Fluorescently Labeled α-Conotoxin TxID, a New Probe for α3β4 Neuronal Nicotinic Acetylcholine Receptors

Abstract

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.

Synthesis, Activity, and Application of Fluorescent Analogs of [D1G, Δ14Q]LvIC Targeting α6β4 Nicotinic Acetylcholine Receptor

“…41 Additionally, TxID-F, as a fluorescent analog of α-CTx TxID, could block α3β4 nAChR with an IC 50 of 73 nM, and it could serve as a fluorescent probe for cell imaging. 29 However, Cy3-RgIA-5727 and TxID-F exhibited poor selectivity. 29,41 Thus, the successful design and application of fluorescent analogs of α-CTxs provide novel pharmacological tools for exploring the function and distribution of nAChRs in specific tissues.…”

“…For example, Cy3-RgIA-5727, as a fluorescent analog of α-CTx RgIA-5474, potently blocked α9α10 nAChR expressed in Xenopus oocytes with an IC 50 of 23 pM, and it could be applied to label the α9α10 nAChR in mouse cochlear tissue . Additionally, TxID-F, as a fluorescent analog of α-CTx TxID, could block α3β4 nAChR with an IC 50 of 73 nM, and it could serve as a fluorescent probe for cell imaging . However, Cy3-RgIA-5727 and TxID-F exhibited poor selectivity. , Thus, the successful design and application of fluorescent analogs of α-CTxs provide novel pharmacological tools for exploring the function and distribution of nAChRs in specific tissues.…”

“…Additionally, TxID-F, as a fluorescent analog of α-CTx TxID, could block α3β4 nAChR with an IC 50 of 73 nM, and it could serve as a fluorescent probe for cell imaging . However, Cy3-RgIA-5727 and TxID-F exhibited poor selectivity. , Thus, the successful design and application of fluorescent analogs of α-CTxs provide novel pharmacological tools for exploring the function and distribution of nAChRs in specific tissues. Fluorescent analogs, especially those with high activity and specificity, show great potential as fluorescent probes in the future.…”

“…For 27 Other α-CTxs, such as MII and TxID (targeting α3β2 and α3β4 nAChRs, respectively), could also connect with different fluorescent molecules, providing pharmacological tools for exploring the functional neuroanatomy of various subtypes of nAChRs. 19,28,29 Scorpion toxins targeting voltage-gated potassium ion (K V ) channels could also be linked to different fluorescent molecules, such as HsTX1[R14A], OSK1, and AgTx2, serving as pharmacological tools for studying K V channels. 30,31 In addition, a labeled toxin for imaging the rat TRPV1 ion channel could be obtained by connecting an agonist double-knot toxin (DkTx) with fluorescent molecules.…”

“…For example, the fluorescent molecule 5-carboxytetramethylrhodamine succinimidyl ester (5-TAMRA-SE) could react with the N-terminus of α-CTx LtIA targeting α3β2 nAChR to obtain a novel fluorescent analog of LtIA . Other α-CTxs, such as MII and TxID (targeting α3β2 and α3β4 nAChRs, respectively), could also connect with different fluorescent molecules, providing pharmacological tools for exploring the functional neuroanatomy of various subtypes of nAChRs. ,, Scorpion toxins targeting voltage-gated potassium ion (K V ) channels could also be linked to different fluorescent molecules, such as HsTX1­[R14A], OSK1, and AgTx2, serving as pharmacological tools for studying K V channels. , In addition, a labeled toxin for imaging the rat TRPV1 ion channel could be obtained by connecting an agonist double-knot toxin (DkTx) with fluorescent molecules . Together, these results indicate that peptide toxins with high activity and selectivity for specific subtypes of ion channels can serve as ideal pharmacological tools for visualizing and imaging ion channels when linked to various fluorescent molecules.…”