We have previously identified adenosine triphosphate (ATP)-gated P2X7 receptor (P2X7R) subunits on mammalian motor nerve terminals (Deuchars et al. 2001). Here we report further on some properties of the P2X7R at the neuromuscular junction.
First, we tested if P2X7R-mediated responses are influenced by a receptor protein tyrosine phosphatase-β (RPTPβ), which is associated with the P2X7R complex (but not the P2X2R) and dephosphorylates the receptor (Kim et al. 2001). Transversus abdominis muscle and intercostal nerves were dissected from humanely killed young adult mice and placed into oxygenated physiological saline, and nerve terminals loaded with RH414 via stimulation of intercostal nerve stumps. Application of the P2X7R agonist benzoyl ATP (BzATP; 30 mM) stimulated vesicle release as demonstrated by nerve terminal destaining. In the presence of RPTPβ inhibitor, bisperoxo-(1,10-phenanthrolone)-oxovanadate (BvP: 100 mM), nerve terminal destaining was potentiated (BzATP, 23 ± 4 %, n = 6; BzATP + BvP, 40 ± 9 %, n = 6; control, 9 ± 1 %, n = 5; mean relative destain ± S.E.M.). The native agonist of the P2X7R, ATP, was also able to produce destaining/vesicle release. 10 mM ATP applied in an essentially calcium/magnesium-free saline solution evoked a degree of destaining greater than that by 30 mM BzATP (42 ± 7 %, n = 5).
Since many (approximately 25 %) motor nerve terminals did not destain with agonist application we conducted immuno-electron microscopy to determine if lack of response was related to absence of P2X7R immunoreactivity. Mouse flexor digitorum brevis muscle was fixed in 4 % paraformaldehyde and 0.2 % glutaraldehyde solution. Subsequently, 50 mm sections were labelled with anti-P2X7R antibodies (Alomone, 1:1000), visualized with diaminobenzidine (DAB). Only a proportion of motor nerve terminals possessed the P2X7R – even in ultra-thin section heterogeneity was apparent within a single field of view. Further examination of DAB-labelled motor nerve terminals subsequent to BzATP stimulation revealed that many appeared depleted of synaptic vesicles, compared with unlabelled terminals. In addition, vesicle depletion was blocked by oxidised ATP (100 mM), Brilliant Blue G (1 mM) and calmidazolium (100 mM).
Taken together, these data suggest that P2X7R are located on only a proportion of mammalian motor nerve terminals, and that these can be activated by pharmacological and native agonists. Activation results in release of vesicles as demonstrated by vital imaging and electron microscopy.
K.J.C. and T.S.M. carried out some of this work as part of BSc projects. This work was funded by Action Research.
All procedures accord with current UK legislation.