Site-Specific Fluorescent Labeling of the Cysteine-Rich Toxin, DkTx, for TRPV1 Ion Channel Imaging and Membrane Binding Studies

Abstract

Peptide toxins secreted by venomous animals bind to mammalian ion channel proteins and modulate their function. The high specificity of these toxins for their target ion channels enables them to serve as powerful tools for ion channel biology. Toxins labeled with fluorescent dyes are employed for the cellular imaging of channels and also for studying toxin-channel and toxin-membrane interactions. Several of these toxins are cysteine-rich, rendering the production of properly folded fluorescently labeled toxins technically challenging. Herein, we evaluate a variety of site-specific protein bioconjugation approaches for producing fluorescently labeled double-knot toxin (DkTx), a potent TRPV1 ion channel agonist that contains an uncommonly large number of cysteines (12 out of a total of 75 amino acids present in the protein). We find that popular cysteine-mediated bioconjugation approaches are unsuccessful as the introduction of a non-native cysteine residue for thiol modification leads to the formation of misfolded toxin species. Moreover, N-terminal aldehyde-mediated bioconjugation approaches are also not suitable as the resultant labeled toxin lacks activity. In contrast to these approaches, C-terminal bioconjugation of DkTx via the sortase bioconjugation technology yields functionally active fluorescently labeled DkTx. We employ this labeled toxin for imaging rat TRPV1 heterologously expressed in Xenopus laevis oocytes, as well as for performing membrane binding studies on giant unilamellar vesicles composed of different lipid compositions. Our studies set the stage for using fluorescent DkTx as a tool for TRPV1 biology and provide an informative blueprint for labeling cysteine-rich proteins.

Dissecting the contributions of membrane affinity and bivalency of the spider venom protein DkTx to its sustained mode of TRPV1 activation

“…We envisioned that the presence of a large number of cysteines in our proposed tetra-knots (24 in each molecule) would render their production challenging due to the likelihood of these cysteines forming non-native mixed disulfides during folding. This apprehension was supported by our recent work on labeling DkTx with a fluorophore that revealed that introducing a non-native cysteine into DkTx results in the formation of misfolded toxin species ( 26 ). Therefore, instead of attempting to produce the tetra-knots by employing our standard protocol for producing DkTx that entails expressing the full-length linear protein in E. coli followed by subjecting it to redox folding conditions, we employed a step-wise approach involving the production of properly folded precursor double-knots followed by their enzymatic ligation to form the desired tetra-knots.…”

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

“…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. 32 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.…”

“…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.…”

Neurotoxin-Derived Optical Probes for Biological and Medical Imaging

“…To visualise the expression and distribution of TRPV1 channels, double-knot-toxin (DkTx) from tarantula Ornithoctonus huwena venom [ 151 ] conjugated with fluorescein via sortase at the C-terminus was applied to Xenopus leaves oocytes transfected with rat TRPV1 channels [ 152 ] (Table 5 ; Fig. 2 ).…”

“…Because DkTx contains a large number of cysteine residues, cysteine-mediated bioconjugation caused a high level of misfolding of the toxin. Nevertheless, the authors report that their approach enabled successful and selective labelling of rat TRPV1 in the oocyte model [ 152 ].…”