mice, in which ACh release is potentiated by the disinhibition of the L-type VDCCs upon the activation of P2X7 receptors, were used. Miniature end-plate potentials (MEPPs) and evoked end-plate potentials (EPPs) were recorded when the motor terminals were loaded with slow or fast Ca(2+) chelators (EGTA-AM or BAPTA-AM, respectively, 50 μM). In WT and Panx1(−/−) mice, EGTA-AM did not change either spontaneous or evoked ACh release, while BAPTA-AM inhibited synaptic transmission by suppressing the quantal content of EPPs throughout the course of the short rhythmic train (50 Hz, 1 s). In the motor synapses of either WT or Panx1(−/−) mice in the presence of BAPTA-AM, the activation of P2X7 receptors by BzATP (30 μM) returned the EPP quantal content to the control level. In the neuromuscular junctions (NMJs) of Panx1(−/−) mice, EGTA-AM completely prevented the BzATP-induced increase in EPP quantal content. After Panx1(−/−) NMJs were treated with BAPTA-AM, BzATP lost its ability to enhance the EPP quantal content to above the control level. Nitrendipine (1 μM), an inhibitor of L-type VDCCs, was unable to prevent this BzATP-induced enhancement of EPP quantal content to the control level. We propose that the activation of P2X7 receptors may provide additional Ca(2+) entry into motor nerve terminals, which, independent of the modulation of L-type VDCC activity, can partially reduce the buffering capacity of Ca(2+) chelators, thereby providing sufficient Ca(2+) signals for ACh secretion at the control level. However, the activity of both Ca(2+) chelators was sufficient to eliminate Ca(2+) entry via L-type VDCCs activated by P2X7 receptors and increase the EPP quantal content in the NMJs of Panx1(−/−) mice to above the control level.