Nicotinic acetylcholine receptors (nAChRs) are essential pentameric ligand-gated ion channels that are distributed throughout the central and peripheral nervous systems and nonneuronal tissues in mammalian species that play critical roles in a variety of neural and mental activities. The α3β2 nAChR subtype participates in pain, addiction to nicotine, and other neurophysiological and pathological activities. Owing to the lack of highly selective pharmacological tools targeting α3β2, related research on its tissue distribution and function has been hindered. α-Conotoxin (α-CTx) LtIA, discovered from Conus literatus in our lab, potently and selectively blocks α3β2 nAChR, providing an important molecular probe to study the α3β2 nAChR structure and function. We used the fluorescent molecule 5-carboxytetramethylrhodamine succinimidyl ester, which can react with the N-terminus of LtIA, to obtain a novel fluorescent analogue of LtIA (LtIA-F). The potency and selectivity of LtIA-F were tested using a two-electrode voltage clamp recording on various nAChRs expressed in Xenopus laevis oocytes. LtIA-F potently inhibited ACh-evoked currents at the α3β2 nAChR, with an IC 50 value of 90.66 nM, displaying a ∼4-fold decrease in potency compared with native LtIA without a change in selectivity. The serum stability results indicated that LtIA-F exhibited stability similar to that of native LtIA. This study on an α-CTx LtIA fluorescent analogue provides a wealth of pharmacological tools to explore the structure−function relationship, distribution, and ligand binding domain of the α3β2 nAChR subtype.
Neuronal nicotinic acetylcholine receptors (nAChRs) are important ion channel membrane proteins that are widely distributed in the central nervous system (CNS) and peripheral nervous system (PNS). As an important member, α3β4 nAChRs are related to pain sensation in PNS and nicotine addiction in CNS. However, research related to the α3β4 nAChRs is greatly limited by the lack of subtype-selective pharmacological tools. The α-conotoxin (α-CTx) TxID from the marine cone snail, Conus textile, is a selective α3β4 nAChR antagonist with relatively high potency. In this study, a fluorescent dye (5-TAMRA SE) was used to label TxID on the N-terminus of α-CTx TxID, and pure TxID-F (fluorescent analogue of TxID) was obtained by HPLC. At the same time, the potency and selectivity of TxID-F were detected by high-performance liquid chromatography (HPLC). Additionally, the potency and selectivity of TxID-F were determined by using a two-electrode voltage-clamp technique on various nAChRs expressed in the Xenopus oocyte expression system. The results obtained by electrophysiology showed that TxID-F maintained the same order of potency (IC50 73 nM) as the native toxin (IC50 25 nM) for the α3β4 nAChR subtype. In addition, the results of fluorescent spectroscopy and circular dichroism showed TxID-F has the same fluorescence as 5-TAMRA SE, as well as similar profiles as TxID. The results of flow cytometry showed that the histogram shifted significantly to the right for the RAW264.7 cells expressing α3β4-containing nAChRs stained with TxID-F and confirmed by live cell imaging. The study of fluorescent-labeled α-CTx TxID provides a rich pharmacological tool to explore the structure–function relationship, distribution, and ligand-binding domain of α3β4 nAChR subtype in the future.
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