Calcium imaging with genetically encoded sensor Case12: Facile analysis of α7/α9 nAChR mutants

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.

Azemiopsin, a Selective Peptide Antagonist of Muscle Nicotinic Acetylcholine Receptor: Preclinical Evaluation as a Local Muscle Relaxant

“…Az also manifested ability to interact with the human neuronal homopentameric α7 nAChR but with a much lower affinity (IC 50 = 2.67 ± 0.02 μM, Figure 2 b). It should be mentioned, that the corresponding cellular calcium responses were provoked by acetylcholine (ACh) at concentrations of 30 μM (EC 100 on muscle nAChR) and 10 μM (EC 50 on α7 nAChR), respectively [ 11 ]. Electrophysiology experiments discovered no influence of Az on ion currents induced by 20 μM nicotine in rat neuronal heteromeric α4β2 nAChR at a concentration up to 50 μM ( Figure 2 c).…”

“…Calcium imaging procedure was performed as published earlier [ 11 ]. Briefly, after removing the growth medium, the transfected Neuro2a cells and cultivated SH-SY5Y cells were washed with a buffer containing 140 mM NaCl, 2 mM CaCl 2 , 2.8 mM KCl, 4 mM MgCl 2 , 20 mM HEPES, 10 mM glucose; pH 7.4.…”

Makaluvamine G from the Marine Sponge Zyzzia fuliginosa Inhibits Muscle nAChR by Binding at the Orthosteric and Allosteric Sites

“…Since severe muscle weakness of affected persons is attributed to Ca 2+ accumulation in the endplates [ 19 ], we decided to test the effectiveness MG via Ca 2+ detection in the fluorescence experiments ( Figure 4 C,D) using the method described in reference [ 20 ].…”

“…Fluorescent detection of cytoplasmic Ca 2+ rise . Calcium imaging was performed following Reference [ 20 ]. Briefly, the cell medium was removed and the cells were washed with the external buffer.…”

Activation of α7 Nicotinic Acetylcholine Receptor Upregulates HLA-DR and Macrophage Receptors: Potential Role in Adaptive Immunity and in Preventing Immunosuppression

“…This effect became more prominent after application of more potent agonist epibatidine (Epi, 10 µM, Figure 3B,K) and α7 nAChR selective agonist PNU 282,987 (1 µ M, Figure 3C,K). It is very hard or even impossible to detect α7 nAChR-mediated calcium response in neurons and neuroblastoma cells in the absence of a selective positive α7 nAChR allosteric modulator PNU 120,596, decreasing the extremely high rate of receptor desensitization [35][36][37]. Applying PNU 120,596 (10µ M) to THP-1ϕ cells, we observed a great increase in the number of nicotine-responsive cells and in the corresponding calcium rise amplitudes ( Figure 3D,L).…”