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omega-Conotoxin block of N-type calcium channels in frog and rat sympathetic neurons
L M Boland1, J A Morrill, B P Bean
1Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115.
Abstract:
Block of N-type Ca channels by omega-conotoxin GVIA (CgTx) was studied in freshly dissociated bullfrog and rat sympathetic neurons. With 2-5 mM Ba as charge carrier, CgTx blocked almost all of the high-threshold Ca channel current recorded in the presence of nimodipine (3 microM) to block L-type Ca channels. Toxin block reversed slowly (time constant approximately 1 hr) in frog cells and even more slowly in rat cells. CgTx block was faster and more potent in rat cells than frog cells. The rate of block was proportional to CgTx concentration, consistent with 1:1 binding of CgTx to channels. When the external Ba concentration was increased, the development of block was slower, consistent with competition between CgTx and Ba for a binding site. The recovery from block was somewhat faster in higher external Ba. Some cells had significant current remaining in saturating concentrations of nimodipine and CgTx, especially with high Ba concentrations in the external solution. The current resistant to nimodipine and CgTx was activated at lower depolarizations than the CgTx-sensitive current and had faster activation and inactivation kinetics, but unlike low-threshold T-type current, the resistant current had rapidly decaying tail currents.
Insights
Omega-conotoxin GVIA (CgTx) effectively blocks N-type calcium channels in frog and rat neurons. Block kinetics and potency differ between species, with some remaining current resistant to both CgTx and nimodipine.
Area of Science:
- Neuroscience
- Pharmacology
- Ion Channel Physiology
Background:
- N-type calcium channels are crucial for neuronal function.
- Omega-conotoxin GVIA (CgTx) is a selective blocker of N-type calcium channels.
- Understanding CgTx block kinetics is important for its therapeutic applications.
Purpose of the Study:
- To investigate the block of N-type calcium channels by CgTx in bullfrog and rat sympathetic neurons.
- To characterize the kinetics and potency of CgTx block.
- To identify potential remaining calcium channel currents resistant to CgTx and nimodipine.
Main Methods:
- Electrophysiological recordings (whole-cell patch-clamp) were performed on dissociated bullfrog and rat sympathetic neurons.
- Barium (Ba) was used as the charge carrier (2-5 mM).
- Nimodipine (3 µM) was used to block L-type calcium channels.
Main Results:
- CgTx effectively blocked high-threshold calcium channel current in the presence of nimodipine.
- Block reversal was slow (time constant ~1 hr in frog, slower in rat).
- CgTx block was faster and more potent in rat neurons compared to frog neurons.
- Block rate was proportional to CgTx concentration, suggesting 1:1 binding.
- Increased external Ba concentration slowed block development, indicating competition for binding.
- Some current remained resistant to both CgTx and nimodipine, with distinct activation and kinetic properties.
Conclusions:
- CgTx is a potent blocker of N-type calcium channels in sympathetic neurons.
- Species-specific differences exist in CgTx block kinetics.
- The findings support a 1:1 binding model for CgTx.
- Competition between CgTx and Ba ions influences block kinetics.
- A distinct population of calcium channels resistant to CgTx and nimodipine may exist.