Characterization of cell membrane fragments containing muscle type nAChR from Tetronarce californica after preparation using high pressure homogenization.

The nicotinic acetylcholine receptor (nAChR) is a pentameric ligand-gated ion channel (pLGIC) commonly used as a model for receptors belonging to the Cys-loop superfamily. Members of pLGICs are standardly used in numerous toxicological investigations e.g., GABA and nAChR in the context of nerve agent poisoning. Organophosphorus compounds inhibit AChE, leading to accumulation of acetylcholine in the synaptic cleft and subsequently to a cholinergic crisis, in part through desensitization of nAChR. Due to the limitations of standard therapy, studies concerning functional ligand-receptor interactions of therapeutically active substances are of high importance. Therefore, we developed a novel method to obtain muscle type nAChR-containing membrane fragments from native tissue using high-pressure homogenization. The obtained microsomal fragments were characterized using Dynamic Light Scattering, laser Doppler electrophoresis and protein concentration. The microsomal membrane fragments were further purified, and the plasma membrane fraction was enriched using different density gradients. K and B values were determined using a scintillation proximity assay (SPA) with [H]epibatidine as reporter ligand. Measurement data showed that the ideal conditions to obtain microsomal membrane fragments with high pressure homogenization were four runs at 400bar. For density gradient centrifugation the under layering of the microsomal membrane fragments (bottom-up method) is to be preferred for further purification. Sucrose seems to be more efficient compared to xylitol or iodixanol density gradients. The nAChR-containing plasma membrane fractions resulting from the developed purification protocol achieve a high degree of quality and reproducibility, making them suitable to model physiological conditions. This system has the potential to be used in both bead- and filtration-based assays probing affinity parameters for ligand binding or functional experiments. The protocol can be easily modified for other LGICs or transmembrane proteins, allowing for further expansion of its use.