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Beta subunit coexpression and the alpha1 subunit domain I-II linker affect piperidine block of neuronal calcium
G W Zamponi1, T W Soong, E Bourinet
1Biotechnology Laboratory, University of British Columbia, Vancouver, Canada.
Abstract:
The effects of local anesthetics were examined on a family of transiently expressed neuronal calcium channels. Fomocaine, a local anesthetic containing a morpholine ring, preferentially blocked alpha1E channels (Ki = 100 microM), and had a lower affinity (3- to 15-fold) for alpha1A, alpha1B, and alpha1C channels. Block was incompletely reversible, followed 1:1 kinetics, and did not affect steady-state inactivation properties. Fomocaine block was sensitive to the concentration of permeant ion and enhanced in the presence of external pore blockers, suggesting a site of action in the conducting pathway. Flecainide, which carries a piperidine ring, and the diphenylbutylpiperidine antipsychotic, penfluridol, caused qualitatively similar block, suggesting that morpholine rings are compatible with the piperidine receptor site. In contrast, procaine, which contains an alkyl chain, caused reversible low affinity block of the different calcium channels (Kd values between 2 and 5 mM) and was least effective on alpha1E and did not compete with fomocaine, suggesting that local anesthetics interact with at least two distinct receptor sites. Compared to coexpression with the Ca channel beta1b subunit, block at the piperidine receptor site was significantly weakened with the beta2a subunit suggesting that the nature of the beta subunit contributes to drug binding. Amino acid changes in the cytoplasmic linker between domains I and II resulted in decreased fomocaine and penfluridol blocking affinity. Furthermore, the blocking affinity observed with alpha1B, was conferred on alpha1A by substitution of the domain I-II linker of alpha1B into alpha1A. Taken together, the data suggest that beta subunit binding and the domain I-II linker contribute to the piperidine receptor site on neuronal calcium channels.
Insights
Local anesthetics like fomocaine selectively block neuronal calcium channels, with binding influenced by channel subunits and specific protein regions. This research identifies key sites for drug interaction on these channels.
Area of Science:
- Neuropharmacology
- Molecular Biology
- Ion Channel Research
Background:
- Neuronal calcium channels are crucial for nerve function.
- Local anesthetics can modulate neuronal activity by interacting with ion channels.
- Understanding these interactions is key to developing targeted therapeutics.
Purpose of the Study:
- To investigate the effects of local anesthetics on neuronal calcium channels.
- To identify the specific binding sites and mechanisms of action for different local anesthetics.
- To explore the role of channel subunits and protein domains in drug interaction.
Main Methods:
- Transient expression of neuronal calcium channel subtypes (alpha1A, alpha1B, alpha1C, alpha1E).
- Electrophysiological recordings to assess channel block by local anesthetics (fomocaine, flecainide, penfluridol, procaine).
- Site-directed mutagenesis to investigate the role of beta subunits and the I-II linker domain.
Main Results:
- Fomocaine preferentially blocked alpha1E channels, with lower affinity for other subtypes.
- Block kinetics and ion dependency suggest an intracellular site of action within the channel pore.
- Flecainide and penfluridol showed similar block patterns to fomocaine, indicating compatibility with the piperidine receptor site.
- Procaine exhibited reversible, low-affinity block, suggesting interaction with a distinct receptor site.
- Beta subunit composition (beta1b vs. beta2a) significantly altered blocking affinity.
- Mutations in the I-II linker domain reduced blocking affinity for fomocaine and penfluridol.
Conclusions:
- Local anesthetics interact with neuronal calcium channels at distinct sites.
- The piperidine receptor site involves interactions with beta subunits and the I-II linker domain.
- These findings provide insights into the molecular basis of local anesthetic action on neuronal calcium channels.