Cell-Free Electrophysiological Functional Measurements with Native Membranes of Torpedo Californica Electric Tissue

Abstract

Objective: Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels which mediate Na+, K+ and Ca2+ conductance. The human muscle subtype α1β1δe is an important target in drug discovery related to anaesthesia or therapeutic approaches of intoxications with organophosphorus compounds. Because of its high degree of homology with the human muscle subtype, αβδγ nAChRs from the Torpedo electric organ are a suitable surrogate. For functional characterisation, cell-free electrophysiological methods based on solid supported membranes (SSM) can be useful in cases where conventional electrophysiology cannot be applied. In this sensor based technique, charge translocation is measured via capacitive coupling of the supporting membrane. To obtain a sufficient current density, multiple parameters have an impact evaluated in this study.Experimental procedures: Crude membranes from frozen electric organ of Torpedo californica were purified with sucrose gradient centrifugation and then adsorbed to SSM varying buffers, average vesicle size and incubation. The activation of nAChR was measured with a SURFE2R workstation allowing automated high throughput measurements. Voltage dependent sodium channels were blocked with ambroxol. Rapid exchange of a non-activating buffer to an activating buffer containing the agonist carbamoylcholine initiated Na+ influx in the membrane vesicles. The results were correlated with the different experimental designs.Results: Depending on the average particle size, zeta potential and incubation, stable and reproducible cholinergic signals were recorded (∼ 1 nA). The use of ambroxol prevented a premature collapse of the sodium gradient. Increasing carbamoylcholine content in the activating solution led to curves with saturation behaviour. However, carbamoylcholine concentrations > 1 mM seemed to induce desensitisation of the receptors.Conclusion: The results presented here demonstrate that functional high-throughput measurements can be performed with native membranes of Torpedo californica electric tissue. Though, maintaining and regeneration of sodium gradient requires sensitive measurements.