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

Abstract

Previously, we have presented evidence for the presence of L-type voltage-dependent Ca2+ channels (VDCC) in 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (BAPTA-AM)-incubated motor nerve terminals (MNTs) of the levator auris muscle of mature mice. The aim of the present 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 quantal content (QC) of transmitter release with the P/Q-type channels fully blocked. The QC was not significantly different under the three experimental conditions tested: incubation with dimethylsulphoxide (DMSO), ethylene-glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid, (acetoxymethyl)ester (EGTA-AM) and BAPTA-AM. After preincubation with OA (1 microM), but not with pervanadate, QC increased substantially in the BAPTA-AM-incubated (up to 400%) MNT, but not in those incubated with DMSO or EGTA-AM. The OA-induced increment of QC was attenuated greatly (approximately 95% reduction) by preincubation with either nitrendipine (10 microM) or calciseptine (300 nM). The effect of OA (1 microM) and pervanadate (0.1 mM) on spontaneous neurotransmitter release was also studied. After preincubation with OA, but not per-vanadate, miniature end-plate potential (MEPP) frequency increased only in the BAPTA-AM-incubated MNT (up to 700% increment). This response was attenuated (by approximately 80%) by nitrendipine (10 microM) or calciseptine (300 nM). In contrast, neither omega-agatoxin IVA (120 nM) nor omega-conotoxin GVIA (1 microM) affected this OA-induced increment significantly. We also evaluated the relationship between QC and extracellular [Ca2+] ([Ca2+]o) in BAPTA-AM-incubated MNT. Under conditions in which only P/Q-type VDCC were available to participate in neurotransmitter release, QC increased as [Ca2+]o was raised from 0.5 to 2 mM. However, when only L-type VDCC were available, QC increased when [Ca2+]o increased from 0.5 to 1 mM, but decreased significantly 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 microM), a potent inhibitor of protein kinase C (PKC) and adenosine 3',5'cyclic monophosphate (cAMP)-dependent protein kinase (PKA), attenuated the OA-induced increment of both QC and MEPP frequency (50% and 70% decrement, respectively), suggesting the participation of at least 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 protein phosphatases are inhibited and intracellular [Ca2+] is buffered by the fast chelator BAPTA.

Iberiotoxin-Induced Block of Ca2+-Activated K+Channels Induces Dihydropyridine Sensitivity of ACh Release from Mammalian Motor Nerve Terminals

“…Furthermore, it has been postulated that at mammalian motor nerve terminals, L-type Ca 2ϩ channels are in close proximity to A 2A adenosine receptors and activation of A 2A receptors during prolonged depolarization most likely unmasks L-type Ca 2ϩ channels via activation of protein kinases (Correia-de-Sá et al, 2000a). More direct evidence also supports the role of protein kinases in activating L-type Ca 2ϩ channels involved in ACh release from mature mammalian motor nerves (Urbano et al, 2001).…”

Acetylcholine Release at Neuromuscular Junctions of Adult Tottering Mice Is Controlled by N-(Cav2.2) and R-Type (Cav2.3) but Not L-Type (Cav1.2) Ca2+Channels

“…These concentrations were chosen based on literature determining their effectiveness at murine neuromuscular junctions (cf. Atchison, 1989;Xu et al, 1998;Santafe et al, 2000;Urbano et al, 2001Urbano et al, , 2003Flink and Atchison, 2002;Kaja et al, 2006).…”

Increased asynchronous release and aberrant calcium channel activation in amyloid precursor protein deficient neuromuscular synapses

“…Aberrant activation of N-or L-type calcium channels has been observed when P/Q type VDCC or the adhesion protein NCAM are disrupted (Pagani et al, 2006;Polo-Parada et al, 2001;Urbano et al, 2001;Urbano et al, 2003). Of note, the N-type Ca 2+ channel is located farther away from the Ca 2+ sensor as compared with the P/Qtype channel (Wu et al, 1999), which increase the probability of Ca 2+ being bound by a calcium buffer like EGTA-AM, and would contribute to a higher extent of asynchronous release and greater synaptic depression at the presynaptic terminals (Hefft and Jonas, 2005;Pagani et al, 2006;Zakharenko et al, 1999).…”

“…Of relevance to the present study, P/Q-type Ca 2+ channels are the predominant VDCCs involved in mediating synaptic transmission at the mature mammalian NMJ. N-and L-type calcium channels become active only under certain non-physiological conditions such as genetic or pharmacological manipulations of VDCCs or diseases (Pagani et al, 2006;Polo-Parada et al, 2001;Urbano et al, 2001;Urbano et al, 2003). Since administration of N-and L-type Ca 2+ channel blockers was more effective on inhibiting the evoked and delayed release in APP −/− NMJs as compared to the wild-type controls, our results indicate that loss of APP leads to ectopic activation of N-and L-type Ca 2+ channels, which contribute, at least in part, to increased asynchronous release and greater depression.…”