Tao Ma,Chenxing Xu,Siyuan Li,Siqi Hu,Kaizhi Ding,Jinpeng Yu,Pengcheng Wei,Dongting Zhangsun,Xiaopeng Zhu,Sulan Luo
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.
Shengrong Pei,Chenxing Xu,Yao Tan,Meiting Wang,Jinpeng Yu,Dongting Zhangsun,Xiaopeng Zhu,Sulan Luo
α6β4* nicotinic acetylcholine receptor (nAChR) (* represents the possible presence of additional subunits) is mainly distributed in the central and peripheral nervous system and is associated with neurological diseases, such as neuropathic pain; however, the ability to explore its function and distribution is limited due to the lack of pharmacological tools. As one of the analogs of α-conotoxin (α-CTx) LvIC from Conus lividus, [D1G, Δ14Q]LvIC (Lv) selectively and potently blocks α6/α3β4 nAChR (α6/α3 represents a chimera). Here, we synthesized three fluorescent analogs of Lv by connecting fluorescent molecules 6-carboxytetramethylrhodamine succinimidyl ester (6-TAMRA-SE, R), Cy3 NHS ester (Cy3, C) and BODIPY-FL NHS ester (BDP, B) to the N-terminus of the peptide and obtained Lv-R, Lv-C, and Lv-B, respectively. The potency and selectivity of three fluorescent peptides were evaluated using two-electrode voltage-clamp recording on nAChR subtypes expressed in Xenopus laevis oocytes, and the potency and selectivity of Lv-B were almost maintained with the half-maximal inhibition (IC50) of 64 nM. Then, we explored the stability of Lv-B in artificial cerebrospinal fluid and stained rat brain slices with Lv-B. The results indicated that the stability of Lv-B was slightly improved compared to that of native Lv. Additionally, we detected the distribution of the α6β4* nAChR subtype in the cerebral cortex using green fluorescently labeled peptide and fluorescence microscopy. Our findings not only provide a visualized pharmacological tool for exploring the distribution of the α6β4* nAChR subtype in various situ tissues and organs but also extend the application of α-CTx [D1G, Δ14Q]LvIC to demonstrate the involvement of α6β4 nAChR function in pathophysiology and pharmacology.
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