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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: J Neurochem. 2010 May 18;114(4):994–1006. doi: 10.1111/j.1471-4159.2010.06819.x

Alexa Fluor 546-ArIB[V11L;V16A]is a potent ligand for selectively labeling α7 nicotinic acetylcholine receptors

Arik J Hone 1,2, Paul Whiteaker 3, Jesse L Mohn 4, Michele H Jacob 4, J Michael McIntosh 1,2,5
PMCID: PMC2936243  NIHMSID: NIHMS209619  PMID: 20492354

Abstract

The α7* nicotinic acetylcholine receptor (nAChR) subtype is widely expressed in the vertebrate nervous system and implicated in neuropsychiatric disorders that compromise thought and cognition. In this report, we demonstrate that the recently developed fluorescent ligand Cy3-ArIB[V11L;V16A] labels α7 nAChRs in cultured hippocampal neurons. However, photobleaching of this ligand during long image acquisition times prompted us to develop a new derivative. In photostability studies, this new ligand, Alexa Fluor 546-ArIB[V11L;V16A], was significantly more resistant to bleaching than the Cy3 derivative. The classic α7 ligand α-bungarotoxin binds to α1* and α9* nAChRs. In contrast, Alexa Fluor 546-ArIB[V11L;V16A] potently (IC50 1.8 nM) and selectively blocked α7 nAChRs but not α1* or α9* nAChRs expressed in Xenopus oocytes. Selectivity was further confirmed by competition binding studies of native nAChRs in rat brain membranes. The fluorescence properties of Alexa Fluor 546-ArIB[V11L;V16A] were assessed using human embryonic kidney-293 cells stably transfected with nAChRs; labeling was observed on cells expressing α7 but not cells expressing α3β2, α3β4, or α4β2 nAChRs. Further imaging studies demonstrate that Alexa Fluor 546-ArIB[V11L;V16A] labels hippocampal neurons from wild type mice but not from nAChR α7 subunit-null mice. Thus, Alexa Fluor 546-ArIB[V11L;V16A] represents a potent and selective ligand for imaging α7 nAChRs.

Keywords: α-conotoxin, fluorescence, α7 nAChR, hippocampal neurons

Introduction

Nicotinic acetylcholine receptors (nAChRs) are ligand gated ion channels that are ubiquitously expressed throughout the central and peripheral nervous systems by both neuronal and non-neuronal cells. These receptors are pentameric assemblies of homologous subunits that combine to form ligand-gated ion channels that, when activated by agonists, open to allow the passage of ions across the cell membrane. Seventeen nAChR subunits have been cloned encoding the α1-α10, β1-β4, δ, ε, and γ subunits. These subunits assemble in various combinations to form heteromeric receptors or, in the case of the α7, α8, and α9 subunits, homomeric receptors (for review see (Albuquerque et al. 2009).

The hippocampus, located in the temporal lobe of the brain, is critically involved in learning and memory. It is a highly organized structure with the principal neurons arranged in a configuration that is known as the tri-synaptic circuit. Included in this circuit is a heterogenous population of GABAergic interneurons. A subpopulation of these interneurons abundantly expresses α7 nAChRs which are involved in modulating the release of GABA (Albuquerque et al. 1998, Alkondon et al. 1999). The hilar region of the dentate gyrus has both excitatory and inhibitory circuit neurons that express α7 nAChRs. Glutamatergic mossy cells are involved in regulating the activity of granule cells and CA3 pyramidal cells and virtually all mossy cells express α7 nAChRs (Frazier et al. 2003). Accumulating evidence suggests that nAChRs play an important role in the activity of the hippocampal tri-synaptic circuit and have been implicated in a number of neuropsychiatric disorders including Alzheimer’s disease and schizophrenia (Mexal et al. 2010, Sinkus et al. 2009, Dziewczapolski et al. 2009, Bencherif & Lippiello 2010). Accordingly, nAChRs have received considerable attention as targets for novel therapeutic compounds aimed at treating these conditions (Marks et al. 2009, Hauser et al. 2009, Buccafusco & Terry 2009). Unfortunately, current probes for detecting α7 nAChRs have serious drawbacks that limit their utility. For example, commercially available α7 antibodes have been shown to produce labeling in α7 knockout mice (Herber et al. 2004, Moser et al. 2007). Historically, ligands derived from α-BgTx have been used for detecting α7 nAChRs. However, it is now widely accepted that this peptide toxin also binds to non-α7 nAChR subtypes including those that contain the α1 and α9 subunits (Elgoyhen et al. 1994, Elgoyhen et al. 2001, Sgard et al. 2002). This is less of a concern for studies that involve the hippocampus because α1 and α9 subunits are not expressed by cells of the brain. However, Franceschini et al. reported significant residual α-BgTx binding to an unknown site in brain tissue from α7 knockout mice (Franceschini et al. 2002). In addition, McCann et al. recently reported that α-BgTx binds to a subset of GABAARs that contain the β3 subunit (McCann et al. 2006). This GABAAR subtype is present in hippocampal neurons (Marchionni et al. 2007) complicating the use of α-BgTx in detecting α7 nAChRs in this tissue. Because of the limitations of α-BgTx and α7 antibodies, the development of novel selective probes for the α7 nAChR subtype is a priority.

We previously developed a fluorescent Cy3 derivative of an α7 selective α-conotoxin (α-CTx) (Hone et al. 2009) and now show for the first time that it can be used to label native α7 nAChRs present in cultured hippocampal neurons. In addition, we report the development of new derivative, Alexa Fluor 546-ArIB[V11L;V16A], that is a more potent blocker and more resistant to photobleaching than the Cy3 ligand. The selectivity of this new ligand for α7 nAChRs was assessed using functional assays in Xenopus laevis oocytes and in radioligand binding studies of native nAChRs. Lastly, we demonstrate the fluorescent peptides ability to produce specific labeling of cultured hippocampal neurons from wild type (wt) mice but not from mice that lack the α7 gene (α7 knockouts).

Materials and Methods

Alexa Fluor 546 NHS ester dye, penicillin/streptomycin, minimum essential medium, G418 sulfate [Geneticin], 0.5% trypsin/EDTA, Hoechst 33342, calcium and magnesium free phosphate buffered saline (PBS), Hank’s Balanced Salt Solution (HBSS), tissue culture grade HEPES solution, heat inactivated horse serum (HS), B27 supplement, Glutamax, 2.5% trypsin, and Neurobasal medium were from Invitrogen (Carlsbad, CA). Cy3 NHS ester dye and [I25I]-α-BgTx (specific activity 2000 Ci/mmol−1) were purchased from General Electric Healthcare (Piscataway, NJ). Heat inactivated fetal bovine serum (FBS) was from HyClone (Logan, UT). α-CTx ArIB[V11L;V16A] and [I25I]-α-CTx MII (specific activity 2200 Ci/mmol) were synthesized as previously described (Whiteaker et al. 2000b). Rat brain regions were purchased from Pel-Freeze Biologicals (Rogers, AR). DNAse I (Type II), α-bungarotoxin (α-BgTx), α-cobratoxin (α-CbTx), acetylcholine chloride (ACh), carbachol, poly-L-lysine hydrobromide, poly-D-lysine hydrobromide, acetonitrile (ACN), trifluoroaceitic acid (TFA), atropine sulphate, bovine serum albumin (BSA), phenylmethylsulfonyl fluoride (PMSF), leupeptin trifluoroacetate, pepstatin A, aprotinin, EDTA, and EGTA, were from Sigma Aldrich (St. Louis, MO). The HEPES used for oocyte electrophysiology experiments was from Research Organics (Cleveland, OH). Amikacin sulphate, sulfamethoxazole, and trimethoprim were purchased from the University of Utah Health Science Center (Salt Lake City, UT). The mouse strains FVB/NJ and C57BL/6J were purchased from Jackson Laboratories (Bar Harbor, ME). CHRNA7 null mice (Orr-Urtreger et al. 1997) were generously provided by M.J. Marks (University of Colorado, Boulder, CO). The stably transfected human embryonic kidney cells (HEK293) cells lines KXα3β2R4, KXα3β4R2, KXα4β2R2, and KXα7R1 were generously provided by K. Kellar (Georgetown University, Washington D.C.).

Conjugation of α-CTx ArIB[V11L;V16A] with NHS ester Dyes

To generate fluorescent conjugates of α-CTx ArIB[V11L;V16A], the peptide (50 nmol) was suspended in 11 μl of a water solution buffered with 227 mM NaHCO3 and adjusted to pH 8.5 with NaOH. NHS ester dye (200 μg) in 14 μl of anhydrous DMSO was added to the peptide solution and incubated at RT for 2 h in the dark with brief vortexing and centrifugation performed at least once during this time period. The reaction was terminated by dilution first with 50 μl of DMSO followed by 150 μl of buffer A (0.1% (vol/vol) trifluoroacetic acid (TFA) in distilled water). The reactants were separated by reverse phase-HPLC (RP-HPLC) using a Vydac C18 column. The buffers used were buffers A and B60 (0.092% (vol/vol) TFA, 60% (vol/vol) ACN and the remainder water). The peptides were eluted using a linear gradient of 20% to 90% B60 over 40 minutes. Yield quantification of the fluorescent conjugates was determined using the Absmax and the molar extinction coefficients for each fluorophore (104,000 M−1cm−1 at 556 nm for Alexa Fluor 546 and 150,000 M−1cm−1 at 550 nm for Cy3) and the Lambert-Beer equation. The masses were verified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) and electrospray mass spectrometry (MS) which was performed at the Salk Institute, La Jolla, CA under the direction of J. Rivier.

Oocyte Electrophysiology

Detailed methods for the expression of nAChRs in Xenopus laevis oocytes have been previously described (Hone et al. 2009). Oocytes were voltage-clamped and exposed to ACh and toxins as previously described (Cartier et al. 1996). Briefly, the oocyte chamber consisting of a cylindrical well (~30 μl in volume) was gravity perfused at a rate of ~2 ml/min with ND96 containing 0.01% (wt/vol) BSA and 1 μM atropine to block potential contaminating signal from endogenous muscarinic receptors. For experiments involving α7 and α9α10, atropine was excluded from the perfusion solution because it has been shown to block these receptor subtypes. Oocytes were exposed once a minute to a 1 sec pulse of ACh. The ACh concentrations used were 200 μM for α7, 50 μM for α1β1δε, and 100 μM for all other subtypes. Detailed methods for application of toxins and data analysis are described in (Hone et al. 2009).

Cell culture

The cells lines KXα3β2R4, KX3β4R2, KXα4β2R2, and KXα7R1 were established previously by stably transfecting human embryonic kidney-293 (HEK293) cells with rat nAChR subunit genes (Xiao et al. 1998, Xiao & Kellar 2004, Xiao et al. 2009). The cells were routinely grown in a selective growth medium (SGM-7) which consists of minimum essential medium containing 2.2 mg/ml NaHCO3, 10% FBS (vol/vol), 100 U/ml penicillin, 100 μg/ml streptomycin, 0.7 mg/ml G418 [Geneticin], and 1 mM carbachol. The cells were grown in 75 cm2 flasks and passaged every 3–4 days when they reached ~80–90% confluency. For fluorescence labeling experiments, cells were plated on 15 mm borosilicate glass coverslips (cat. #64-0703 Warner Instruments, Hamden, CT) treated with 0.1 mg/ml poly-L-lysine and grown in 12 well Falcon tissue culture plates (cat. #353503, Fisher Scientific, Pittsburg, PA). Cells were suspended at RT in SGM-7 at a density of 8 × 104/ml. One ml of the cell suspension was added to each well plus 500 μl of SGM-7 and cultured at 37°C in 95% air and 5% CO2 for 2–3 days prior to use. Cells were washed three times with room temperature PBS (pH 7.5) then exposed to a PBS solution containing 2 μM Hoechst 33342 and either 100 nM or 1 μM Alexa Fluor 546-ArIB[V11L;V16A] for 20–45 min (depending on the concentration used) at 4°C. Non-specific binding was minimized by including 10% (vol/vol) FBS in the PBS solution. In KXα7R1 cells, non-specific binding was determined by pre-incubating the cells with 2 μM α-CbTx for 20 minutes at 4°C then with 100 nM Alexa Fluor 546-ArIB[V11L;V16A] in the presence of 2 μM α-CbTx. Cells were washed three times with 4°C PBS and the coverslips were then placed in a 60 mm Petri dish with a borosilicate glass bottom and immediately imaged using epifluorescence microscopy. The photobleaching rates of fluorescent conjugates were determined by continually illuminating a region of interest (a labeled KXα7R1cell) and measurements of fluorescence intensity were taken every 3 s for 120 s at 340×280 resolution with 4×4 binning. The fluorescence intensity for each time point was normalized to the initial value and curve-fit with a single exponential equation using GraphPad Prism. The microscope used was a Nikon Eclipse FN1 equipped with an epiilluminator, a 40x NIR Apo water dipping objective numerical aperture 0.80, and a filter set appropriate for Hoechst 33342, Cy3, and Alexa Fluor 546 fluorophores. The images were captured at 640×480 resolution with 2×2 binning using a Nikon CCD camera cooled to −10°C. The light source for all microscopy experiments was a Lamda LS xenon arc lamp with a 300 watt bulb (Sutter Instruments, La Jolla, CA). Images were adjusted for brightness and contrast using NIH ImageJ software (Bethesda, MD).

Displacement Binding Assays

Brain Membranes

To prepare brain membranes, rat brain regions were obtained from Pel-Freez (Rogers, AR). Choices of brain regions were made as appropriate for each nAChR subtype being investigated. For experiments measuring monoiodinated α-BgTx ([125I]-α-BgTx) binding to α7 nAChRs, hippocampus was used. For experiments assessing inhibition of monoiodinated [125I]-Epibatidine ([125I]-Epi) binding to α4β2* nAChRs, cortex was used, and displacement of monoiodinated [125I]-α-CTx MII binding was investigated using pooled olfactory tubercle, striatum, and superior colliculus membranes. Brain region samples were homogenized in ice-cold hypotonic buffer (14.4 mM NaCl, 0.2 mM KCl, 0.2 mM CaCl2, 0.1 mM MgSO4, and 2 mM HEPES, pH 7.5) using a glass-Teflon tissue grinder. Particulate fractions were collected by centrifugation at 25,000 × g for 15 min at 4°C (Eppendorf 5417 C centrifuge; Eppendorf North America, New York, NY). The pellets were resuspended in fresh homogenization buffer, incubated on ice for 10 min, and then harvested by centrifugation as described above. Each pellet was washed twice more by resuspension/centrifugation before storage (in pellet form in homogenization buffer) at −70°C until use.

Binding Assay

The selectivity of Alexa Fluor 546-ArIB[V11L;V16A] for native α7 nAChRs was assessed by a panel of displacement binding assays. Binding to α7 nAChRs was measured by displacement of [125I]-α-BgTx binding from hippocampal membranes as described by Whiteaker et al. (Whiteaker et al. 2008), using a Packard Cobra counter (PerkinElmer Life and Analytical Sciences). Results for inhibition of [125I]-α-BgTx binding were calculated using a one-site fit: B = B0/(1+I/IC50), where B is ligand bound at inhibitor concentration I, Bo is the binding in the absence of inhibitor, and IC50 is the concentration of inhibitor required to reduce binding to 50% of Bo. Values for Ki (inhibition binding constant) were derived by the method Cheng-Prusoff method (Cheng & Prusoff 1973). Binding to non-α7 nAChRs was measured using similar techniques, but with the following differences. Displacement of 500 pM [125I]-α-CTx MII from pooled membranes from the striatum, superior colliculus, and olfactory tubercules, was measured using a 2 h initial incubation period, and a five minute period for dissociation of non-specific binding (Salminen et al. 2005). Inhibition of 200 pM [125I]-Epi from cortical membranes was also assessed using a 2 h incubation period, and assays were filtered immediately, without application of a non-specific binding dissociation period (Whiteaker et al. 2000a).

Hippocampal Cultures

Primary neuronal cultures were prepared from rat hippocampus as previously described (Tretter et al. 2008). The hippocampus was chosen because it is relatively large and easy to dissect. Furthermore, the expression of α7 nAChRs by cultured hippocampal neurons has been extensively characterized (Fayuk & Yakel 2007, Kawai et al. 2002, Massey et al. 2006). Briefly, hippocampi were isolated from E18 rat brains and dissociated after trypsinization. Cells were plated on poly-L-lysine-coated glass coverslips in 35 mm Petri dishes with Neurobasal medium containing 5% (vol/vol) FBS which was replaced after 6 h by Neurobasal medium containing B27 without FBS. Cultures were grown at a densityof 1 ×105 cells per 35 mm dish. The neurons were fed twice a week by replacing 33% of the medium. After 18 days in vitro, the neurons were fixed with 4% (wt/vol) paraformaldehyde for 20 min, rinsed, then blocked with 5% (vol/vol) normal goat serum (NGS) + 0.3% (vol/vol) Triton X-100 in PSB for 1 hr. Neurons were stained with either 1 μM or 10 μM Cy3-ArIB[V11L;V16A] and primary antibodies towards either SV2 (Developmental Studies Hybridoma Bank, Iowa City, IA) or PSD-95 (Upstate Biotechnology, Lake Placid, NY) in 2%(vol/vol) NGS + 0.3% (vol/vol) Triton X-100 for 1 hr, then washed 3x with 0.3% (vol/vol) Triton X-100 in PBS. Neurons were then stained with appropriate Alexa Fluor 488-conjugated secondary antibodies (Molecular Probes, Eugene, OR) for 45 min. Non-specific binding of Cy3-ArIB[V11L;V16A] was assessed by pre-incubating the neurons with 10 μM α-BgTx for 60 min prior to and during exposure to Cy3-ArIB[V11L;V16A]. All steps were performed at RT. Images were obtained using a Zeiss Axioscope microscope (Zeiss, Thornwood, NY) with a Q-Imaging Retiga 200R Fast 1394 black and white CCD camera (Surrey, British Columbia, Canada) and Nikon Instruments NIS Elements software (Melville, NY) as described in Rosenberg et al. (Rosenberg et al. 2008).

Five postnatal date 0 (P0) mouse pups per isolation were sacrificed with CO2 and the brains removed and placed in ice cold HBSS (buffered with 10 mM HEPES and adjusted to pH 7.4 with NaOH). The hippocampi were dissected out, cut into small pieces with iridectomy scissors, and exposed to HBSS containing 0.25% (vol/vol) trypsin in a 15 ml conical tube. After a 20 minute incubation period at 37°C, the hippocampal pieces were washed twice for 5 min with HBSS to remove the trypsin from the tissue and then suspended in 2.5 ml of HBSS containing 10 μg/ml of DNAse I Type II in the presence of 5 mM MgCl2. The hippocampi were then mechanically dissociated with a normal Pasteur pipette followed by one with an opening that had been reduced to approximately half the diameter by flaming. The cell suspension was centrifuged for 2 minutes at 200 × g, aspirated, and resuspended in 10 ml of Neurobasal medium containing 10% (vol/vol) HS, 2% (vol/vol) B27, 100 U/ml penicillin, 100 μg/ml streptomycin, and 2 mM Glutamax. The cell suspension was passed through a 70 μm cell strainer to remove large pieces of debris and any tissue that had not been completely dissociated. The cells were counted using a hemocytometer and the density adjusted to ~2.0 × 105 cells/ml. Cell viability was assessed using the trypan blue exclusion test. One ml of the cell suspension was added to each well of a 12 well BD Falcon culture plate (cat. #353503 Fisher Scientific, Pittsburg, PA) that contained a 15 mm borosilicate glass coverslip (cat. #64-0703 Warner Instruments, Hamden, CT). The coverslips were first flamed using 95% EtOH and treated with 0.2 mg/ml poly-D-lysine. The cells were cultured at 37°C in 95% air and 5% CO2 for 15–21 days prior to use. Fifty percent of the medium was replaced every 4 days with the Neurobasal growth medium containing 2% (vol/vol) B27, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.5 mM Glutamax. On day 6 in vitro, some cultures received 5 μM cytosine-β-D-arabinofuranoside to inhibit the proliferation of non-neuronal cells.

Fluorescence microscopy of living neurons

To label α7 nAChRs, the coverslips of neurons were placed in individual 35 mm Petri dishes containing 2 ml of RT labeling solution (140 mM NaCl, 5 mM KCl, 2 mM CaCl2 1 mM MgCl2, 5% (vol/vol) HS, 10 μg/ml each of aprotinin, leupeptin trifluoroacetate, and pepstatin A, and 10 mM HEPES pH 7.4). The neurons were exposed to labeling solution containing Alexa Fluor 546-ArIB[V11L;V16A] and Hoechst 33342 for 20 minutes at RT and subsequently washed by exchanging the labeling solution six times over five min with labeling solution but without fluorescent peptide or nuclear stain. In some experiments, the labeling was performed at 4°C to evaluate the possibility of cellular uptake of the fluorescent peptide by endocytotic mechanisms. To determine non-specific binding, the neurons were pre-incubated with solution containing 10 μM α-BgTx for 20 minutes followed by Alexa Fluor 546-ArIB[V11L;V16A] for 20 minutes in the presence of the competing ligand. The cell nuclei were labeled by exposing them to 2 μM Hoechst 33342 during the labeling step with Alexa Fluor 546-ArIB[V11L;V16A]. Non-specific binding was minimized by including 5% (vol/vol) HS in all labeling and wash solution. Image acquisition and processing procedures were identical as described for imaging of the HEK293 cells and in addition, differential interference contrast (DIC) microscopy was used to examine the morphology of the neurons under study

Results

Cy3-ArIB[V11L;V16A] detects native α7 nAChRs on cultured hippocampal neurons

α-CTx ArIB is a peptide that was synthesized based on the predicted sequence of a gene isolated from the vermivorous marine snail Conus arenatus. Select substitutions were made in the amino acid sequence to produce ArIB[V11L;V16A] that potently and selectively blocks rat α7 nAChRs (IC50= 0.4 nM) expressed in Xenopus laevis oocytes (Whiteaker et al. 2007). We previously demonstrated that ArIB[V11L;V16A] could be conjugated with Cy3 NHS ester to produce a fluorescent ligand, Cy3-ArIB[V11L;V16A], that labels α7 nAChRs in a stably transfected HEK293 cell line (Hone et al. 2009). Here we demonstrate that this ligand labels native α7 nAChRs in cultured rat hippocampal neurons. Hippocampal neurons were fixed, permeabilized, and labeled with Cy3-ArIB[V11L;V16A] (Supp. Fig. 1a). The punctate labeling of α7 nAChR clusters on processes was significantly reduced by exposing the neurons to 10 μM α-BgTx for 60 min prior to and during exposure to Cy3-ArIB[V11L;V16A]. However, some residual intracellular staining in the soma remained (Supp. Fig. 1b). To determine whether α7 nAChRs have a synaptic localization, we examined their distribution relative to two different synaptic markers, synaptic vesicle protein 2 (SV2) and postsynaptic density protein (PSD-95). SV2 is a membrane glycoprotein localized to secretory vesicles and is a marker for presynaptic nerve terminals (Buckley & Kelly 1985). As illustrated in Fig. 1, labeling of α7 nAChRs (Fig. 1a) matches closely that of SV2 (Fig. 1b) and showed close juxtaposition and partial overlap with SV2 (Fig. 1c). α7 nAChRs (Fig. 1d) and PSD-95 (Fig. 1e) labeling also showed close proximity (Fig. 1f). The distribution of α7 nAChRs, detected by Cy3-ArIB[V11L;V16A], is consistent with a synaptic/perisynaptic localization on cultured hippocampal neurons.

Fig. 1.

Fig. 1

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Cy3-ArIB[V11L;V16A] labels α7 nAChRs in cultured rat hippocampal neurons. The staining pattern of α7 nAChRs (a) and SV2 (b) showed close juxtaposition and partial overlap (c, merged image). α7 nAChRs (d) and PSD-95 (e) also showed close proximity (f, merged image). White outlined boxes in (c) and (f) are shown digitally magnified below. Images were taken at 63x magnification; scale bar is 25 μm.

Generation of Alexa Fluor 546-ArIB[V11L;V16A]

During the course of the development and testing of Cy3-ArIB[V11L;V16A], we noticed reductions in fluorescence intensity during the acquisition of z-series images likely due to photobleaching of the fluorophore. We therefore sought to develop a second-generation probe with more photostable properties. The Alexa Fluor series of fluorophores developed by Molecular Probes (Eugene, OR) is reported to possess enhanced fluorescence characteristics compared to other fluorophores (Berlier et al. 2003, Panchuk-Voloshina et al. 1999). We reasoned that an Alexa Fluor 546-tagged ArIB[V11L;V16A] might possess superior properties and also enhance the ability to study α7 nAChRs in native systems where α7 nAChRs may be sparsely expressed.

The amino acid sequence of ArIB[V11L;V16A] and the structure of Alexa Fluor 546 NHS ester are shown in supplemental Fig. 2. ArIB[V11L;V16A] was reacted with Alexa Fluor 546 NHS as described in Materials and Methods. The RP-HPLC separation of conjugation products from the reactants is shown in Fig. 2. The absorbance peaks of the chromatogram correspond to the unconjugated peptide (peak 1), the fluorescent conjugate (peaks 2–4), and the unconjugated dye (peaks 5–7). The conjugation of the peptide with Alexa Fluor 546 NHS ester resulted in a heterogeneous product owing to the isomeric nature of the dye. Since the three major isomers have considerable overlap with respect to the retention time, each isomer was re-purified by an additional RP-HPLC. The monoisotopic mass of each isomer was determined by MALDI-TOF MS. The calculated monoisotopic mass of the conjugate is 3251.08 Da. The observed masses were: 3251.93 (isomer 1), 3251.54 (isomer 2), and 3251.70 (isomer 3). In each case, there was also a secondary mass ~ 42 Da less than the parent mass. This smaller mass may represent product breakdown under MALDI MS conditions. Consistent with this interpretation, the smaller mass was not detected with electrospray MS.

Fig. 2.

Fig. 2

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RP-HPLC separation of Alexa Fluor 546-ArIB[V11L;V16A] from the reactants. The labeled absorbance peaks correspond to unconjugated α-CTx ArIB[V11L;V16A] (1), fluorescent conjugate isomers 1–3 (2–4), and unconjugated Alexa Fluor 546 NHS ester dye (5–7).

Alexa Fluor 546-ArIB[V11L;V16A] selectively blocks α7 nAChRs expressed in Xenopus laevis oocytes

Xenopus laevis oocytes were used to heterologously express nAChRs in order to assess the activity of the fluorescent conjugate. Initially, the three conjugate isomers were tested separately for their ability to block α7 nAChR responses to ACh and were determined to have similar activities (data not shown). All subsequent experiments were performed using the three isomers pooled together. As shown in Fig. 3, Alexa Fluor 546-ArIB[V11L;V16A] potently blocked the α7 subtype with an IC50 value of 1.8 (1.2–2.7) nM. The selectivity profile was determined by testing the fluorescent conjugate for activity on a panel of nAChR subtypes. At the highest concentration tested, (10 μM or >5,000 times the IC50 for α7 nAChRs), significant block was only observed for α3β2, α3β4, and α6/α3β2β3 nAChR subtypes. After a 5 minute static bath exposure to 10 μM Alexa Fluor 546-ArIB[V11L;V16A], the responses, expressed as a percent of control, were 26.6 ± 0.8% for α3β2, 60.2 ± 5.6% for α3β4, and 34.8 ± 1.9% for α6/α3β2β3 (Fig. 3). This suggests that the IC50 for each of these three subtypes will be in the vicinity of 10 μM, the highest concentration tested. In addition, the off-rate kinetics of Alexa Fluor 546-ArIB[V11L;V16A] at these three subtypes and the α7 subtype were very different. The oocytes were exposed to a static bath application of 1 μM Alexa Fluor 546-ArIB[V11L;V16A] for 5 min then perfused with ND96 alone and the responses monitored for recovery from block. α7 responses recovered very slowly (Fig. 4a) whereas the responses of α3β2 (Fig. 4b), α3β4 (Fig. 4c), and α6/α3β2β2 (Fig. 4d) nAChRs fully recovered in less than 60 sec. The slow reversibility of the interaction between Alexa Fluor 546-ArIB[V11L;V16A] and α7 nAChRs allows for extensive washing during labeling experiments that would eliminate any toxin bound to non-α7 nAChRs while reducing non-specific binding.

Fig. 3.

Fig. 3

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Concentration-response analysis of Alexa Fluor 546-ArIB[V11L;V16A] activity on nAChRs expressed in Xenopus laevis oocytes. Alexa Fluor 546-ArIB[V11L;V16A] blocks rα7 with an IC50 of 1.8 (1.2–2.7) nM, nH = 1.3 (0.85–1.7). Solid lines indicate concentration-response data for Alexa Fluor 546-ArIB[V11L;V16A] binding to rα7, mα1β1δε, rα2β2, α2β4, α3β2, α3β4, α4β2, rα4β4, rα6/α3β2β3, rα6/α3β4, and rα9α10. Data points for mα1β1δε, rα2β2, α2β4, α4β2, rα4β4, rα6/α3β4, and rα9α10 at the 10 μM concentration are, for clarity, shown separated to avoid overlap. Each data point with error bars represents the mean ± SEM from n= 4 for α7 and α6/α3β2β3 and 3 for all others. r, rat; m, mouse; nH, Hill slope; ( ), 95% confidence interval.

Fig. 4.

Fig. 4

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Alexa Fluor 546-ArIB[V11L;V16A] potently blocks α7 but not other nAChR subtypes. Representative trace recordings of Xenopus laevis oocytes expressing cloned nAChRs. After a stable ACh baseline was achieved, the oocytes were exposed to 1 μM Alexa Fluor 546-ArIB[V11L;V16A] for 5 min in a static bath then washed with ND96 and the responses to ACh monitored for recovery. The recovery rate for α7 nAChR responses (a) was very slow and recovered to 5.5 ± 0.9% after 20 min of wash. In contrast, α3β2 (b), α3β4 (c), and α6/α3β2β3 (d) responses rapidly recovered from block; only responses 3–5 are shown. Arrows in b–d indicate response after 5 min of wash. ±, SEM from n=3 for α3β2, α3β4, and α6/α3β2β3, and 4 for α7; r, rat.

Alexa Fluor 546-ArIB[V11L;V16A] displaces [125I]-α-BgTx but not [125]-α-CTx MII or [125I]-Epi from rat brain membranes

We used a panel of binding assays to assess the affinity of Alexa Fluor 546-ArIB[V11L;V16A] for native rat nAChRs. [125I]-α-BgTx is selective for α7 nAChRs when used to label brain nAChRs because α1* and α9*, two other nAChR subtypes to which α-BgTx binds, are not expressed in brain tissue (Elgoyhen et al. 1994, Elgoyhen et al. 2001, Sgard et al. 2002). [125]-α-CTx MII selectively binds to α3* and α6* containing nAChR subtypes while, in cortex, [125I]-Epi primarily detects the α4β2 subtype (Whiteaker et al. 2000b, Perry & Kellar 1995, Davila-Garcia et al. 1997). Alexa Fluor 546-ArIB[V11L;V16A] displaced [125I]-α-BgTx from hippocampal membranes with an IC50 of 7.9 ± 0.81 nM (Fig. 5a). This corresponds to a Ki of 1.8 ± 0.6 nM using the Cheng-Prusoff method. No displacement of [125]-α-CTx MII or [125I]-Epi by Alexa Fluor 546-ArIB[V11L;V16A] was observed using a concentration up to 1 μM (Fig. 5b). These results, taken together with those from the Xenopus oocyte experiments, indicate that Alexa Fluor 546-ArIB[V11L;V16A] selectively binds to α7 nAChRs.

Fig. 5.

Fig. 5

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Alexa Fluor 546-ArIB[V11L;V16A] competes with [125I]-α-BgTx but not [125I]-Epi or [125I]-α-CTx MII for binding sites from rat brain membranes. Competition binding assay was performed as described in Materials and Methods and the data were fit using a single-site model. (a), Alexa Fluor 546-ArIB[V11L;V16A] potently displaced [125I]-α-BgTx with a Ki of 1.8 ± 0.6 nM, nH =1.9 ± 0.5. (b), No displacement of [125I]-α-CTx MII or[125I]-Epi was observed using a concentration of Alexa Fluor 546-ArIB[V11L;V16A] up to 1 μM. nH, Hill slope; each data point represents the mean ± SEM from 4 individual determinations.

Alexa Fluor 546-ArIB[V11L;V16A] labels HEK293 cells expressing α7 nAChRs

Alexa Fluor 546-ArIB[V11L;V16A] is a relatively hydrophobic ligand, a property that can produce undesired non-specific binding. Additionally the peptide could bind to unknown sites on nAChRs which would not have been detected in the functional or radioligand binding assays. To address these concerns, we used HEK293 cell lines that stably express rat α7, α3β2, α3β4, and α4β2 nAChRs to directly examine the binding of the fluorescent ligand. Application of 100 nM Alexa Fluor 546-ArIB[V11L;V16A] to KXα7R1 produced punctate labeling which appeared to be confined to the cell surface membrane (Fig. 6a). The labeling could be prevented by preincubating KXα7R1 cells with 2 μM α-CbTx. (Fig. 6b). Based on kinetic data from the oocyte experiments, under the conditions used, minimal to no binding to α3β2, α3β4 or α4β2 receptors would be expected. Consistent with this prediction, application of Alexa Fluor 546-ArIB[V11L;V16A] at 100 nM to KXα3β2R4, KXα3β4R2, and KXα4β2R2 cells failed to produce labeling (data not shown). Application of a higher concentration (1 μM) to KXα3β2R4 (Fig. 6c) or KXα3β4R2 (Fig. 6d) cells also failed to produce labeling and no increase in non-specific binding was observed.

Fig. 6.

Fig. 6

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Alexa Fluor 546-ArIB[V11L;V16A] selectively labels HEK293 cells expressing α7 but not α3β2 or α3β4 nAChRs. (a), KXα7R1 cells were labeled with Alexa Fluor 546-ArIB[V11L;V16A] (red). The labeling was abolished by pre-incubating the cells with 2 μM α-CbTx (b). No labeling was observed on KXα3β2R4 (c) or KXα3β4R2 (d) cells. The cells in all images were counter stained with Hoechst 33342 (blue) to label the nuclei. All images were captured with a cooled CCD camera at 40x magnification; scale bar is 25 μm.

Next, we assessed the photostability of the new ligand. KXα7R1 cells were labeled with either Alexa Fluor 546-ArIB[V11L;V16A] or the Cy3 derivative. Labeled cells were continuously illuminated and measurements of fluorescence intensity were taken every 3 sec. The Alexa 546-conjugate was significantly more resistant to photobleaching than the Cy3-conjugate (Fig. 7).

Fig. 7.

Fig. 7

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Alexa Fluor 546-ArIB[V11L;V16A] is more resistant to photobleaching than Cy3-ArIB[V11L;V16A]. KXα7R1 cells were labeled with either ◆ Alexa Fluor 546-ArIB[V11L;V16A] or ○ Cy3-ArIB[V11L;V16A]. The cells were continuously illuminated and measurements of fluorescence intensity were taken every 3 sec as described in the Materials and Methods. Each data point represents the mean ± SEM from 3 individual cells.

Alexa Fluor 546-ArIB[V11L;V16A labels living hippocampal neurons from wild type but not α7 knockout mice

Primary hippocampal neuron cultures from three different genetic mouse strains were used to examine the binding of Alexa Fluor 546-ArIB[V11L;V16A]. Initially, we examined cultures obtained using wild type (wt) FVB/NJ mice. We observed intense punctate labeling on both the soma and neurites of a subpopulation of neurons (Fig. 8a and b). Based on the labeling pattern found here and previous identification of hippocampal neuron subtypes that express functional α7 nAChRs (Buhler & Dunwiddie 2002, Szabo et al. 2008, Zhao et al. 2009), we predict that these cells are interneurons. Preincubating the cultured neurons with 10 μM α-BgTx reduced the intensity of the labeling to a level indistinguishable from background (Fig. 8c). Although it appeared that block of Alexa Fluor 546-ArIB[V11L;V16A] by α-BgTx was virtually complete, small amounts of fluorescence remained which likely represents non-specific binding. However, it is also possible that some specific binding of Alexa Fluor 546-ArIB[V11L;V16A] to α7 nAChRs could remain if α-BgTx does not occupy each of the potential binding sites on the homopentameric α7 nAChR (Palma et al. 1996). We employed a genetic approach to assess this possibility by comparing the binding to hippocampal neurons from wt C57BL/6J versus α7 knockout mice. As observed in cultured neurons from FVB/NJ mice, a subpopulation of neurons from wt C57BL/6J were labeled after exposure to Alexa Fluor 546-ArIB[V11L;V16A] (Fig. 8d). In contrast, no neurons were labeled in cultures from α7 knockout mice (Fig. 8e). The distribution of non-specific binding in cultures from α7 knockouts was similar to that observed in wt cultures that had been pre-incubated with α-BgTx. We examined 105 neurons from α7 knockout mice and did not observe any neurons with labeling similar to those from wt mice.

Fig. 8.

Fig. 8

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Alexa Fluor 546-ArIB[V11L;V16A] labels α7 nAChRs in live cultured hippocampal neurons from wt mice but not from α7 knockouts. (a), A 1 μm interval z-series of a single hippocampal neuron from wt FVB/NJ mice labeled with Alexa Fluor 546-ArIB[V11L;V16A]. Note that the labeling is confined to the cell surface and has the appearance of discrete puncta. (b), Image of cultured hippocampal neurons from wt FVBN/J mice labeled with Alexa Fluor 546-ArIB[V11L;V16A]. (c), The labeling was significantly reduced when the neurons were pre-incubated with 10 μM α-BgTx before and during exposure to Alexa Fluor 546-ArIB[V11L;V16A]. (d), Image of hippocampal neurons from wt C57B6/J mice labeled with Alexa Fluor 546-ArIB[V11L;V16A]. (e), No labeling was observed on neurons from α7 knockouts (as negative controls to show specificity). Images in (b-e) are of a single focal plane merged with the corresponding image of the nuclei stained with Hoechst 33342 (blue). Neurons that are not labeled by Alexa Fluor 546-ArIB[V11L;V16A] are indicated by arrowheads. The remaining nuclei correspond to non-neuronal cells present in the cultures and appear as faint or out of focus due to being in a different focal plane relative to the neurons. Images were captured at 40x magnification; scale bar is 25 μm for all images.

Discussion

In this work, we demonstrate that the recently described ligand Cy3-ArIB[V11L;V16A] specifically labels α7 nAChRs expressed by cultured rat hippocampal neurons (Fig 1a–f). The labeling could be blocked by preincubating the neurons with excess α-BgTx (Supp. Fig. 1a and b). However, some residual intracellular staining of the soma remained possibly reflecting non-specific binding or incomplete block under the experimental conditions used in this study. This residual Cy3-ArIB[V11L;V16A] staining of the soma was frequent in neurons that had been fixed and permeabilized but was rarely observed in living neurons exposed to Alexa Fluor 546-ArIB[V11L;V16A].

α7 nAChRs have a relatively high permeability to calcium (Fucile et al. 2003, Zhao et al. 2003) and activation of these receptors on hippocampal neurons can substantially increase intracellular calcium concentrations (Khiroug et al. 2003, Fayuk & Yakel 2007). Such activity in dendrites and presynaptic terminals could facilitate depolarization and calcium-dependent release of neurotransmitter, respectively. Indeed, at the mossy fiber-CA3 pyramidal cell synapse, α7 nAChRs are expressed presynaptically and facilitate the release of glutamate (Sharma et al. 2008, Zhong et al. 2008). Further, α7 nAChRs function on dendrites and soma of GABAergic inhibitory interneurons in the hippocampus to modulate the activity of these interneurons (Szabo et al. 2008, Buhler & Dunwiddie 2002, Placzek et al. 2009). We used antibodies directed against PSD-95 and SV2 to examine the distribution of α7 nAChRs relative to presynaptic and postsynaptic markers in cultured rat hippocampal neurons. We observed partial overlap and close juxtaposition of α7 nAChR labeling at both SV2 and PSD-95 sites (Fig. 1a–f), providing strong support for the efficacy of fluorescently tagged ArIB[V11L;V16A] detection of native α7 nAChRs at and near synaptic junctions. Moreover, some labeling was detected at sites lacking SV2 and PSD-95 staining. α7 nAChRs are not exclusively confined to either pre- or postsynaptic sites, but also accumulate at perisynaptic sites, similar to the distribution observed for GABAA-Rs on cultured hippocampal neurons (Kannenberg et al. 1999). In conjunction with the live imaging experiments where α7 nAChR labeling was observed on somata and neurites, our results suggest that α7 nAChRs are expressed at multiple sites on cultured hippocampal neurons.

During our studies with Cy3-ArIB[V11L;V16A], we observed photobleaching during long exposure times. We therefore developed a new derivative namely, Alexa Fluor 546-ArIB[V11L;V16A]. This new ligand selectively blocks α7 nAChRs expressed in Xenopus oocytes with nanomolar potency (Fig. 3). In addition, there are substantial differences in the kinetics of recovery from toxin block that are nAChR subtype dependent. Recovery from block of α7 nAChRs by Alexa Fluor 546-ArIB[V11L;V16A] is very slow (5.5 ± 0.9% recovery after 20 min of wash, Fig 4a) whereas the block of other receptor subtypes is rapidly reversible (Fig. 4b–d). These substantial differences have practical implications in that a 5 min wash of the tissue preparation will reverse any binding that may occur to non-α7 subtypes. Alexa Fluor 546-ArIB[V11L;V16A] binding to native nAChRs was examined through competition binding assays using brain membranes. Alexa Fluor 546-ArIB[V11L;V16A] potently displaced [125I]-α-BgTx but failed to displace [125I]-α-CTx MII from α3* and α6* nAChRs or [125I]-Epi from β2* and β4* nAChRs in rat brain membranes (Fig. 5a and b). Binding of Alexa Fluor 546-ArIB[V11L;V16A] to nAChRs expressed by stably transfected HEK293 cells was used to assess the fluorescent properties of the ligand and assess the degree of non-specific binding. Specific fluorescent labeling was detected on cells expressing α7 nAChRs but not on cells expressing α3β2, α3β4, or α4β2 nAChRs. Interestingly, only a minority of KXα7R1 cells had detectable amounts of labeling (Fig. 6a). The remaining cells may express some α7 nAChRs but they are likely few in number and at low density thereby escaping detection under the imaging conditions used. Additionally, there may be differences in receptor expression that is cell cycle dependent.

Both Cy3-ArIB[V11L;V16A] and the new ligand, Alexa 546-ArIB[V11L;V16A], fluorescently labeled neurons from native tissue known to abundantly express α7 nAChRs. Under the conditions used in the present study, both ligands performed well. However, the Alexa Fluor 546 derivative had the potential advantage of being more photostable (Panchuk-Voloshina et al. 1999). Indeed, the Alexa Fluor 546 derivative was significantly more resistant to photobleaching compared to the Cy3 derivative (Fig. 7). Thus, for conditions where long image acquisition times are required (e.g. during long duration z-series acquisitions or examination of tissue with moderate receptor abundance), the Alexa Fluor 546 derivative is the superior ligand.

Hippocampal neurons are known to express several nAChR subtypes. Single-cell RT-PCR, immunoprecipitation, and immunohistochemistry experiments indicate the presence of nAChR subunits α2-α5, α7, and β2-β4 (Gahring et al. 2004, Sudweeks & Yakel 2000, Mao et al. 2008). Among the possible subunit combinations, those that contain α4 and α7 are the most abundant subtypes expressed by hippocampal neurons but some neurons have been reported to express receptors with an α3β4-like pharmacology (Alkondon & Albuquerque 2002). In cultured neurons from wt mouse hippocampus, punctate labeling was observed on a subpopulation of neurons. This labeling corresponds to α7 nAChRs based on the pharmacological profile of Alexa Fluor 546-ArIB[V11L;V16A] as determined by oocyte electrophysiology, competition binding assays, displacement by competitive ligands, and the absence of labeled hippocampal neurons from α7 knockout mice. The distribution of α7 nAChRs appeared to be concentrated on neurons having an interneuron-like morphology with multiple neurites extending from the soma. In contrast, unlabeled neurons were large triangular or tear drop-shaped with only a few neurites extending from the soma (characteristics of pyramidal neurons). This is consistent with reports that pyramidal neurons are generally unresponsive to nicotinic agonists (McQuiston & Madison 1999, Frazier et al. 2003, Frazier et al. 1998) (but see (Alkondon et al. 1997)) and likely express few α7 nAChRs. Previous reports suggest that in cultured hippocampal neurons, GABAergic interneurons express the highest levels of α7 nAChRs (Kawai et al. 2002, Massey et al. 2006) and therefore the neurons identified in the present study are presumed to be GABAergic as well. However, as mentioned earlier, glutamatergic mossy cells of the hilar/dentate gyrus region also express α7 nAChRs but these neurons constitute a small minority of cells in the hippocampus and their frequency in culture has not been extensively investigated.

Alexa Fluor 546-ArIB[V11L;V16A] represents a unique fluorescent ligand that selectively labels α7 nAChRs. This selectively provides a useful advantage over derivatives of α-BgTx for detecting α7 nAChRs in tissues that express multiple nAChR subtypes. This is of particular relevance to studies that involve dorsal root ganglia and immune cells where there is substantial expression of both α7 and α9* nAChRs (Biallas et al. 2007, Grau et al. 2007, Haberberger et al. 2004, Peng et al. 2004, Wang et al. 2003) In addition, although α1* receptors are predominantly expressed in muscle, in certain neuromuscular pathologies α7 nAChRs have also been reported (Tsuneki et al. 2003). Thus, the development of Alexa Fluor 546-ArIB[V11L;V16A] is a significant addition to the pharmacopeia of nAChR ligands.

Supplementary Material

Supp Fig s1

Supplemental Fig. 1 Cy3-ArIB[V11L;V16A] labels α7 nAChRs on fixed cultured rat hippocampal neurons. In (a), neurons were labeled with 1 μM Cy3-ArIB[V11L;V16A]. In (b), neurons were preincubated with 10 μM α-BgTx prior to and during exposure to 1 μM Cy3-ArIB[V11L;V16A] to assess non-specific binding. α-BgTx significantly reduced the number of Cy3-ArIB[V11L;V16A] puncta on processes indicating that these puncta correspond to α7 nAChR clusters. While also reduced, some residual staining of the cell soma was detected. Images were captured at 40x magnification; scale bar is 25 μm.

Supp Fig s2

Supplemental Fig. 2 The amino acid sequence of ArIB[V11L;V16A] showing the disulfide bonds between cysteine pairs (a) and the structure of Alexa Fluor 546 NHS ester dye (b).

Acknowledgments

Work supported by NIH grants MH53631 and GM48677 to J.M.M, DA12242 to P.W., and NS21725, DC008802 and P30 NS047243 to M.H.J. We thank M. Marks for providing the α7 knockout mice (supported by P30 DA015663) and Y. Xiao and K. Kellar for the HEK293 cell lines.

Abbreviations

ACh

acetylcholine

nAChR

nicotinic acetylcholine receptor

CA

cornus ammonis

α-BgTx

α-bungarotoxin

[125I]-α-BgTx

monoiodinated α-bungarotoxin

[125I]-Epi

monoiodinated epibatidine

[125I]-α-CTx MII

monoiodinated α-conotoxin MII

α-CbTx

α-cobratoxin

α-CTx

α-conotoxin

RP-HPLC

reverse phase-HPLC

MALDI-TOF MS

matrix assisted laser desorption ionization-time of flight mass spectrometry

CCD

charge-coupled device

human embryonic kidney-293

HEK293

wild type

wt

*

denotes the possible presence of additional subunits

Footnotes

The authors declare there are no conflicts of interest.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig s1

Supplemental Fig. 1 Cy3-ArIB[V11L;V16A] labels α7 nAChRs on fixed cultured rat hippocampal neurons. In (a), neurons were labeled with 1 μM Cy3-ArIB[V11L;V16A]. In (b), neurons were preincubated with 10 μM α-BgTx prior to and during exposure to 1 μM Cy3-ArIB[V11L;V16A] to assess non-specific binding. α-BgTx significantly reduced the number of Cy3-ArIB[V11L;V16A] puncta on processes indicating that these puncta correspond to α7 nAChR clusters. While also reduced, some residual staining of the cell soma was detected. Images were captured at 40x magnification; scale bar is 25 μm.

NIHMS209619-supplement-Supp_Fig_s1.tif (15MB, tif)
Supp Fig s2

Supplemental Fig. 2 The amino acid sequence of ArIB[V11L;V16A] showing the disulfide bonds between cysteine pairs (a) and the structure of Alexa Fluor 546 NHS ester dye (b).

NIHMS209619-supplement-Supp_Fig_s2.tif (4.6MB, tif)

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