Probes for Neurotransmitter Receptors—Section 16.2

Page Contents

• α-Bungarotoxin Probes for Nicotinic Acetylcholine Receptors
• Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit
• BODIPY FL Prazosin for α1-Adrenergic Receptors
• BODIPY TMR-X Muscimol for GABA_A Receptors
• Fluorescent Angiotensin II for AT1 and AT2 Receptors
• Naloxone Fluorescein for µ-Opioid Receptors
• Probes for Amino Acid Neurotransmitter Receptors
• Probes for Other Receptors
• Data Table
• Ordering Information

Fluorescent receptor ligands provide a sensitive means of identifying and localizing various cellular receptors, ion channels and ion carriers. Many of these site-selective fluorescent probes may be used on live or fixed cells, as well as in cell-free extracts. The high sensitivity and selectivity of these fluorescent probes make them especially good candidates for measuring low-abundance receptors. Various methods for further amplifying detection of these receptors are discussed in Ultrasensitive Detection Technology—Chapter 6 and Antibodies, Avidins and Lectins—Chapter 7.

This section is devoted to our probes for neurotransmitter receptors. Additional fluorescently labeled receptor ligands (including low-density lipoproteins, epidermal growth factors, transferrin and fibrinogen conjugates and chemotactic peptides) are described in Probes for Following Receptor Binding and Phagocytosis—Section 16.1, along with other probes for studying receptor-mediated endocytosis. Probes for Ion Channels and Carriers—Section 16.3 describes a variety of probes for Ca²⁺, Na⁺, K⁺ and Cl^– ion channels and carriers. Probes for Signal Transduction—Chapter 17 focuses on reagents for investigating events—such as calcium regulation, kinase, phosphatase and phospholipase activation, and lipid trafficking—that occur downstream from the receptor–ligand interaction (Figure 16.2.1).

Fluorescent α-Bungarotoxins

Nicotinic acetylcholine receptors (nAChRs) are neurotransmitter-gated ion channels that produce an increase in Na⁺ and K⁺ permeability, depolarization and excitation upon activation by acetylcholine (Figure 16.2.1). α-Bungarotoxin is a 74–amino acid (~8000 dalton) peptide containing 5 lysine residues and 10 cysteine residues paired in 5 disulfide bridges. Extracted from Bungarus multicinctus venom, α-bungarotoxin binds with high affinity to the α-subunit of the nAChR of neuromuscular junctions. We provide an extensive selection of fluorescent α-bungarotoxin conjugates (Labeled and unlabeled alpha-bungarotoxins—Table 16.4) to facilitate visualization of nAChRs with a variety of instrumentation. We attach approximately one fluorophore to each molecule of α-bungarotoxin, thus retaining optimal binding specificity. The labeled bungarotoxins are then chromatographically separated from unlabeled molecules to ensure adequate labeling of the product.

Alexa Fluor 488 α-bungarotoxin (B13422) has fluorescence spectra similar to those of fluorescein α-bungarotoxin (F1176) and is therefore suitable for use with standard fluorescein optical filter sets. Tetramethylrhodamine α-bungarotoxin (T1175) has been the preferred red-orange–fluorescent probe for staining the nAChR (). We not only offer the red-orange–fluorescent Alexa Fluor 555 α-bungarotoxin (B35451), but also the red-fluorescent Alexa Fluor 594 α-bungarotoxin (B13423), which has a longer-wavelength emission maximum and therefore offers better spectral separation from green-fluorescent dyes in multicolor experiments. Our two longest-wavelength conjugates—Alexa Fluor 647 α-bungarotoxin (B35450) and Alexa Fluor 680 α-bungarotoxin (B35452)—are spectrally separated from both green-fluorescent and orange-fluorescent dyes, allowing researchers to easily perform three- and four-color experiments.

Fluorescent α-bungarotoxins have been used in a variety of informative investigations to:

• Correlate receptor clustering during neuromuscular development with tyrosine phosphorylation of the receptor.
• Detect reinnervation of adult muscle after nerve damage and to identify and visualize endplates.
• Document nAChR cluster formation after myoblast fusion.
• Label proteins fused to the BBS expression tag (a 13–amino acid sequence excerpted from the nAChR) in situ.
• Monitor nAChR-mediated responses in neuromuscular damage and degeneration models.

Biotinylated α-Bungarotoxin

Nicotinic AChRs can also be labeled with biotinylated α-bungarotoxin (B1196), which is then localized using fluorophore- or enzyme-labeled avidin, streptavidin or NeutrAvidin biotin-binding protein conjugates, or NANOGOLD and Alexa Fluor FluoroNanogold streptavidin (Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6, Molecular Probes avidin, streptavidin, NeutrAvidin and CaptAvidin conjugates—Table 7.9). Based on the intracellular dissociation of biotinylated α-bungarotoxin and streptavidin, researchers were able to distinguish new, pre-existing and recycled pools of nAChR at the synapses of live mice by sequentially labeling with biotinylated α-bungarotoxin and fluorescent streptavidin conjugates. Complexation of biotinylated α-bungarotoxin with Qdot nanocrystal–streptavidin conjugates (Qdot Nanocrystals—Section 6.6 ) enables single-molecule detection of nAChR. The nanocrystal labeling methodology allows detection and tracking of diffuse, nonclustered nAChRs, whereas dye-labeled α-bungarotoxin conjugates primarily detect nAChR clusters. In addition, the biotinylated toxin can be employed for affinity isolation of the nAChR using a streptavidin or CaptAvidin agarose (S951, C21386; Avidin, Streptavidin, NeutrAvidin and CaptAvidin Biotin-Binding Proteins and Affinity Matrices—Section 7.6) column.

Unlabeled α-Bungarotoxin

In addition to the fluorescent and biotinylated derivatives, we have unlabeled α-bungarotoxin (B1601), which has been shown to be useful for radioiodination. Unlabeled α-bungarotoxin has also been employed for ELISA testing of nAChR binding, as well as for investigating the function of the α-bungarotoxin–binding component (α-BgtBC) in vertebrate neurons.

The action of acetylcholine (ACh) at neuromuscular junctions is regulated by acetylcholinesterase (AChE), the enzyme that hydrolyzes ACh to choline and acetate. The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit (A12217) provides an ultrasensitive method for continuously monitoring AChE activity and for detecting ACh in a fluorescence microplate reader or fluorometer. Other potential uses for this kit include screening for AChE inhibitors and measuring the release of ACh from synaptosomes. The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit can also be used for the ultrasensitive, specific assay of free choline, classified as an essential nutrient in foods.

In this assay, AChE activity is monitored indirectly using the Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), a highly sensitive and stable fluorogenic probe for H₂O₂ that is also useful in assaying other enzymes and analytes (Substrates for Oxidases, Including Amplex Red Kits—Section 10.5). First, AChE converts the acetylcholine substrate to choline. Choline is in turn oxidized by choline oxidase to betaine and H₂O₂, the latter of which, in the presence of horseradish peroxidase, reacts with the Amplex Red reagent to generate the red-fluorescent product resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1) with excitation/emission maxima of ~570/585 nm (). Experiments with purified AChE from electric eel indicate that the Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit can detect AChE levels as low as 0.002 U/mL using a reaction time of only one hour (Figure 16.2.2). In our laboratories, we have been able to detect acetylcholinesterase activity from a tissue sample with total protein content as low as 200 ng/mL or 20 ng/well in a microplate assay. By providing an excess of AChE in the assay, the kit can also be used to detect acetylcholine levels as low as 0.3 µM, with a detection range between 0.3 µM and ~100 µM acetylcholine (Figure 16.2.3). The Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂ for use as a positive control
• Concentrated reaction buffer
• Choline oxidase from Alcaligenes sp.
• Acetylcholine (ACh)
• Acetylcholinesterase (AChE) from electric eel
• Detailed protocols (Amplex Red Acetylcholine/Acetylcholinesterase Assay Kit)

Each kit provides sufficient reagents for approximately 500 assays using a fluorescence microplate reader and a reaction volume of 200 µL per assay.

Prazosin is a high-affinity antagonist for the α₁-adrenergic receptor. The green-fluorescent BODIPY FL prazosin (B7433) can be used to localize the α₁-adrenergic receptor on cultured cortical neurons and in vascular smooth muscle cells from α₁-adrenergic receptor–knockout mice. BODIPY FL prazosin has also been successfully employed in multidrug resistance (MDR) transporter activity assays.

Muscimol is a powerful agonist of the GABA_A receptor and has been widely used to reversibly inactivate localized groups of neurons. Using red-fluorescent BODIPY TMR-X muscimol (M23400), researchers can correlate the distribution of muscimol with its pharmacological effects and detect the presence of GABA_A receptors on cell surfaces.

Angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe) stimulates smooth muscle contraction and plays an important role in blood pressure control and in water and salt homeostasis. These effects are exerted via two G-protein–coupled receptor subtypes, referred to as AT1 and AT2. Our N-terminal–labeled fluorescein and Alexa Fluor 488 analogs of angiotensin II (A13438, A13439) are useful tools for imaging the distribution of these receptors, as well as for flow cytometric analysis of angiotensin II endocytosis. These fluorescent peptides have been characterized for purity by HPLC and mass spectrometry and generally display selectivity for AT1 over AT2 binding.

The µ-opioid receptor plays a critical role in analgesia. Among the antagonists that have been used to define and characterize these receptors are naloxone, a drug used to counteract the effects of opioid overdose, and naltrexone, a drug used in the treatment of opioid addiction. Naloxone fluorescein (N1384) has been reported to bind to the µ-opioid binding site with high affinity, permitting receptor visualization in transfected Chinese hamster ovary (CHO) cells. Flow cytometry analysis of the binding of naloxone fluorescein to NMDA and µ-opioid receptors (which was displaced by NMDA and met-enkephalin, respectively) has been used to deduce the effects of operant conditioning on visual cortex receptor pattern.

Caged Amino Acid Neurotransmitters

When illuminated with UV light or by multiphoton excitation, caged amino acid neurotransmitters are converted into biologically active amino acids that rapidly initiate neurotransmitter action. Thus, these caged probes provide a means of controlling the release—both spatially and temporally—of agonists for kinetic studies of receptor binding or channel opening.

The different caging groups confer special properties on these photoactivatable probes (Properties of six different caging groups—Table 5.2). We synthesize two caged versions of L-glutamic acid (C7122, G7055), as well as caged carbachol (N-(CNB-caged) carbachol, C13654) and caged γ-aminobutyric acid (O-(CNB-caged) GABA, A7110), all of which are biologically inactive before photolysis.O-(CNB-caged) GABA (A7110) and γ-(CNB-caged) L-glutamic acid (G7055), which exhibit fast uncaging rates and high photolysis quantum yields, have been used to investigate the activation kinetics of GABA receptors and glutamate receptors, respectively. N-(CNB-caged) L-glutamic acid (C7122) does not hydrolyze in aqueous solution because it is caged on the amino group, thus enabling researchers to use very high concentrations without risk of light-independent glutamic acid production.

Anti–NMDA Receptor Antibodies

N-methyl-D-aspartate (NMDA) receptors constitute cation channels of the central nervous system that are gated by the excitatory neurotransmitter L-glutamate. We offer affinity-purified rabbit polyclonal antibodies to NMDA receptor subunits 2A, 2B and 2C (A6473, A6474, A6475). The anti–NMDA receptor subunit 2A and 2B antibodies were generated against fusion proteins containing amino acid residues 1253–1391 of subunit 2A and 984–1104 of subunit 2B, respectively. These two antibodies are active against mouse, rat and human forms of the antigens and are specific for the subunit against which they were generated. In contrast, the anti–NMDA receptor subunit 2C antibody was generated against amino acid residues 25–130 of subunit 2C and recognizes the 140,000-dalton subunit 2C, as well as the 180,000-dalton subunit 2A and subunit 2B from mouse, rat and human. These three affinity-purified antibodies are suitable for immunohistochemistry (), Western blots, enzyme-linked immunosorbent assays (ELISAs) and immunoprecipitations.

Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit

The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (A12221) provides an ultrasensitive method for continuously detecting glutamic acid or for monitoring glutamate oxidase activity in a fluorescence microplate reader or a fluorometer. In this assay, L-glutamic acid is oxidized by glutamate oxidase to produce α-ketoglutarate, NH₃ and H₂O₂. L-Alanine and L-glutamate–pyruvate transaminase are also included in the reaction. Thus, the L-glutamic acid is regenerated by transamination of α-ketoglutarate, resulting in multiple cycles of the initial reaction and a significant amplification of the H₂O₂ produced. Hydrogen peroxide reacts with the Amplex Red reagent in a 1:1 stoichiometry in a reaction catalyzed by horseradish peroxidase (HRP) to generate the highly fluorescent product resorufin (R363, Introduction to Enzyme Substrates and Their Reference Standards—Section 10.1). Because resorufin has absorption/emission maxima of ~571/ 585 nm, there is little interference from autofluorescence in most biological samples.

If the concentration of L-glutamic acid is limiting in this assay, then the fluorescence increase is proportional to the initial L-glutamic acid concentration. The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit allows detection of as little as 10 nM L-glutamic acid in purified systems using a 30-minute reaction time (Figure 16.2.4). If the reaction is modified to include an excess of L-glutamic acid, then this kit can be used to continuously monitor glutamate oxidase activity. For example, purified L-glutamate oxidase from Streptomyces can be detected at levels as low as 40 µU/mL (Figure 16.2.5). The Amplex Red reagent has been used to quantitate the activity of glutamate-producing enzymes in a high-throughput assay for drug discovery. The Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit contains:

• Amplex Red reagent
• Dimethylsulfoxide (DMSO)
• Horseradish peroxidase (HRP)
• H₂O₂
• Concentrated reaction buffer
• L-Glutamate oxidase from Streptomyces sp.
• L-Glutamate–pyruvate transaminase from pig heart
• L-Glutamic acid
• L-Alanine
• Detailed protocols (Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit)

Each kit provides sufficient reagents for approximately 200 assays using a fluorescence microplate reader and a reaction volume of 100 µL per assay.

The Molecular Probes Handbook discusses a diverse array of receptor probes, including fluorescent derivatives of:

• Low-density lipoprotein (LDL)
• Lipopolysaccharides
• Epidermal growth factor (EGF)
• Transferrin
• Fibrinogen
• Gelatin and collagen
• Ovalbumin and bovine serum albumin
• Casein
• Histone H1
• Subunit B of cholera toxin
• Chemotactic peptide
• Insulin

These ligands are all transported into the cell by receptor-mediated endocytosis. Additional information about these probes, as well as membrane and fluid-phase markers, can be found in Probes for Following Receptor Binding and Phagocytosis—Section 16.1.