Two-photon uncaging: New prospects in neuroscience and cellular biology.

An uncaging process refers to a fast and efficient release of a biomolecule after photochemical excitation from a photoactivatable precursor. Two-photon excitation produces excited states identical to standard UV excitation while overcoming major limitations when dealing with biological materials, like spatial resolution, tissue penetration and toxicity and has therefore been applied to the uncaging of different biological effectors. A literature survey of two-photon uncaging of biomolecules is described in this article, including applications in cellular- and neurobiology.

Affinity labeling of cysteine-mutants evidences contact residues in modeled receptor binding sites.

To investigate the topology of binding sites in two ionotropic receptors, we have initiated a strategy combining affinity labeling with cysteine-scanning mutagenesis. For the GABAA receptor we have used reactive derivatives of non-competitive blockers (NCBs) to explore interacting positions in its channel. The polypeptide positions of the M2 segment of the alpha1 subunit which we mutated into cysteine were selected for their established accessibility, as determined by the substituted-cysteine accessibility method (SCAM). Using the Xenopus oocyte expression system, we show that receptors containing mutations V257C and S272C are inactivated by several reactive NCBs. These position-selective inactivations lead to an analysis of NCB binding in the channel. For the NMDA receptor glycine-binding site, the prototype antagonist L-701,324 was derivatized at different positions with different reactive groups. The receptor positions to mutate into cysteine were selected after a 3-D homology model. The observed receptor inactivations are mutant- and probe-selective, leading to an unambiguous chemical docking of the antagonist pharmacophore and supporting the model. The site-specificity of the inactivating reactions is assessed by protection experiments and by mutant to wild-type (WT) comparisons. The scope and limitations of the method are briefly discussed.

Cysteine mutants as chemical sensors for ligand-receptor interactions.

The incorporation of cysteine residues into membrane receptors by mutagenesis has enabled the development of engineered proteins. Chemical modification of the mutant receptor using a wide range of biochemical and biophysical probes has facilitated functional studies of ligand-receptor interactions. In particular, the substituted-cysteine accessibility method (SCAM) represents a successful example of how to probe transmembrane receptor domains after chemical modification of the mutants with sulfydryl-reacting molecules. We propose an extension of this methodology using site-specific affinity probes that react with cysteine mutants to gain reliable structural information on the binding of a ligand in its receptor site.

Dynamic deconvolution of a pre-equilibrated dynamic combinatorial library of acetylcholinesterase inhibitors.

A dynamic combinatorial library composed of interconverting acylhydrazones has been generated and screened towards inhibition of acetylcholinesterase from the electric ray Torpedo marmorata. Starting from a small set (13) of initial hydrazide and aldehyde building blocks, a library containing possibly 66 different species was obtained in a single operation. Of all possible acylhydrazones formed, active compounds containing two terminal cationic recognition groups separated by an appropriate distance, permitting two-site binding, could be rapidly identified by using a dynamic deconvolution--screening procedure, based on the sequential removal of starting building blocks. A very potent bis-pyridinium inhibitor (K(i)=1.09 nM, alphaK(i)=2.80 nM) was selected from the process and the contribution of various structural features to inhibitory potency was evaluated.

Photoaffinity labeling of Torpedo nicotinic receptor with the agonist [3H]DCTA: identification of amino acid residues which contribute to the binding of the ester moiety of acetylcholine.

Torpedo marmorata acetylcholine binding sites were photolabeled using 360 nm light, at equilibrium in the desensitized state, with the agonist [3H]DCTA utilizing the CeIV/glutathione procedure described previously (Grutter, et al. (1999) Biochemistry 38, 7476-7484). Photoincorporation of [3H]DCTA was concentration-dependent with a maximum of 7.5% specific labeling on the alpha-subunit and 1.2% on the gamma-subunit. The apparent dissociation constants for labeling of the alpha- and gamma-subunits were 2.2 +/- 1.1 and 3.6 +/- 2.8 microM, respectively. The alpha-chains isolated from receptor-rich membranes photolabeled in the absence or in the presence of carbamylcholine were cleaved with CNBr using an efficient "in gel" procedure. The resulting peptide fragments were purified by HPLC and further submitted to trypsinolysis. The digest was analyzed by HPLC leading to a single radioactive peak which, by microsequencing, revealed two sequences extending from alpha Lys-179 and from alpha His-186, respectively. Radioactive signals could be unambiguously attributed to positions corresponding to residues alpha Tyr-190, alpha Cys-192, alpha Cys-193, and alpha Tyr-198. These four identified [3H]DCTA-labeled residues, which have been also labeled with other affinity and photoaffinity probes including the agonist [3H]nicotine, belong to loop C of the ACh binding site. The chemical structure of [3H]DCTA, together with its well-defined and powerful photochemical reactivity, provides convincing evidence that loop C-labeled residues are primarily involved in the interaction with the ester moiety of acetylcholine.

Molecular investigations on the nicotinic acetylcholine receptor: conformational mapping and dynamic exploration using photoaffinity labeling.

The nicotinic acetylcholine receptor (nAChR) is a well-understood member of the ligand-gated ion channels superfamily. The members of this signaling proteins group, including 5HT3, GABA(A), glycine, and ionotropic glutamate receptors, are thought to share common secondary, tertiary, and quaternary structures on the basis of a very high degree of sequence similarity. Despite the absence of X-ray crystallographic data, considerable progress on structural analysis of nAChR was achieved from biochemical, mutational, and electron microscopy data allowing the emergence of a three-dimensional image. Photoaffinity labeling and site-directed mutagenesis gave information on the tertiary structure with respect to the agonist/antagonist binding sites, the ion channel, and its selectivity filter. nAChR is an allosterical protein that undergoes interconversion among several conformational states. Time-resolved photolabeling was used in an attempt to elucidate the structural changes that occur in nAChR on neurotransmitter activation. Tertiary and quaternary rearrangements were found in the cholinergic binding pocket and in the channel lumen, but the structural determinant and the functional link between the binding of agonist and the channel gating remain unknown. Time-resolved photolabeling of the functional activated A state using photosensitive agonists might help in understanding the dynamic process leading to the interconversion of the different states.

Evaluation and biological properties of reactive ligands for the mapping of the glycine site on the N-methyl-D-aspartate (NMDA) receptor.

The glycine-binding site of the N-methyl-D-aspartate (NMDA) receptor, given its potential as pharmacological target, has been thoroughly studied by structure-activity relationships, which has made possible its description in terms of spatial limits and interactions of various types. A structural model, based on mutational analysis and sequence alignements, has been proposed. Yet, the amino acid residues responsible for the interactions with the ligand have not been unambiguously characterized. To evidence nucleophilic pocket-lining residues, we have designed and synthesized reactive glycine-site ligands derived from 3-substituted 4-hydroxy-quinolin-2(1H)-ones by introducing various electrophilic groups at different positions of the molecule. These ligands were found to have high affinity at the glycine site and to be functional antagonists by inhibiting glycine/glutamate-induced currents in transfected oocytes. The correlation between their potency and their substitution pattern was strictly consistent with previously established structure-activity relationships. Most ligands displayed intrinsic reactivity toward cysteine, but none inactivated wild-type receptors. This is consistent with the model since it indicates the absence of exposed cysteine in the glycine-binding site. A strategy of cysteine incorporation by point mutations at selected polypeptide positions will create unambiguously localized targets for our reactive probes.

Irreversible site-directed labeling of the 4-aminobutyrate binding site by tritiated meta-sulfonate benzene diazonium. Contribution of a nucleophilic amino acid residue of the alpha1 subunit.

Tritiated meta-sulfonate benzene diazonium ([3H]MSBD), a molecule structurally related to 4-aminobutyrate (GABA), which presents a reactivity toward nucleophilic amino acid residues, was synthesized to investigate the GABA binding site on the GABAA receptor. Irreversible labeling reactions using [3H]MSBD were performed on purified GABAA receptors isolated from cow brain membranes and labeled receptors were analyzed by SDS/PAGE. [3H]MSBD was found to be specifically incorporated into proteins in the 45-60 kDa molecular mass range which were identified as alpha1 subunits and beta2/beta3 subunits by immunoprecipitation with subunit-specific antibodies. The specific immunoprecipitation of alpha and beta subunits confirms that binding of [3H]MSBD occurs at the boundary of these subunits. These labeling results confirm the involvement of nucleophilic residues from the beta subunit but reveal also the contribution of yet unidentified nucleophilic residues on the alpha subunit for the GABA binding site.

Interaction of non-competitive blockers within the gamma-aminobutyric acid type A chloride channel using chemically reactive probes as chemical sensors for cysteine mutants.

Selected channel-lining cysteine mutants from the M2 segment of rat alpha1 gamma-aminobutyric acid (GABA) type A receptor subunit, at positions 257, 261, 264, and 272 were co-expressed with beta1 and gamma2 subunits in Xenopus oocytes. They generated functional receptors displaying conductance and response to both GABA and picrotoxinin similar to the wild type alpha1beta1gamma2 receptor. Three chemically reactive affinity probes derived from non-competitive blockers were synthesized to react with the engineered cysteines: 1) dithiane bis-sulfone derivative modified by an isothiocyanate function (probe A); 2) fiprole derivatives modified by an alpha-chloroketone (probe B) and alpha-bromoketone (probe C) moiety. These probes blocked the GABA-induced currents on all receptors. This blockade could be fully reversed by a washing procedure on the wild type, the alpha1T261Cbeta1gamma2 and alpha1L264Cbeta1gamma2 mutant receptors. In contrast, an irreversible effect was observed for all three probes on both alpha1V257Cbeta1gamma2 and alpha1S272Cbeta1gamma2 mutant receptors. This effect was probe concentration-dependent and could be abolished by picrotoxinin and/or t-butyl bicyclophosphorothionate. These data indicate a major interaction of non-competitive blockers at position 257 of the presumed M2 segment of rat alpha1 subunit but also suggest an interaction at the more extracellular position 272.

Functional characterization and potential applications for enhanced green fluorescent protein- and epitope-fused human M1 muscarinic receptors.

Four recombinant human M1 (hM1) muscarinic acetylcholine receptors (mAChRs) combining several modifications were designed and overexpressed in HEK293 cells. Three different fluorescent chimera were obtained through fusion of the receptor N terminus with enhanced green fluorescent protein (EGFP), potential glycosylation sites and a large part of the third intracellular (i3) loop were deleted, a hexahistidine tag sequence was introduced at the receptor C terminus, and, finally, a FLAG epitope was either fused at the receptor N terminus or inserted into its shortened i3 loop. High expression levels and ligand binding properties similar to those of the wild-type hM1 receptor together with confocal microscopy imaging demonstrated that the recombinant proteins were correctly folded and targeted to the plasma membrane, provided that a signal peptide was added to the N-terminal domain of the fusion proteins. Their functional properties were examined through McN-A-343-evoked Ca2+ release. Despite the numerous modifications introduced within the hM1 sequence, all receptors retained nearly normal abilities (EC50 values) to mediate the Ca2+ response, although reduced amplitudes (Emax values) were obtained for the i3-shortened constructs. Owing to the bright intrinsic fluorescence of the EGFP-fused receptors, their detection, quantitation, and visualization as well as the selection of cells with highest expression were straightforward. Moreover, the presence of the different epitopes was confirmed by immunocytochemistry. Altogether, this work demonstrates that these EGFP- and epitope-fused hM1 receptors are valuable tools for further functional, biochemical, and structural studies of muscarinic receptors.

Nicotinic acetylcholine receptor probed with a photoactivatable agonist: improved labeling specificity by addition of CeIV/glutathione. Extension to laser flash photolabeling.

The molecular structure of Torpedo marmorata acetylcholine binding sites has been investigated previously by photoaffinity labeling. However, besides the nicotine molecule [Middleton et al. (1991) Biochemistry 30, 6987-6997], all other photosensitive probes used for this purpose interacted only with closed receptor states. In the perspective of mapping the functional activated state, we synthesized and developed a new photoactivatable agonist of nAChR capable of alkylation of the acetylcholine (ACh) binding sites, as reported previously [Kotzyba-Hibert et al. (1997) Bioconjugate Chem. 8, 472-480]. Here, we describe the setup of experimental conditions that were made in order to optimize the photolabeling reaction and in particular its specificity. We found that subsequent addition of the oxidant ceric ion (CeIV) and reduced glutathione before the photolabeling step lowered considerably nonspecific labeling (over 90% protection with d-tubocurarine) without affecting the binding properties of the ACh binding sites. As a consequence, irradiation at 360 nm for 20 min in these new conditions gave satisfactory coupling yields (7.5%). A general mechanism was proposed to explain the successive reactions occurring and their drastic effect on the specificity of the labeling reaction. Last, these incubation conditions can be extended to nanosecond pulsed laser photolysis leading to the same specific photoincorporation as for usual irradiations (8.5% coupling yield of ACh binding sites, 77% protection with carbamylcholine). Laser flash photocoupling of a diazocyclohexadienoyl probe on nAChR was achieved for the first time. Taken together, these data indicate that future investigation of the molecular dynamics of allosteric transitions occurring at the activated ACh binding sites should be possible.

Fluorescent muscarinic EGFP-hM1 chimeric receptors: design, ligand binding and functional properties.

We describe the construction, expression and characterization of recombinant proteins comprising the enhanced green fluorescent protein (EGFP) fused to the amino-terminal part of the muscarinic hM1 receptor together or not with an additional hexahistidine tag placed at the C-terminal end of the receptor. Expression of the fluorescent proteins reaches levels identical to those of the wt hM1 receptor, provided that fusion takes place at the very N-terminal end of the receptor. Also correct protein folding and targeting to plasma membrane is obtained upon addition of a signal peptide promoting amino-terminal domain translocation through the membrane. Ligand binding properties of--and activation of the calcium release response by--the fusion proteins are almost identical to those of the wild-type muscarinic receptor, indicating that such fluorescently-labelled receptors are valuable model systems for further functional, biochemical and structural studies.

Reactive affinity probes for the mapping of the glycine-binding site of the NMDA receptor NR1 subunit.

The glycine co-agonist binding site of the NMDA receptor is a target for the prevention and treatment of neurotoxic and neurodegenerative conditions. Until now, the interactions taking place at this site, and its structure, have been investigated by ligand structure-activity relationships and by site-directed mutagenesis. On the basis of a structural model which is currently proposed for this site, we have designed and synthesized six affinity markers by substituting electrophilic reactive groups in the 4, the 7 and the 3' positions of L 701,324, a high-affinity glycine site antagonist. These compounds compete with 3H-DCKA binding to rat brain membranes at equilibrium with nanomolar to low-micromolar affinities, and antagonize glycine-evoked currents in oocytes transfected with wild-type NR1-NR2B. However, they do not induce a time-shift in binding equilibria, and do not inactivate irreversibly the glycine evoked currents. Since they react only with cysteine at physiological pH, we conclude that there is no such residue in the site, in agreement with the model. Our affinity markers therefore represent potential topological probes for NMDA receptors with sequence positions related to the glycine-binding site mutated into cysteine.

Trp82 and Tyr332 are involved in two quaternary ammonium binding domains of human butyrylcholinesterase as revealed by photoaffinity labeling with [3H]DDF.

Purified butyrylcholinesterase (BuChE) was photolabeled by [3H]-p-N, N-dimethylamino benzene diazonium ([3H]DDF) to identify the quaternary ammonium binding sites on this protein [Ehret-Sabatier, L. , Schalk, I., Goeldner, M., and Hirth, C. (1992) Eur. J. Biochem. 203, 475-481]. The covalent photoincorporation occurs with a stoichiometry of one mole of probe per mole of inactivated site and could be fully prevented by several cholinergic inhibitors such as tacrine or tetramethylammonium. After complete deglycosylation of the enzyme using N-glycosidase F, the alkylated protein was trypsinolyzed and the digests were analyzed by HPLC coupled to ES-MS. A direct comparison of tryptic fragments from labeled and unlabeled BuChE allowed us to identify the tryptic peptide Tyr61-Lys103 as carrying the probe. Purification of the labeled peptides by anion-exchange chromatography gave a major radioactive peak which was further fractionated by reversed-phase HPLC leading to three, well-resolved, radioactive peaks. Microsequencing revealed that two of these peaks contained an overlapping sequence starting at Tyr61, while the third peak contained a sequence extending from Thr315. Radioactive signals could be unambiguously attributed to positions corresponding to residues Trp82 and Tyr332. This labeling study establishes the existence of two different binding domains for quaternary ammonium in BuChE and exemplifies additional cation/pi interactions in cholinergic proteins. This work strongly supports the existence of a peripheral anionic site in BuChE, implying residue Tyr332 as a key element.

Pharmacological and structural integrity of muscarinic M2 acetylcholine receptors produced in Sf9 insect cells.

Muscarinic acetylcholine receptors (human m2 subtype), expressed in Sf9 cells, using the baculovirus system, were purified and found to display the expected ligand binding properties, whether membrane-bound or affinity-purified. The purified recombinant receptors were specifically photolabelled with p-N,N-[3H]dimethylamino and p-N,N-[3H]dibutylamino benzene diazonium derivatives. Electrophoretic patterns for covalent radioactive incorporation of the probes were essentially similar to those for [3H]propylbenzilylcholine mustard-labelled receptor sites but were dependent on the infection time of Sf9 cells. Pharmacological properties of the recombinant receptors being unaltered did not reflect structural integrity of the protein as substantial proteolytic fragmentation was detected at a prolonged infection time, i.e., at the highest level of expression. Selection of overexpression conditions, as illustrated here for muscarinic receptors, thus requires not only pharmacological controls, but also analysis of the covalently labelled protein under strongly dissociating conditions.

Nicotinic acetylcholine receptor labeled with a tritiated, photoactivatable agonist: a new tool for investigating the functional, activated state.

Upon agonist activation, the nicotinic acetylcholine receptor undergoes allosteric transitions leading to channel opening and sodium ion influx. The molecular structure of the agonist binding site has been mapped previously by photoaffinity labeling, but most photosensitive probes used for this purpose interact only with closed receptor states (resting or desensitized). We have synthesized two novel photoactivatable 4-diazocyclohexa-2,5-dienone derivatives as cholinergic agonist candidates, with the objective of identifying structural changes at the acetylcholine binding site associated with receptor activation. One of these ligands, 9b, is a functional agonist at muscle acetylcholine receptors in human TE 671 cells. In photolabeling experiments with 9b, up to 35% inactivation of agonist binding sites was observed at Torpedo acetylcholine receptors. Tritiated 9b was synthesized, and photolabeling was found to occur mainly on the alpha-subunit in a partially protectable manner. This novel radiolabeled photoprobe appears to be suitable for future investigation of the molecular dynamics of allosteric transitions occurring at the active acetylcholine receptor binding site.

[3H](azidophenyl)ureido taxoid photolabels peptide amino acids 281-304 of alpha-tubulin.

The taxoid binding site on porcine brain tubulin was covalently labeled, in the presence or absence of Taxotere, with the photoaffinity reagent [3H]-p-(azidophenyl)ureido taxoid derivative [3H]TaxAPU [Combeau, C., Commercon, A., Mioskowski, C., Rousseau, B., Aubert, F., & Goeldner, M. (1994) Biochemistry 33, 6676-6683]. After disulfide reduction and carboxymethylation, the alkylated tubulin samples were treated with trypsin and the mixtures of peptides were first fractionated by gel filtration over Sephadex G50. Anion exchange chromatography of the radioactive areas showed, for one area, three major radioactive signals which were further analyzed by reversed phase C18 HPLC, leading to well-resolved radioactive peaks. Microsequencing of these different peaks gave a complete sequence of a tryptic fragment on alpha-tubulin (alpha-281-304) and two partial peptide sequences of a tryptic fragment on beta-tubulin (beta-217-229) in addition to sequences of mixture of peptides. The radioactive signals were lost while concentrating the samples for microsequencing, preventing the identification of the modified amino acids. These results identify the first peptide on alpha-tubulin which binds to the taxoids and confirm the involvement of both alpha- and beta-tubulin in the taxoid binding site.

Covalent labeling of muscarinic acetylcholine receptors by tritiated aryldiazonium photoprobes.

p-dimethylamino (A) and p-dibutylamino (B) benzenediazonium salts, previously characterized as efficient labels of membrane-bound and solubilized muscarinic receptor sites, are endowed with overall interesting photochemical and alkylating properties that allow their use as structural probes of the muscarinic ligand binding domain to be considered. Under reversible binding conditions, these antagonists display no binding selectivity towards the 5 muscarinic acetylcholine receptor (mAChR) subtypes. They were used here, in a tritiated form, as photoaffinity labels of purified muscarinic receptors from porcine striatum, and their irreversible binding was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. When irradiated under energy transfer conditions, [3H]A and [3H]B were both found to covalently label purified muscarinic receptor sites in a light-dependent and atropine-protectable manner. The electrophoretic migration properties of the alkylated sites were similar to those of [3H]propylbenzilylcholine mustard (PrBCM)-labeled mAChRs. Specific radioactive incorporation showed a clear dependency on probe concentration. Labeling efficiency was rather high, with up to 30% and even 60% of the receptor population being photolabeled by [3H]A and [3H]B, respectively. These two photoactivatable ligands have proven to be powerful tools for the structural analysis of other cholinergic targets (acetylcholinesterase and the nicotinic acetylcholine receptor) by allowing the characterization of a number of different residues belonging to their acetylcholine-binding domain. Altogether, these results reinforce the interest of our site-directed labeling approach because [3H]A- and [3H]B-alkylated mAChRs may now be considered as suitable materials to investigate the muscarinic receptor-binding pocket through peptide mapping, sequence analyses, and identification of radiolabeled amino acid residues.