A Fluorescent Inhibitory Peptide Identifies α6β4 nAChR Distribution in the Rat Urinary Tract

α6β4 nicotinic acetylcholine receptors (nAChRs) represent potential therapeutic targets for pain management; however, studies on the distribution and function of these receptors have been constrained by the scarcity of pharmacological tools. An analogue of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC, selectively inhibits heterologous rat α6/α3β4 nAChRs (where α6/α3 denotes the chimeric subunit) and represents a promising pharmacological tool. However, its limited stability may restrict further applications. Rational design based on the [D1G, Δ14Q]LvIC analogue led to the identification of a key mutant, [d-Lys¹¹]LvIC, with a half-maximal inhibitory concentration (IC₅₀) of 8.2 nM. The mutant retained the desired activity and substantial selectivity for 11 other nAChR subtypes while demonstrating superior stability in rat serum. Based on these favorable properties, we conjugated the analogue to a BODIPY-FL (BDP) fluorophore to obtain a high-fidelity fluorescent probe, enabling the investigation of α6β4 nAChRs tissue distribution in rats. The fluorescent probe, [d-Lys¹¹]LvIC-BDP, was preliminarily applied to detect a reduction in α6β4 nAChRs expression in a cyclophosphamide (CYP)-induced rat model of interstitial cystitis/bladder pain syndrome (IC/BPS). The development of this fluorescent probe provides an ideal tool for investigating the tissue distribution and functional characterization of α6β4 nAChRs.