Ca(2+)-dependent and -independent components of transmitter release at the frog neuromuscular junction

Abstract

1. When a Ca2+ chelator, bis (O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), was loaded into the presynaptic nerve terminal of the frog neuromuscular junction (NMJ), facilitation, measured as an increase in endplate potential (EPP) amplitudes during a train of ten stimulations at 100 Hz, was greatly decreased within 20 min of BAPTA-AM (the acetoxymethyl ester of BAPTA) perfusion, and remained at a constant low level thereafter, suggesting that [Ca2+]i at the presynaptic nerve terminal was buffered by BAPTA. 2. Detailed examination of the two components of facilitation of EPP amplitude in the BAPTA-loaded NMJs showed that the fast component was lost almost completely, while the slow component was unaffected by loaded BAPTA. Augmentation and potentiation were also unaffected by BAPTA. 3. Under external Ca(2+)-free conditions (with 1 mM-EGTA), both augmentation and potentiation of miniature endplate potential (MEPP) frequency were clearly observed after tetanic stimulation in the normal NMJ, and were also unaffected by loaded BAPTA. 4. The above findings strongly support the residual Ca2+ hypothesis for the fast component of facilitation, and suggest that the three slower processes (the slow component of facilitation, augmentation and potentiation) occur independently of [Ca2+]i. This Ca2+ independence was supported by the fact that facilitation and potentiation have multiplicative effects on the amount of release. 5. The quantal content of the first EPP in the train remained unchanged throughout the time course of BAPTA loading for most NMJs. This suggests that [Ca2+]i immediately adjacent to Ca2+ channels at the active zone triggers transmitter release and is little affected by loaded BAPTA. 6. MEPP frequency was almost unchanged during BAPTA loading, suggesting that the basal [Ca2+]i remained unchanged close to the dissociation constant of BAPTA for Ca2+ (108 nM). 7. The slow component of facilitation had a multiplicative relationship with augmentation and potentiation, suggesting that the underlying mechanism for the slow component of facilitation differs from that for augmentation and potentiation.