Promiscuous and Reversible Blocker of Presynaptic Calcium Channels in Frog and Crayfish Neuromuscular Junctions From Phoneutria nigriventer Spider Venom

2003 ◽  
Vol 90 (5) ◽  
pp. 3529-3537 ◽  
Author(s):  
Lanfranco R. P. Troncone ◽  
John Georgiou ◽  
Shao-Ying Hua ◽  
Donald Elrick ◽  
Ivo Lebrun ◽  
...  
Keyword(s):  
Transmitter Release ◽  
Neuromuscular Junctions ◽  
Therapeutic Agents ◽  
Channel Blockers ◽  
Spontaneous Release ◽  
Presynaptic Calcium ◽  
P Type ◽  
Partial Blockade ◽  
Phoneutria Nigriventer ◽  
Evoked Transmitter Release

Peptide channel blockers found in venoms of many predators are useful pharmacological tools and potential therapeutic agents. The venom of the Brazilian spider Phoneutria nigriventer contains a fraction, ω-phonetoxin-IIA (ω-Ptx-IIA, 8360 MW), which blocks Ca2+ channels. At frog neuromuscular junctions (NMJ) bathed in normal Ca2+ (1.8 mM) saline, ω-Ptx IIA did not affect spontaneous transmitter release but reversibly reduced evoked transmitter release by 75 and 95% at 12 and 24 nM, respectively. In contrast, toxin effects were irreversible in low-Ca2+ (0.5 mM) saline. Ca2+ imaging in normal-Ca2+ saline showed that ω-Ptx-IIA partially blocked stimulus-dependent presynaptic Ca2+ signals, and the blockade was almost completely reversible. Increases in spontaneous release frequency induced by high extracellular K+ were blocked by ω-Ptx-IIA. Therefore ω-Ptx-IIA blocks N-type Ca2+ channels, which admit Ca2+ that triggers transmitter release at the frog NMJ. Additional evidence predicts that ω-Ptx-IIA binds to N-type Ca2+ channels at a different site from that of ω-Conotoxin-GVIA. ω-Ptx-IIA also gave a low-affinity partial blockade of transmitter release and presynaptic Ca2+ signals at crayfish NMJs where P-type channels are blocked by ω-agatoxin-IVA. The Ca2+-dependent reversibility and promiscuity of this toxin may make it highly useful experimentally and therapeutically.

Structural and functional changes of frog neuromuscular junctions in high calcium solutions

1971 ◽  
Vol 178 (1053) ◽  
pp. 407-415 ◽  
Keyword(s):  
Transmitter Release ◽  
Synaptic Vesicles ◽  
Neuromuscular Junctions ◽  
Motor Nerve ◽  
Secretory Response ◽  
Spontaneous Release ◽  
Functional Changes ◽  
High Calcium ◽  
Release Of Acetylcholine ◽  
Evoked Transmitter Release

When frog muscles are exposed for several hours to a solution of isotonic calcium chloride, the secretory response of the motor nerve terminals to imposed depolarization ultimately fails and the rate of spontaneous release of acetylcholine also declines towards zero. The failure of depolarization-evoked transmitter release is irreversible while spontaneous release reappears, though in highly abnormal fashion, when the muscle is returned to a normal ionic medium. Examination of motor end-plates, during various stages of calcium treatment, shows that there is gradual intra-axonal agglutination of synaptic vesicles which is only very incompletely reversible. This effect is presumably the consequence of gradual entry and intracellular accumulation of calcium ions. Analogous treatment with isotonic magnesium, while resulting in immediate loss of evoked transmitter release, does not lead to progressive agglutination of synaptic vesicles, nor to irreversible impairment of the secretory response of the nerve terminal. The possible relations between structural and functional changes during calcium and magnesium treatment are discussed.

Different VAMP/Synaptobrevin Complexes for Spontaneous and Evoked Transmitter Release at the Crayfish Neuromuscular Junction

1998 ◽  
Vol 80 (6) ◽  
pp. 3233-3246 ◽  
Author(s):  
Shao-Ying Hua ◽  
Dorota A. Raciborska ◽  
William S. Trimble ◽  
Milton P. Charlton
Keyword(s):  
Neuromuscular Junction ◽  
Transmitter Release ◽  
Neurotransmitter Release ◽  
Action Potentials ◽  
Neuromuscular Junctions ◽  
Spontaneous Release ◽  
Intracellular Ca ◽  
Crayfish Neuromuscular Junction ◽  
Evoked Transmitter Release

Hua, Shao-Ying, Dorota A. Raciborska, William S. Trimble, and Milton P. Charlton. Different VAMP/synaptobrevin complexes for spontaneous and evoked transmitter release at the crayfish neuromuscular junction. J. Neurophysiol. 80: 3233–3246, 1998. Although vesicle-associated membrane protein (VAMP/synaptobrevin) is essential for evoked neurotransmitter release, its role in spontaneous transmitter release remains uncertain. For instance, many studies show that tetanus toxin (TeNT), which cleaves VAMP, blocks evoked transmitter release but leaves some spontaneous transmitter release. We used recombinant tetanus and botulinum neurotoxin catalytic light chains (TeNT-LC, BoNT/B-LC, and BoNT/D-LC) to examine the role of VAMP in spontaneous transmitter release at neuromuscular junctions (nmj) of crayfish. Injection of TeNT-LC into presynaptic axons removed most of the VAMP immunoreactivity and blocked evoked transmitter release without affecting nerve action potentials or Ca2+ influx. The frequency of spontaneous transmitter release was little affected by the TeNT-LC when the evoked transmitter release had been blocked by >95%. The spontaneous transmitter release left after TeNT-LC treatment was insensitive to increases in intracellular Ca2+. BoNT/B-LC, which cleaves VAMP at the same site as TeNT-LC but uses a different binding site, also blocked evoked release but had minimal effect on spontaneous release. However, BoNT/D-LC, which cleaves VAMP at a different site from the other two toxins but binds to the same position on VAMP as TeNT, blocked both evoked and spontaneous transmitter release at similar rates. The data indicate that different VAMP complexes are employed for evoked and spontaneous transmitter release; the VAMP used in spontaneous release is not readily cleaved by TeNT or BoNT/B. Because the exocytosis that occurs after the action of TeNT cannot be increased by increased intracellular Ca2+, the final steps in neurotransmitter release are Ca2+ independent.

α-Latrocrustatoxin Increases Neurotransmitter Release by Activating a Calcium Influx Pathway at Crayfish Neuromuscular Junction

1999 ◽  
Vol 82 (6) ◽  
pp. 3550-3562 ◽  
Author(s):  
Donald B. Elrick ◽  
Milton P. Charlton
Keyword(s):  
Transmitter Release ◽  
Neuromuscular Junctions ◽  
Calcium Influx ◽  
Fold Increase ◽  
Amplitude Distribution ◽  
Nerve Terminals ◽  
Ionophore A23187 ◽  
Channel Blockers ◽  
Binding Characteristics ◽  
Black Widow Spider Venom

α-latrocrustatoxin (α-LCTX), a component of black widow spider venom (BWSV), produced a 50-fold increase in the frequency of spontaneously occurring miniature excitatory postsynaptic potentials (mEPSPs) at crayfish neuromuscular junctions but did not alter their amplitude distribution. During toxin action, periods of high-frequency mEPSP discharge were punctuated by periods in which mEPSP frequency returned toward control levels. EPSPs were increased in amplitude during periods of enhanced mEPSP discharge. α-LCTX had no effect when applied in Ca2+-free saline, but subsequent addition of Ca2+ caused an immediate enhancement of mEPSP frequency even when α-LCTX was previously washed out of the bath with Ca2+-free saline. Furthermore removal of Ca2+from the saline after α-LCTX had elicited an effect immediately blocked the action on mEPSP frequency. Thus α-LCTX binding is insensitive to Ca2+, but toxin action requires extracellular Ca2+ ions. Preincubation with wheat germ agglutinin prevented the effect of α-LCTX but not its binding. These binding characteristics suggest that the toxin may bind to a crustacean homologue of latrophilin/calcium-independent receptor for latrotoxin, a G-protein-coupled receptor for α-latrotoxin (α-LTX) found in vertebrates. α-LCTX caused “prefacilitation” of EPSP amplitudes, i.e., the first EPSP in a train was enhanced in amplitude to a greater degree than subsequent EPSPs. A similar alteration in the pattern of facilitation was observed after application of the Ca2+ionophore, A23187, indicating that influx of Ca2+ may mediate the action of α-LCTX. In nerve terminals filled with the Ca2+ indicator, calcium green 1, α-LCTX caused increases in the fluorescence of the indicator that lasted for several minutes before returning to rest. Neither fluorescence changes nor toxin action on mEPSP frequency were affected by the Ca2+ channel blockers ω-agatoxin IVA or Cd2+, demonstrating that Ca2+ influx does not occur via Ca2+ channels normally coupled to transmitter release in this preparation. The actions of α-LCTX could be reduced dramatically by intracellular application of the Ca2+ chelator, bis-( o-aminophenoxy)- N,N,N′,N′-tetraacetic acid. We conclude that induction of extracellular Ca2+influx into nerve terminals is sufficient to explain the action of α-LCTX on both spontaneous and evoked transmitter release at crayfish neuromuscular junctions.

scholarly journals Post-tetanic decay of evoked and spontaneous transmitter release and a residual-calcium model of synaptic facilitation at crayfish neuromuscular junctions.

1983 ◽  
Vol 81 (3) ◽  
pp. 355-372 ◽  
Author(s):  
R S Zucker ◽  
L O Lara-Estrella
Keyword(s):  
Transmitter Release ◽  
Calcium Concentration ◽  
Neuromuscular Junctions ◽  
Slow Component ◽  
Nonlinear Relationship ◽  
Time Constants ◽  
Excitatory Junction Potential ◽  
Excitatory Junction Potentials ◽  
Presynaptic Calcium ◽  
Junction Potential

The post-tetanic decay in miniature excitatory junction potential (MEJP) frequency and in facilitation of excitatory junction potentials (EJPs) was measured at crayfish neuromuscular junctions. A 2-s tetanus at 20 Hz caused the MEJP frequency to increase an average of 40 times and the EJP amplitude to increase an average of 13 times. Both MEJP frequency and EJP facilitation decayed with two time constants. The fast component of MEJP frequency decay was 47 ms, and that of EJP facilitation was 130 ms. The slow component of MEJP frequency decay was 0.57 s, and that of EJP facilitation was approximately 1 s. These results were consistent with the predictions of a residual calcium model, with a nonlinear relationship between presynaptic calcium concentration and transmitter release.

Sign in / Sign up

Export Citation Format

Share Document