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 (OP). Due to its high degree of homology with the human muscle subtype, αβdγ 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. Previous studies of the bispyridinium non-oxime MB327 demonstrated promising effects in improving OP-impaired neuromuscular neurotransmission. Therefore, the functional interaction of MB327 with nAChR was investigated 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 (Nanion Technologies, Munich) 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. Experimental approaches with various concentrations of carbamoylcholine and MB327 were performed.

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. The agonist carbamoylcholine at concentrations above 1 mM seemed to induce desensitisation, a conformational state of the receptor without response. In the presence of 1 µM MB327, no carbamoylcholine-induced desensitisation was observed. However, MB327 without agonist did not lead to an intrinsic effect.

Conclusion:

The results presented here demonstrate that functional high-throughput measurements can be performed with native membranes of Torpedo californica electric tissue. It is suggested that MB327 interacts in a manner of positive allosteric modulation. Further research is necessary to verify this hypothesis.