Molecular Probes for the Visualization of Nicotinic Acetylcholine Receptors Based on Snake Three-Finger Toxins and Red Fluorescent Protein

Abstract— Objective: The visualization of macromolecular complexes is an essential task in modern bioorganic chemistry. This study presents the development of fluorescent molecular probes based on snake toxins for targeting nicotinic acetylcholine receptors (nAChRs). Chimeric constructs were produced by fusing snake toxins with a red fluorescent protein and evaluated for binding affinity and imaging capability. Methods: Three chimeric proteins were engineered by fusing snake toxins (α-bungarotoxin, α-cobratoxin, or neurotoxin NT-II) with the red fluorescent protein mKate2. These constructs were expressed in a bacterial system and purified by size-exclusion chromatography. The binding affinity of the probes to nAChRs was evaluated using competitive radioligand assay with radiolabeled α-bungarotoxin. Fluorescence microscopy was used to visualize receptor binding on human neuroblastoma SH-SY5Y cells. Results and Discussion: The chimeric fluorescent probes exhibited a high binding affinity for nAChRs derived from the electric organ of Torpedo californica, with half-maximal inhibitory concentration (IC₅₀) values in the nanomolar range. This demonstrates that the fusion of the snake toxins with mKate2 does not impair receptor binding. Furthermore, the probes enabled successful visualization of acetylcholine receptors on the surface of SH-SY5Y cells, confirming their functionality as imaging tools. The use of these constructs provides a non-radioactive, highly specific method for receptor localization in biological systems. Conclusions: The developed fluorescent molecular probes based on snake neurotoxins and mKate2 are effective tools for high-affinity binding and visualization of nAChRs. These chimeric proteins offer a promising approach for non-radioactive imaging in neurochemical and pharmacological studies.