Fluorescent Agonists for the Torpedo Nicotinic Acetylcholine Receptor

Krieger, Florian; Mourot, Alexandre; Aráoz, Rómulo; Kotzyba‐Hibert, Florence; Molgó, Jordi; Bamberg, Ernst; Goeldner, Maurice

doi:10.1002/cbic.200700757

Cited by 21 publications

(14 citation statements)

References 42 publications

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“…Few GPCR ligands bearing fluorogenic dyes have been reported to date 7. In addition, they suffer from limited efficacy because of nonspecific interactions8 or the requirement to perform studies on isolated membranes (not living cells) 9. Moreover, all these examples were limited to blue dyes, whereas red dyes are advantageous for cellular studies because of lower sample photodamage, light scattering, and autofluorescence.…”

Section: Methodsmentioning

confidence: 99%

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

et al. 2014

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Classical fluorescence-based approaches to monitor ligand-protein interactions are generally hampered by the background signal of unbound ligand, which must be removed by tedious washing steps. To overcome this major limitation, we report here the first red fluorescent turn-on probes for a G protein-coupled receptor (oxytocin receptor) at the surface of living cells. The peptide ligand carbetocin was conjugated to one of the best solvatochromic (fluorogenic) dyes, Nile Red, which turns on emission when reaching the hydrophobic environment of the receptor. We showed that the incorporation of hydrophilic octa(ethylene glycol) linker between the pharmacophore and the dye minimized nonspecific interaction of the probe with serum proteins and lipid membranes, thus ensuring receptor-specific turn-on response. The new ligand was successfully applied for background-free imaging and quantification of oxytocin receptors in living cells.

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Section: Methodsmentioning

confidence: 99%

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

et al. 2014

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“…Stage V-VI Xenopus oocytes free of follicular cells were maintained in Barth's solution supplemented with antibiotics (15). Transplantation of Torpedo α1 2 βγδ nAChR used microinjection, into the oocyte cytoplasm, of a membrane suspension (50 nL at 3.5 mg/mL protein) from a Nanoliter2000 Micro4 Controller mounted on a microscope (36). Expression of human α4β2 nAChR used microinjection, into the oocyte nucleus, of a 1:1 mixture of cDNA (50 nL at 0.28 μg/μl) encoding α4 and β2 subunits in a pRc/CMV expression vector and a Nanoliter2000 microinjector (15).…”

Section: Methodsmentioning

confidence: 99%

Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism

Bourne

Radić

Aráoz

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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147

194

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Spirolide and gymnodimine macrocyclic imine phycotoxins belong to an emerging class of chemical agents associated with marine algal blooms and shellfish toxicity. Analysis of 13-desmethyl spirolide C and gymnodimine A by binding and voltage-clamp recordings on muscle-type α1 2 βγδ and neuronal α3β2 and α4β2 nicotinic acetylcholine receptors reveals subnanomolar affinities, potent antagonism, and limited subtype selectivity. Their binding to acetylcholine-binding proteins (AChBP), as soluble receptor surrogates, exhibits picomolar affinities governed by diffusion-limited association and slow dissociation, accounting for apparent irreversibility. Crystal structures of the phycotoxins bound to Aplysia-AChBP (≈2.4Å) show toxins neatly imbedded within the nest of aromatic side chains contributed by loops C and F on opposing faces of the subunit interface, and which in physiological conditions accommodates acetylcholine. The structures also point to three major features: (i) the sequence-conserved loop C envelops the bound toxins to maximize surface complementarity; (ii) hydrogen bonding of the protonated imine nitrogen in the toxins with the carbonyl oxygen of loop C Trp147 tethers the toxin core centered within the pocket; and (iii) the spirolide bisspiroacetal or gymnodimine tetrahydrofuran and their common cyclohexene-butyrolactone further anchor the toxins in apical and membrane directions, along the subunit interface. In contrast, the sequence-variable loop F only sparingly contributes contact points to preserve the broad receptor subtype recognition unique to phycotoxins compared with other nicotinic antagonists. These data offer unique means for detecting spiroimine toxins in shellfish and identify distinctive ligands, functional determinants and binding regions for the design of new drugs able to target several receptor subtypes with high affinity.acetylcholine binding protein | marine phycotoxins | nicotinic acetylcholine receptor | pharmacological and structural analyses | seafood poisoning

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“…The α1 2 βγδ nAChR-rich membranes were prepared at 4 °C from freshly dissected and sliced Torpedo electric organs by procedures previously described, 65 resuspended in 5 mM glycine, and stored aliquotted at −80 °C until use. Microtransplantation of Torpedo nAChR used microinjection into the oocyte cytoplasm of a membrane suspension (50 nL at 3.5 mg/mL protein) from a Nanoliter2000 Micro4 controller mounted on a microscope.…”

Section: Methodsmentioning

confidence: 99%

Total Synthesis of Pinnatoxins A and G and Revision of the Mode of Action of Pinnatoxin A

Aráoz¹,

Servent

Molgó³

et al. 2011

J. Am. Chem. Soc.

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125

141

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Pinnatoxins belong to an emerging class of potent marine toxins of the cyclic imine group. Detailed studies of their biological effects have been impeded by unavailability of the complex natural product from natural sources. This work describes the development of a robust, scalable synthetic sequence relying on a convergent strategy that delivered a sufficient amount of the toxin for detailed biological studies and its commercialization for use by other research groups and regulatory agencies. A central transformation in the synthesis is the highly diastereoselective Ireland–Claisen rearrangement of a complex α,α-disubstituted allylic ester based on a unique mode for stereoselective enolization through a chirality match between the substrate and the lithium amide base. With synthetic pinnatoxin A, a detailed study has been performed that provides conclusive evidence for its mode of action as a potent inhibitor of nicotinic acetylcholine receptors selective for the human neuronal α7 subtype. The comprehensive electrophysiological, biochemical, and computational studies support the view that the spiroimine subunit of pinnatoxins is critical for blocking nicotinic acetylcholine receptor subtypes, as evidenced by analyzing the effect of a synthetic analogue of pinnatoxin A containing an open form of the imine ring. Our studies have paved the way for the production of certified standards to be used for mass-spectrometric determination of these toxins in marine matrices and for the development of tests to detect these toxins in contaminated shellfish.

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Fluorescent Agonists for the Torpedo Nicotinic Acetylcholine Receptor

Cited by 21 publications

References 42 publications

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

Red Fluorescent Turn‐On Ligands for Imaging and Quantifying G Protein‐Coupled Receptors in Living Cells

Structural determinants in phycotoxins and AChBP conferring high affinity binding and nicotinic AChR antagonism

Total Synthesis of Pinnatoxins A and G and Revision of the Mode of Action of Pinnatoxin A

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