Ancestral Reconstruction Approach to Acetylcholine Receptor Structure and Function.

Area of Science:

• Biochemistry
• Evolutionary Biology
• Neuroscience

Background:

• Acetylcholine receptors (AChRs) are crucial pentameric ligand-gated ion channels with diverse evolutionary origins.
• Muscle-type AChRs are heteropentameric complexes composed of α, β, δ, and ɛ subunits.

Purpose of the Study:

• To reconstruct and functionally characterize an ancestral β subunit of muscle-type AChRs.
• To investigate the evolutionary origins of AChR structure and function by creating hybrid receptors.

Main Methods:

• Amino acid sequence reconstruction of an ancestral β subunit shared by humans and cartilaginous fishes.
• Co-expression of the resurrected ancestral β subunit with human α, δ, and ɛ subunits.
• Electrophysiological recordings (whole-cell and single-channel) to assess receptor function.

Main Results:

• The ancestral β subunit successfully formed human/ancestral hybrid AChRs despite 132 amino acid substitutions.
• Hybrid receptors exhibited reduced acetylcholine potency.
• Single-channel recordings showed decreased conductance and open probability in hybrid receptors.

Conclusions:

• The resurrected ancestral β subunit is capable of forming functional hybrid AChRs, providing insights into AChR evolution.
• This approach offers a promising strategy for identifying the molecular basis of AChR structure and function evolution.