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Mutations at two distinct sites within the channel domain M2 alter calcium permeability of neuronal alpha 7 nicotinic
D Bertrand1, J L Galzi, A Devillers-Thiéry
1Département de Physiologie, Centre Médical Universitaire (Faculté de Médecine), Geneva, Switzerland.
Abstract:
The relative permeability for sodium, potassium, and calcium of chicken alpha 7 neuronal nicotinic receptor was investigated by mutagenesis of the channel domain M2. Mutations in the "intermediate ring" of negatively charged residues, located at the cytoplasmic end of M2 (site 1), reduce calcium permeability without significantly modifying other functional properties (activation and desensitization) of the receptor; a similar change of ion selectivity is also noticed when mutations at site 1 are done in the context of a receptor mutant that conducts ions in a desensitized state. Moreover, mutations of two adjacent rings of leucines at the synaptic end of M2 (site 2) have multiple effects. They abolish calcium permeability, increase the apparent affinity for acetylcholine by 10- to 100-fold, augment Hill numbers (up to 4.6-5.0) of acetylcholine dose-response relationships, slow rates of ionic response onset, and lower the extent of desensitization. Mutations at these two topographically distinct sites within M2 selectively alter calcium transport without affecting the relative permeabilities for sodium and potassium.
Insights
Mutations in the M2 channel domain of chicken alpha 7 nicotinic receptors selectively alter calcium permeability. These findings provide insights into ion channel selectivity and function.
Area of Science:
- Neuroscience
- Molecular Biology
- Biophysics
Background:
- Neuronal nicotinic acetylcholine receptors (nAChRs) are crucial for neurotransmission.
- The M2 channel domain of nAChRs forms the ion-conductive pore and determines ion selectivity.
Purpose of the Study:
- To investigate the role of specific residues within the M2 domain of chicken alpha 7 nAChRs in determining relative ion permeability, particularly for calcium.
- To elucidate how mutations at distinct sites within the M2 domain affect receptor function, including ion selectivity, activation, and desensitization.
Main Methods:
- Site-directed mutagenesis of the M2 channel domain of chicken alpha 7 nAChRs.
- Electrophysiological recordings to assess relative ion permeability (sodium, potassium, calcium).
- Analysis of acetylcholine dose-response relationships and receptor desensitization kinetics.
Main Results:
- Mutations in the 'intermediate ring' (site 1) at the cytoplasmic end of M2 reduced calcium permeability without affecting activation or desensitization.
- Mutations in leucine-rich rings (site 2) at the synaptic end of M2 abolished calcium permeability and altered acetylcholine binding affinity and dose-response relationships.
- Mutations at both sites selectively impacted calcium transport, leaving sodium and potassium permeability largely unaffected.
Conclusions:
- The M2 domain of chicken alpha 7 nAChRs contains distinct regions that differentially regulate calcium permeability.
- Specific mutations within the M2 domain can selectively alter calcium ion flux, offering a tool to probe nAChR channel function and ion selectivity.