α7- and α9-Containing Nicotinic Acetylcholine Receptors in the Functioning of Immune System and in Pain

Nicotinic acetylcholine receptors (nAChRs) present as many different subtypes in the nervous and immune systems, muscles and on the cells of other organs. In the immune system, inflammation is regulated via the vagus nerve through the activation of the non-neuronal α7 nAChR subtype, affecting the production of cytokines. The analgesic properties of α7 nAChR-selective compounds are mostly based on the activation of the cholinergic anti-inflammatory pathway. The molecular mechanism of neuropathic pain relief mediated by the inhibition of α9-containing nAChRs is not fully understood yet, but the role of immune factors in this process is becoming evident. To obtain appropriate drugs, a search of selective agonists, antagonists and modulators of α7- and α9-containing nAChRs is underway. The naturally occurring three-finger snake α-neurotoxins and mammalian Ly6/uPAR proteins, as well as neurotoxic peptides α-conotoxins, are not only sophisticated tools in research on nAChRs but are also considered as potential medicines. In particular, the inhibition of the α9-containing nAChRs by α-conotoxins may be a pathway to alleviate neuropathic pain. nAChRs are involved in the inflammation processes during AIDS and other viral infections; thus they can also be means used in drug design. In this review, we discuss the role of α7- and α9-containing nAChRs in the immune processes and in pain

pdb

PDB file WT.pdb contains the last frame from 10 ns molecular dynamics simulation alpha7-AChBP chimera (PDB 3SQ6). Only two adjacent subunits were subjected to simulations. PDB files with names E185V, E189G, F187S, L119D, Q117T, R186I, S184N and Y118W contain the last frame from molecular dynamics simulation of alpha7-AChBP chimera with the respective mutations (residues numbered according to human alpha7 nAChR). PDB files F104.pdb and V104_mutant.pdb contain structures in which 104th residue of alpha7 AChBP chimera was changed either to phenylalanine (as in actual human alpha7 nAChR) or to valine (as in alpha9 nAChR)

Data from: Calcium imaging with genetically encoded sensor Case12: facile analysis of α7/α9 nAChR mutants

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

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

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

Electrophysiology and Ca_imaging normalized data

The file contains the normalized electrophysiological (TEVC (two-electrode voltage clamp)) and calcium imaging (Ca-img) data for oocyte and Neuro2a cells responses, respectively. WT human α7 nAChR (nicotinic acetylcholine receptor) and mouse muscle nAChR were expressed in oocytes and Neuro2a cells. Besides, their mutants (at positions 117-119, 184, 185, 187 and 189 in α7 nAChR and at positions 153 and 190 in muscle nAChR) were heterologously expressed as well and their responses were estimated accordingly. Responses to different concentrations of acetylcholine (Ach) and epibatidine (Epi) were measured. For calcium imaging studies two calcium sensors (genetically encoded Case12 and low-molecular weight Fluo-4) were used. Calcium imaging analysis was carried out in the presence of a positive allosteric modulator PNU120596

Photos 101-115

Photos 101-115 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 and Alexa Fluor 555-α-bungarotoxin fluorescence in Neuro2a cells expressing human α7 nAChR (nicotinic acetylcholine receptor); Photos 201-210 – Cytochemical fluorescent and bright field images were generated for quantification of Alexa Fluor 555-α-bungarotoxin fluorescence and background fluorescence (in green channel) in Neuro2a cells expressing human α7 nAChR; Photos 301-316 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 fluorescence and background fluorescence (in red channel) in Neuro2a cells expressing human α7 nAChR; Photos 401-413 – Cytochemical fluorescent and bright field images were generated for quantification of background fluorescence (in green and red channels) in Neuro2a cells expressing human α7 nAChR; Photos 501-505 – Cytochemical fluorescent and bright field images were generated for quantification of Case12 and Alexa Fluor 555-α-bungarotoxin fluorescence in Neuro2a cells expressing mouse muscle (WT nAChR; Photos 604-612 – Cytochemical fluorescent and bright field images were generated for quantification of Alexa Fluor 555-α-bungarotoxin fluorescence and background fluorescence (in green channel) in Neuro2a cells expressing mouse muscle (WT) nAChR; Photos 701-710 – Cytochemical fluorescent and bright field images were generated for quantification of TMRE (Tetramethylrhodamine, ethyl ester; 20 nM) labeling of Neuro2a cells expressing human α7 nAChR; Photos 801-805 – Cytochemical fluorescent and bright field images were generated for quantification of PI (Propidium iodide; 50 ng/ml) labeling of Neuro2a cells expressing human α7 nACh

Expression of the fluorescent calcium ion sensor Case12 in Neuro2a cells correlates with cell viability markers.

<p>Cytochemistry of Neuro2a cells transfected with plasmids coding human α7 nAChR, the chaperone NACHO, and the calcium sensor Case12 revealed that 93.5±0.7% (mean±SEM) of Case12-positive cells (<i>green</i>) were labeled with a cell viability marker 20 nM tetramethylrhodamine ethyl ester (TMRE, top panel, <i>red</i>, n = 3,1240 cells). Case12 fluorescence was absent in non-viable Neuro2a cells stained with the DNA-binding reagent propidium iodide (50 ng/ml, bottom panel, <i>red</i>, <i>arrow heads</i>, n = 3, 1561 cells). Scale bars, 60 μm.</p

Agonist affinity to WT and mutant α7 nAChRs measured by calcium imaging of cell populations, and electrophysiology with <i>Xenopus oocytes</i>.

<p>Agonist affinity to WT and mutant α7 nAChRs measured by calcium imaging of cell populations, and electrophysiology with <i>Xenopus oocytes</i>.</p

Video_Calcium response of N2A cells_a7 nAChR

Videos show calcium response of Neuro2a cells expressing human α7 nAChR (nicotinic acetylcholine receptor), a chaperone NACHO and a fluorescent genetically-encoded calcium sensor Case12 to different concentration of acetylcholine in the presence of a positive allosteric modulator PNU120596. Controls in the presence of α-cobratoxin are presented as well

Cytochemistry and calcium imaging of Neuro2a cells expressing WT and G153S, Y190F mutant muscle nAChRs.

<p>(a, b) Cytochemical labeling of WT muscle nAChR with Alexa Fluor 555-α-bungarotoxin (50 nM, αBgt) in Neuro2a cells. (a) Bright field image, (b) fluorescent image. Scale bar, 50 μm. (c) Pie charts represent percentage of transfected Neuro2a cells labeled with Alexa Fluor 555-α-bungarotoxin (αBgt) in the absence (n = 3, 413 total cells and 311 cells labeled with αBgt) or in the presence of Case12 (n = 3, 1233 total cells, 1005 cells expressing Case12, and 834 cells labeled with αBgt), respectively. (d, e) Dose-response curves of the [Ca²⁺]_i rise amplitude in cells expressing WT and G153S, Y190F mutant muscle nAChRs in response to different concentrations of acetylcholine. The protein calcium sensor Case12 (d) and the fluorescent dye Fluo-4 (e) were used to register changes in [Ca²⁺]_i. Each plot point reflects data obtained from 4 independent experiments (mean ± SEM).</p