Abstract
Elucidation of the structural basis of pharmacological differences for highly homologous α7 and α9 nicotinic acetylcholine receptors (nAChRs) may shed light on their involvement in different physiological functions and diseases. Combination of site-directed mutagenesis and electrophysiology is a powerful tool to pinpoint the key amino-acid residues in the receptor ligand-binding site, but for α7 and α9 nAChRs it is complicated by their poor expression and fast desensitization. Here, we probed the ligand-binding properties of α7/α9 nAChR mutants by a proposed simple and fast calcium imaging method. The method is based on transient co-expression of α7/α9 nAChR mutants in neuroblastoma cells together with Ric-3 or NACHO chaperones and Case12 fluorescent calcium ion sensor followed by analysis of their pharmacology using a fluorescence microscope or a fluorometric imaging plate reader (FLIPR) with a GFP filter set. The results obtained were confirmed by electrophysiology and by calcium imaging with the conventional calcium indicator Fluo-4. The affinities for acetylcholine and epibatidine were determined for human and rat α7 nAChRs, and for their mutants with homologous residues of α9 nAChR incorporated at positions 117–119, 184, 185, 187, and 189, which are anticipated to be involved in ligand binding. The strongest decrease in the affinity was observed for mutations at positions 187 and 119. The L119D mutation of α7 nAChR, showing a larger effect for epibatidine than for acetylcholine, may implicate this position in pharmacological differences between α7 and α9 nAChRs.
Highlights
Homopentameric α7 nicotinic acetylcholine receptors (α7 nAChRs) are ligand-gated ion channels (LGIC) characterized by a high calcium ion permeability [1] and a very fast desensitization rate [2]
For efficient testing of α7/α9 nAChR mutant pharmacology, we have developed a calcium imaging technique based on the transient co-expression of α7 nAChR mutants, a chaperone (Ric-3 or NACHO), and the genetically-encoded calcium sensor Case12
Some residues, such as L119 and F187 might play a crucial role in epibatidine binding to the receptor due to direct influence on the complex structure, contributing to pharmacological differences between α7 and α9 nAChRs
Summary
Homopentameric α7 nicotinic acetylcholine receptors (α7 nAChRs) are ligand-gated ion channels (LGIC) characterized by a high calcium ion permeability [1] and a very fast desensitization rate [2]. Being present on both neuronal and non-neuronal cells, α7 nAChRs modulate different cellular processes, such as release of neurotransmitters, cytokines and neurotrophic factors, as well as downstream signaling, gene expression etc. Calcium imaging test for α7/α9 nAChR mutants.
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