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Interactions of the neurotoxin apamin with a Ca2+-activated K+ channel in primary neuronal cultures
Abstract:
Mono[125I]iodoapamin bound to specific sites on cultured rat embryonic neurons. The dissociation constant for the receptor-neurotoxin complex measured at equilibrium was 60-120 pM at pH 7.2 and 4 degrees C, with a maximal binding capacity of 3-8 fmol/mg of cell protein. Apamin inhibited calcium ionophore-induced 86Rb+ release from cell cultures. The dose effect curve for this pharmacological test corresponded closely to the displacement of 125I-apamin by native apamin in binding experiments. Formation of the 125I-apamin receptor complex requires exogenous K+. Reduced binding in the absence of K+ was due to diminished binding capacity rather than a lower affinity. The apamin receptor seems to be associated with a cell surface K+ site which shows 50% occupancy at 1.6 mM, and which could be involved in the regulation of channel activity. Apamin sites were present at the earliest developmental stage tested and their number did not evolve during 8 days in culture. In the same period, however, alpha-scorpion toxin binding increased by a factor of 10. The ontogenesis of Ca2+-activated K+ channels does not seem to occur in parallel with that of voltage-sensitive Na+ channels.
Insights
Apamin specifically binds to neuronal receptors, inhibiting potassium (K+) release and indicating a link between apamin receptors and cell surface K+ sites involved in channel regulation. These sites are present early in development but do not mature alongside voltage-sensitive sodium channels.
Area of Science:
- Neuroscience
- Cell Biology
- Pharmacology
Background:
- Apamin is a neurotoxin known to interact with neuronal targets.
- Understanding the specific binding sites and functional roles of neurotoxins is crucial for elucidating neuronal signaling pathways.
Purpose of the Study:
- To characterize the binding of mono-[125I]iodoapamin to cultured rat embryonic neurons.
- To investigate the relationship between apamin binding and potassium (K+) channel activity.
- To explore the developmental expression of apamin receptors in relation to other ion channels.
Main Methods:
- Radioligand binding assays using mono-[125I]iodoapamin to determine receptor affinity and density.
- Pharmacological experiments measuring 86Rb+ release to assess apamin's effect on K+ efflux.
- Comparative analysis of apamin and alpha-scorpion toxin binding during neuronal development.
Main Results:
- Mono-[125I]iodoapamin bound to specific sites on neurons with high affinity (60-120 pM dissociation constant).
- Apamin inhibited calcium ionophore-induced 86Rb+ release, correlating with binding displacement data.
- Apamin receptor complex formation required exogenous K+, suggesting an association with K+ sites regulating channel activity.
- Apamin receptor density remained constant during 8 days in culture, while alpha-scorpion toxin binding increased significantly.
Conclusions:
- Apamin receptors are present on early embryonic neurons and are functionally linked to K+ channel activity.
- The apamin receptor appears associated with a cell surface K+ site involved in channel regulation.
- The developmental pattern of Ca2+-activated K+ channels differs from that of voltage-sensitive Na+ channels.