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Effects of Ca2+ channel blocker neurotoxins on transmitter release and presynaptic currents at the mouse
E Katz1, D A Protti, P A Ferro
1Instituto de Biología Celular, Facultad de Medicina, Universidad de Buenos Aires, Paraguay, Argentina.
Abstract:
1. The effects of the voltage-dependent calcium channel (VDCC) blockers omega-agatoxin IVA (omega-AgaIVA), omega-conotoxin GVIA (omega-CgTx), omega-conotoxin MVIIC (omega-MVIIC) and omega-conotoxin MVIID (omega-MVIID) were evaluated on transmitter release in the mouse diaphragm preparation. The effects of omega-AgaIVA and omega-MVIIC were also evaluated on the perineurial calcium and calcium-dependent potassium currents, ICa and IK(Ca), respectively, in the mouse levator auris preparation. 2. The P- and Q-type VDCC blocker omega-AgaIVA (100 nM) and P- Q- and N-type channel blockers omega-MVIIC (1 microM) and omega-MVIID (3 microM) strongly reduced transmitter release (> 80-90% blockade) whereas the selective N-type channel blocker omega-CgTx (5 microM) was ineffective. 3. The process of release was much more sensitive to omega-MVIIC (IC50 = 39 nM) than to omega-MVIID (IC50 = 1.4 microM). After almost completely blocking transmitter release (quantal content approximately 0.3% of its control value) with 3 microM omega-MVIIC, elevating the external [Ca2+] from 2 to 10 mM induced an increase of approximately 20 fold on the quantal content of the endplate potential (e.p.p.) (from 0.2 +/- 0.04 to 4.8 +/- 1.4). 4. Nerve-evoked transmitter release in a low Ca(2+)-high Mg2+ medium (low release probability, quantal content = 2 +/- 0.1) had the same sensitivity to omega-AgaIVA (IC50 = 16.8 nM) as that in normal saline solutions. In addition, K(+)-evoked transmitter release was also highly sensitive to the action of this toxin (IC50 = 11.5 nM; 100 nM > 95% blockade). The action of omega-AgaIVA on transmitter release could be reversed by toxin washout if the experiments were carried out at 31-33 degrees C. Conversely, the effect of omega-AgaIVA persisted even after two hours of toxin washout at room temperature. 5. Both the calcium and calcium-dependent potassium presynaptic currents, ICa and IK(Ca), respectively, were highly sensitive to low concentrations (10-30 nM) of omega-AgaIVA. The ICa and the IK(Ca) were also strongly reduced by 1 microM omega-MVIIC. The most marked difference between the action of these two toxins was the long incubation times required to achieve maximal effects with omega-MVIIC. 6. In summary these results provide more evidence that synaptic transmission at the mammalian neuromuscular junction is mediated by Ca2+ entry through P- and/or Q-type calcium channels.
Insights
Voltage-dependent calcium channel (VDCC) blockers, specifically omega-agatoxin IVA and omega-conotoxin MVIIC, significantly inhibit transmitter release at the mouse neuromuscular junction. These findings support the role of P- and/or Q-type calcium channels in synaptic transmission.
Area of Science:
- Neuroscience
- Pharmacology
- Cell Biology
Background:
- Synaptic transmission relies on calcium influx through voltage-dependent calcium channels (VDCCs).
- Specific VDCC subtypes mediate neurotransmitter release at the neuromuscular junction, but their precise roles require further elucidation.
Purpose of the Study:
- To investigate the effects of various VDCC blockers on transmitter release in the mouse diaphragm.
- To examine the impact of specific blockers on calcium and calcium-dependent potassium currents in the mouse levator auris.
Main Methods:
- Electrophysiological recordings in mouse diaphragm and levator auris preparations.
- Application of VDCC blockers: omega-agatoxin IVA (omega-AgaIVA), omega-conotoxin GVIA (omega-CgTx), omega-conotoxin MVIIC (omega-MVIIC), and omega-conotoxin MVIID (omega-MVIID).
- Measurement of transmitter release, presynaptic calcium currents (ICa), and calcium-dependent potassium currents (IK(Ca)).
Main Results:
- Omega-AgaIVA (P/Q-type) and omega-MVIIC/MVIID (P/Q/N-type) significantly reduced transmitter release (>80-90% blockade).
- Selective N-type blocker omega-CgTx was ineffective, indicating N-type channels are not primary mediators of release.
- Release was more sensitive to omega-MVIIC (IC50 = 39 nM) than omega-MVIID (IC50 = 1.4 microM).
- Both ICa and IK(Ca) were sensitive to omega-AgaIVA and omega-MVIIC, with omega-MVIIC requiring longer incubation times.
Conclusions:
- Synaptic transmission at the mammalian neuromuscular junction is primarily mediated by Ca2+ entry through P- and/or Q-type calcium channels.
- Omega-AgaIVA and omega-MVIIC are potent inhibitors of transmitter release, highlighting the importance of P/Q-type channels.
- The differential sensitivity and kinetics of blockers provide insights into channel subtypes involved in neurotransmission.