L-type Ca2+ Channels at Low External Calcium Differentially Regulate Neurotransmitter Release in Proximal-Distal Compartments of the Frog Neuromuscular Junction.

L-type Ca2+ channels (LTCCs) are key elements in electromechanical coupling in striated muscles and formation of neuromuscular junctions (NMJs). However, the significance of LTCCs in regulation of neurotransmitter release is still far from understanding. Here, we found that LTCCs can increase evoked neurotransmitter release (especially asynchronous component) and spontaneous exocytosis in two functionally different compartment of the frog NMJ, namely distal and proximal parts. The effects of LTCC blockage on evoked and spontaneous release as well as timing of exocytotic events were prevented by inhibition of either protein kinase C (PKC) or P2Y receptors (P2Y-Rs). Hence, endogenous signaling via P2Y-R/PKC axis can sustain LTCC activity. Application of ATP, a co-neurotransmitter able to activate P2Y-Rs, suppressed both evoked and spontaneous exocytosis in distal and proximal parts. Surprisingly, inhibition of LTCCs (but not PKC) decreased the negative action of exogenous ATP on evoked (only in distal part) and spontaneous exocytosis. Lipid raft disruption suppressed (1) action of LTCC antagonist on neurotransmitter release selectively in distal region and (2) contribution of LTCCs in depressant effect of ATP on evoked and spontaneous release. Thus, LTCCs can enhance and desynchronize neurotransmitter release at basal conditions (without ATP addition), but contribute to ATP-mediated decrease in the exocytosis. The former action of LTCCs relies on P2Y-R/PKC axis, whereas the latter is triggered by exogenous ATP and PKC-independent. Furthermore, relevance of lipid rafts for LTCC function as well as LTCCs for ATP effects is different in distal and proximal part of the NMJ.

Cadmium desynchronizes neurotransmitter release in the neuromuscular junction: Key role of ROS.

Cd2+ is one of the most widespread environmental pollutants and its accumulation in central and peripheral nervous systems leads to neurotoxicity as well as aggravation of common neurodegenerative diseases. Mechanism of the Cd2+ toxicity is far from being resolved. Here, using microelectrode recordings of postsynaptic responses and fluorescent redox indicators we studied the effect of Cd2+ in the submicromolar range on timing of neurotransmitter release and oxidative status in two functionally different compartments of the same frog motor nerve terminal. Cd2+ (0.1-1 µM) acting as typical voltage-gated Ca2+channel (VGCC) antagonist decreased neurotransmitter release in both distal and proximal parts of the nerve terminal, but in contrast to the VGCC blockers Cd2+(0.1-0.5 µM) desynchronized the release selectively in the distal region. The latter action of Cd2+ was completely prevented by inhibitor of NADPH-oxidase and antioxidants, including mitochondrial specific, as well as redox-sensitive TRPV1 channel blocker. Cd2+ markedly increased levels of mitochondrial reactive oxygen species (ROS) in both the distal and proximal compartments of the nerve terminal, which was associated with lipid peroxidation mainly in the distal region. Zn2+, whose transport systems translocate Cd2+, markedly enhanced the effects of Cd2+ on both the mitochondrial ROS levels and timing of neurotransmitter release. Furthermore, in the presence of Zn2+ ions, Cd2+ also desynchronized the neurotransmitter release in the proximal region. Thus, in synapses Cd2+ at very low concentrations can increase mitochondrial ROS, lipid peroxidation and disturb the timing of neurotransmitter release via a ROS/TRPV-dependent mechanism. Desynchronization of neurotransmitter release and synaptic oxidative stress could be early events in Cd2+ neurotoxicity.

ATP Reduces the Entry of Calcium Ions into the Nerve Ending by Blocking L-type Calcium Channels.

At neuromuscular junctions, ATP inhibits both the evoked and spontaneous acetylcholine release and inward calcium current operating via presynaptic P2Y receptors. It was shown in the experiments with the frog neuromuscular synapse using specific calcium-sensitive dye Oregon Green Bapta 1 that exogenous ATP reduces the amplitude of calcium transient, which reflects the changes in the entry of calcium ions in response to the nerve pulse. The depressing effect of ATP on the transient was prevented by suramin, the blocker of P2 receptors. Nitrendipine, a specific blocker of L-type calcium channels, per se decreased the calcium transient amplitude and significantly attenuated the effect of ATP on the calcium signal. Contrariwise, the preliminary application of ATP to the neuromuscular junction completely eliminated the depressing effect of nitrendipine on the calcium response. The obtained data suggest that an essential component in the inhibitory action of ATP on the calcium transient amplitude is provided by reduction of the entry of calcium ions into a frog nerve ending via L-type voltage-gated calcium channels.

Presynaptic nicotinic cholinoreceptors modulate velocity of the action potential propagation along the motor nerve endings at a high-frequency synaptic activity.

Experiments on frog neuromuscular junctions have demonstrated that asynchrony of the acetylcholine quantal release forming the multi-quantal evoked response at high-frequency synaptic activity is caused, in particular, by a decrease in velocity of the action potential propagation along the non-myelinated nerve endings, which is mediated by activation of the α7 and α4ß4 nicotinic cholinoreceptors.