Calcium binding of presynaptic protrusions as revealed by X-ray spectrum averaging in the rat neuromuscular junction.

Presynaptic calcium binding sites were demonstrated by means of X-ray microprobe analysis in rat neuromuscular junctions subjected to perfusion with a calcium containing (5 mM) aldehyde fixative. Type A calcium binding sites are triangular structures facing the junctional folds, identical with presynaptic protrusions of the active zone. Since, because of low concentration and disturbing effects, calcium peaks cannot be detected if using the conventional single shot analysis, the spectrum averaging technique was used. While gradual rising of the calcium peak from the background can be established in the course of averaging 9 spectra obtained from several Type A sites, spectra obtained from indifferent areas of the presynaptic membrane exhibited a less intensive phenomenon. The results are in agreement with previous data obtained by means of electron cytochemical methods, suggesting that Type A sites may play an important role in the regulation of calcium influx to the intraaxonal area. Junctional folds on the postsynaptic membrane may funnel calcium ions exactly to these restricted places of the nerve membrane and, at the arrival of nerve action potential, calcium ions may enter directly to the active zone. This way, a relatively small amount of calcium is sufficient to release neurotransmitter from the terminal.