Coupling of L-type calcium channels to neurotransmitter release at mouse motor nerve terminals

Previously, we have presented evidence for the presence of L-type voltage-d ependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-te traacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve termi nals (MNTs) of the levator auris muscle of mature mice. The aim of the pres ent work was to study the coupling of these L-type VDCC to neurotransmitter release by inhibiting protein phosphatases. We thus studied the effects of the protein phosphatase inhibitors okadaic acid (OA) and pervanadate on qu antal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimenta l conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-gl ycerol-bis(beta -aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethy l)ester (ECTA-AM) and BAPTA-AM. After preincubation with OA (1 muM), but no t with pervanadate, QC increased substantially in the BAPTA-AM-incubated (u p to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-ind uced increment of QC was attenuated greatly (similar to 95% reduction) by p reincubation with either nitrendipine (10 muM) or calciseptine (300 nM). Th e effect of OA (1 muM) and pervanadate (0.1 mM) on spontaneous neurotransmi tter release was also studied. After preincubation with OA, but not pervana date, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by similar to 80%) by nitrendipine (10 muM) or calciseptine (300nM). In c ontrast, neither omega -agatoxin IVA (120 nM) nor omega -conotoxin GVIA (1 muM) affected this OA-induced increment significantly. We also evaluated th e relationship between QC and extracellular [Ca2+] ([Ca2+](i)) in BAPTA-AM- incubated MNT Under conditions in which only P/Q-type VDCC were available t o participate in neurotransmitter release, QC increased as [Ca2+](0) was ra ised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC in creased when [Ca2+], increased from 0.5 to 1 mM, but decreased significantl y at 2 mM. The mean latency for P/Q-type VDCC-mediated EPP was 1.7-1.9 ms; for L-type VDCC-mediated EPP, 1.9-2.5 ms. The rise time of the L-type VDCC mediated EPP was significantly slower than that mediated by P/Q-type VDCC. Preincubation with H-7 (100 CIM), a potent inhibitor of protein kinase C (P KC) and adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinas e (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at l east these two protein kinases in the coupling of L-type VDCC. In summary, our results show coupling of L-type VDCC to neurotransmitter release when p rotein phosphatases are inhibited and intracellular [Ca2+] is buffered by t he fast chelator BAPTA.