Effects of Ca 2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse neuromuscular junction (original) (raw)
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The Journal of Physiology, 1995
The effects of the calcium channel blockers, funnel-web spider toxin (FTX), w-agatoxin IVA (w-Aga IVA) and w-conotoxin GVIA (w-CgTX), were tested on transmitter release and presynaptic currents in frog motor nerve endings. 2. Evoked transmitter release was blocked by FTX (IC50= 0o02 #l ml-') and w-CgTX (1 #M) but was not affected by w-Aga IVA (0'5/M). When FTX (0 1 #l ml-') was assayed on spontaneous release either in normal Ringer solution or in low Ca2+-high Mg2+ solution, it was found not to affect miniature endplate potential (MEPP) amplitude but to increase MEPP frequency by-2-fold in both conditions. 3. Presynaptic calcium currents (Ica), measured by the perineurial technique in the presence of 10 mm tetraethylammonium chloride (TEA) and 200 juM BaCl2 to block K+ currents, were blocked by w-CgTX (5 ,UM), partially blocked by FTX (1 1d ml-') and not affected by w-Aga IVA (0 5,UM). 4. The presynaptic calcium-activated potassium current (IK(ca)) measured by the perineurial technique in the presence of 0 F5jM 3,4-aminopyridine (DAP) to block voltage-dependent K+ currents, was strongly affected by charybdotoxin (ChTX) (300 nM) and completely abolished by BaCl2 (200 juM). This current was also blocked by w-CgTX (5 FM) and by CdC12 (200 FM) but was not affected by FTX (1 Fl ml-'). The blockade by w-CgTX could not be reversed by elevating [Ca]o to 10 mM. 5. The results suggest that in frog synaptic terminals two w-CgTX-sensitive populations might coexist. The transmitter release process seems to be mediated by calcium influx through a w-CgTXand FTX-sensitive population.
The Journal of physiology, 1996
1. The involvement of the different types of voltage-dependent calcium channels (VDCCs) in synaptic transmission at the mature and newly formed mammalian neuromuscular junction was studied by evaluating the effects of L-, P/Q- and N-type VDCC antagonists on transmitter release in normal and reinnervating levator auris preparations of adult mice. 2. Nerve-evoked transmitter release was blocked by omega-agatoxin IVA (omega-AgaIVA), a P/Q-type VDCC blocker, both in normal and reinnervating endplates (100 nM omega-AgaIVA caused > 90% inhibition). The N-type VDCC antagonist omega-conotoxin GVIA (omega-CgTX; 1 and 5 microM), as occurs in normal preparations, did not significantly affect this type of release during reinnervation. Nitrendipine (1-10 microM), an L-type VDCC blocker, strongly antagonized release in reinnervating muscles (approximately 40-69% blockade) and lacked any effect in normal preparations. 3. In reinnervating muscles, spontaneous release was not dependent on Ca2+ en...
Proceedings of the National Academy of Sciences, 1992
We have studied the effect of the purified toxin from the funnel-web spider venom (FTX) and its synthetic analog (sFTX) on transmitter release and presynaptic currents at the mouse neuromuscular junction. FTX specifically blocks the a-conotoxinand dihydropyridine-insensitive P-type voltage-dependent Ca2+ channd (VDCC) in cerebellar Purkinje cells. Mammalian neuromuscular transmission, which is insensitive to N-or L-type Ca2+ channel blockers, was effectively abolished by FTX and sFTX. These substances blocked the muscle contraction and the neurotransmitter release evoked by nerve stimulation. Moreover, presynaptic Ca2+ currents recorded extracellularly from the interior of the perineural sheaths of nerves innervating the mouse levator auris muscle were specifically blocked by both natural toxin and synthetic analogue. In a parallel set of experiments, K+-induced Ca'5 uptake by brain synaptosomes was also shown to be blocked or greatly diminished by FIX and sFVX. These results indicate that the predominant VDCC in the motor nerve terminals, and possibly in a significant percentage of brain synapses, is the P-type channel.
Neuroscience, 1989
The dihydropyridine, Bay K 8644, was applied in vitro to mouse phrenic nerve-diaphragm muscle preparations. The drug increased both spontaneous and evoked release of acetylcholine from the motor nerve terminal in a concentration- and time-dependent manner. The rise in miniature endplate potential frequency, however, was the result of an increased intraterminal mobilization of free calcium, rather than well-established activation of voltage-dependent calcium channels. This view is supported by the following observations: (1) an increase in frequency was apparent in Ca2+-free medium; (2) Bay K 8644 is known to require a moderate depolarization to affect Ca2+ channels, but no membrane depolarization was detected; and (3) exposure to low Ca2+ and high Mg2+ medium did not diminish the effect on miniature endplate potential frequency. In a medium containing low Ca2+ and high Mg2+, Bay K 8644 increased quantal content of the evoked endplate potentials to a greater degree and with a faster ...
Voltage-dependent P/Q-type calcium channels at the frog neuromuscular junction
Physiological research / Academia Scientiarum Bohemoslovaca, 2011
It is well known that antagonists of N-type voltage-gated calcium channels inhibit the evoked quantal release of acetylcholine in amphibian neuromuscular synapses. This, however, does not exclude the functional expression of other types of voltage-gated calcium channels in these nerve terminals. Using immunocytochemistry, we detected the expression of the alpha1A subunit of P/Q-type calcium channels (that is otherwise typical of mammalian motor nerve endings) in the frog neuromuscular junction. In addition, we demonstrated that the P/Q-type channel blocker omega-agatoxin IVA (20 nM) reduced the action potential-induced calcium transient and significantly decreased both spontaneous and evoked mediator release. Our data indicates the functional expression of P/Q-type calcium channels in the frog motor nerve ending which participate in acetylcholine release.
Pflügers Archiv : European journal of physiology, 1997
The identity of the voltage-dependent calcium channels (VDCC), which trigger the Ca2+-gated K+ currents (IK(Ca)) in mammalian motor nerve terminals, was investigated by means of perineurial recordings. The effects of Ca2+ chelators with different binding kinetics on the activation of IK(Ca) were also examined. The calcium channel blockers of the P/Q family, omega-agatoxin IVA (omega-Aga-IVA) and funnel-web spider toxin (FTX), have been shown to exert a strong blocking effect on IK(Ca). In contrast, nitrendipine and omega-conotoxin GVIA (omega-CgTx) did not affect the Ca2+-activated K+ currents. The intracellular action of the fast Ca2+ buffers BAPTA and DM-BAPTA prevented the activation of the IK(Ca), while the slow Ca2+ buffer EGTA was ineffective at blocking it. These data indicate that P/Q-type VDCC mediate the Ca2+ influx which activates IK(Ca). The spatial association between Ca2+ and Ca2+-gated K+ channels is discussed, on the basis of the differential effects of the fast and ...
Neurochemistry International, 2005
The present study shows that ω-agatoxin-TK, a toxin of the venom of Agelenopsis aperta, which is 10 times more concentrated than the P/Q type Ca2+ channel blocker, ω-agatoxin-IVA in the venom, inhibits the high K+ depolarisation-induced rise in internal Ca2+ (Cai, as determined with fura-2) dose dependently in cerebral (striatal and hippocampal) isolated nerve endings, with calculated IC50's of about 60 nM. The maximal inhibition exerted by ω-agatoxin-TK in striatal synaptosomes (61 ± 11%) is 10% larger than in hippocampal synaptosomes, suggesting a larger population of ω-agatoxin-TK-sensitive Ca2+ channels in striatal than in hippocampal nerve endings. The N-type Ca2+ channel blocker, ω-conotoxin-GVIA (1 μM), inhibits part of the ω-agatoxin-TK-insensitive rise in Cai induced by high K+. In contrast to the inhibition exerted by ω-agatoxin-TK on the Cai response to high K+, ω-agatoxin-TK failed to inhibit the tetrodotoxin-sensitive elevations in Cai and in internal Na+ (Nai, as determined with SBFI) induced by veratridine, indicating that the Ca2+ influx activated by veratridine does not involve ω-agatoxin-TK-sensitive channels. High K+ does not increase Nai. In [3H]Glu preloaded hippocampal synaptosomes super-fused with low Na+ Krebs Ringer HEPES (a condition that guarantees the elimination of neurotransmitter transporters-mediated release), the release of [3H]Glu induced by high K+ is absolutely dependent on the entrance of external Ca2+. This exocytotic release of [3H]Glu attained in the absence of a chemical Na+ gradient is inhibited with the same potency and efficacy by ω-agatoxin-TK and by ω-agatoxin-IVA, which is known to differ from ω-agatoxin-TK in its amino terminal moiety. These results indicate that ω-agatoxin-TK represents a good pharmacological tool to study P/Q type Ca2+ channel-mediated responses in cerebral nerve endings.