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【关键词】 Subcellular
Neuronal nicotinic acetylcholine (ACh) receptors are ligand-gated, cation-selective ion channels. Nicotinic receptors containing 4, 6, β2, and β3 subunits are expressed in midbrain dopaminergic neurons, and they are implicated in the response to smoked nicotine. Here, we have studied the cell biological and biophysical properties of receptors containing 6 and β3 subunits by using fluorescent proteins fused within the M3-M4 intracellular loop. Receptors containing fluorescently tagged β3 subunits were fully functional compared with receptors with untagged β3 subunits. We find that β3- and 6-containing receptors are highly expressed in neurons and that they colocalize with coexpressed, fluorescent 4 and β2 subunits in neuronal soma and dendrites. Förster resonance energy transfer (FRET) reveals efficient, specific assembly of β3 and 6 into nicotinic receptor pentamers of various subunit compositions. Using FRET, we demonstrate directly that only a single β3 subunit is incorporated into nicotinic acetylcholine receptors (nAChRs) containing this subunit, whereas multiple subunit stoichiometries exist for 4- and 6-containing receptors. Finally, we demonstrate that nicotinic ACh receptors are localized in distinct microdomains at or near the plasma membrane using total internal reflection fluorescence (TIRF) microscopy. We suggest that neurons contain large, intracellular pools of assembled, functional nicotinic receptors, which may provide them with the ability to rapidly up-regulate nicotinic responses to endogenous ligands such as ACh, or to exogenous agents such as nicotine. Furthermore, this report is the first to directly measure nAChR subunit stoichiometry using FRET and plasma membrane localization of 6- and β3-containing receptors using TIRF.
6 nicotinic ACh receptor subunits are expressed in several catecholaminergic nuclei in the central nervous system, including retinal ganglion cells (Gotti et al., 2005b), locus coeruleus (Léna et al., 1999), and dopaminergic neurons located in the substantia nigra and ventral tegmental area (Whiteaker et al., 2000; Zoli et al., 2002; Champtiaux et al., 2003). Ligand-binding studies using the 6-specific probe -conotoxin MII suggest that many 6* (* indicates that other subunits may be present in the receptor) receptors are located on presynaptic terminals in the superior colliculus and striatum (Whiteaker et al., 2000). Indeed, this binding activity disappears in the brains of 6 knockout mice (Champtiaux et al., 2002). This strikingly specific expression pattern could indicate a unique function for 6* receptors, and 6* receptors are candidate drug targets for diseases or disorders such as Parkinson's disease or nicotine addiction (Quik and McIntosh, 2006).
Functional, voltage-clamped 6-dependent responses are elusive in heterologous expression systems such as Xenopus laevis oocytes (Kuryatov et al., 2000; Broadbent et al., 2006), but native 6* receptors are readily studied using synaptosome preparations from brain tissue (Whiteaker et al., 2000; Grady et al., 2002; Champtiaux et al., 2003; Gotti et al., 2005a). Indeed, -conotoxin MII-sensitive receptors are pharmacologically and stoichiometrically distinct from -conotoxin MII-resistant receptors in mediating [3H]dopamine release from striatal synaptosomes (Grady et al., 2002; Salminen et al., 2007). Recent studies using 4 and β3 knockout mice demonstrate the existence of functional 6β2, 6β2β3, 64β2, and 64β2β3 receptors (Salminen et al., 2007). It is noteworthy that native 64β2β3 receptors have the highest affinity (EC50 = 0.23 ± 0.08 µM) for nicotine of any nicotinic receptor reported to date. Because nicotine is likely to be present at concentrations 0.5 µM in the cerebrospinal fluid of smokers (Rowell, 2002), only those receptors with the highest affinity for nicotine, including some 4* and 6* receptors, are likely to be important in nicotine addiction. Although previous studies offer major conceptual advances in our understanding of 6* receptors in the brain, there is a lack of information regarding the subcellular localization and biophysical properties of 6 subunits.
β3 subunits are expressed in most of the same locations as 6, including midbrain dopaminergic neurons projecting to the striatum (Zoli et al., 2002). β3 knockout mice demonstrate that β3 subunits are important for the biogenesis of 6* receptors in the brain (Cui et al., 2003; Gotti et al., 2005a). This is corroborated by studies in X. laevis oocytes and tissue culture cells (Kuryatov et al., 2000). β3 also increases 6-specific binding activity in HEK293 cells (Tumkosit et al., 2006). Uncertainty exists, however, because others have reported that β3 incorporation into nAChRs acts as a dominant negative (Boorman et al., 2003; Broadbent et al., 2006), suppressing ACh-evoked responses by an incompletely understood gating mechanism. This effect occurred apparently without significantly altering the surface expression of nAChRs. What is clear is that β3 acts more like a muscle β subunit than a "typical" neuronal β subunit; it does not participate in forming the :non- interface that comprises the neuronal ligand binding site, and other β subunits, either β2 or β4, must be present to form functional nicotinic receptors (Broadbent et al., 2006). This presents a problem both for basic and therapeutic-oriented research on β3* receptors, because there are no pharmacological ligands that can visually or functionally isolate β3-specific actions in cell culture systems or intact brain tissue. Given the precise localization and unique functional properties of β3* receptors, potential for therapeutic intervention that would be afforded by β3-specific probes, and involvement in nicotine addiction (Bierut et al., 2007), it is important to develop and exploit tools to study β3.
We have sought to compare characteristics of 6 and β3 with the better understood 4 and β2 subunits. We previously generated fluorescently labeled 4 and β2 subunits, and we used these subunits to study assembly, trafficking, and nicotine-dependent up-regulation of 4β2 receptors (Nashmi et al., 2003). We have now fluorescently labeled 6 and β3 subunits by inserting a yellow fluorescent protein (YFP) or cyan fluorescent protein (CFP) in the M3-M4 intracellular loop. With this approach, one can optically monitor functional nicotinic ACh receptors containing these subunits in live cells and in real time. We measured 1) functional responses using two-electrode voltage-clamp and patch-clamp electrophysiology, 2) subcellular distribution and colocalization in neurons using confocal microscopy and spectral imaging, 3) receptor assembly and subunit stoichiometry using Förster resonance energy transfer (FRET), and 4) plasma membrane localization and distribution patterns using total internal reflection fluorescence (TIRF) microscopy.
Reagents. Unless otherwise noted, all chemicals were from Sigma-Aldrich (St. Louis, MO). DNA oligonucleotides for PCR and site-directed mutagenesis were synthesized by Integrated DNA Technologies, Inc. (Coralville, IA). Restriction enzymes for molecular biology were purchased from Roche Diagnostics (Indianapolis, IN) or New England Biolabs (Ipswich, MA). Glass-bottomed dishes (35 mm) coated with L-polylysine were purchased from MatTek (Ashland, MA).
Cell Culture and Transfection. N2a cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbecco's modified Eagle's medium (high glucose with 4 mM L-glutamine; Invitrogen, Carlsbad, CA)/Opti-MEM (Invitrogen) mixed at a ratio of 1:1 and supplemented with 10% fetal bovine serum (Invitrogen), penicillin (Mediatech, Herndon, VA), and streptomycin (Invitrogen). N2a cells were transfected in DMEM without serum or antibiotics. Transfection was carried out using Lipofectamine/PLUS (Invitrogen) according to the manufacturer's instructions and with the following modifications. For a 35-mm dish, 1 to 2 µg of total plasmid DNA was mixed with 100 µl of DMEM and 6 µl of PLUS reagent. DMEM/DNA was combined with a mixture of 100 µl of DMEM and 4 µl of Lipofectamine reagent. Rat hippocampal neurons were dissociated and plated on glass-bottomed imaging dishes as described previously (Slimko et al., 2002). For primary neuron transfection, Lipofectamine 2000 (Invitrogen) was used in conjunction with Nupherin (BIOMOL Research Laboratories, Plymouth Meeting, PA) as described below. In brief, in total 1 µg of DNA was incubated with 20 µg of Nupherin in 400 µl of Neurobasal medium without phenol red (Invitrogen), whereas 10 µl of Lipofectamine 2000 was mixed in 400 µl of Neurobasal medium (Invitrogen). After 15 min, the two solutions were combined and incubated for 45 min. Neuronal cultures in 35-mm glass-bottomed culture dishes were incubated in the resulting 800-µl mixture for 120 min, followed by removal of transfection media and refeeding of the original, pretransfection culture media.
Plasmids and Molecular Biology. Mouse 4 and β2 nAChR cDNAs in pCI-neo, both untagged and modified with YFP or CFP fluorescent tags, have been described previously (Nashmi et al., 2003). Mouse 3 and β3 nAChR cDNAs in pCDNA3.1 were a generous gift of Jerry Stitzel (Institute for Behavioral Genetics, University of Colorado, Boulder, CO). A full-length mouse 6 I.M.A.G.E. cDNA (ID no. 4501558) was obtained from Open Biosystems (Huntsville, AL). A modified 6 cDNA was constructed that 1) lacked the 5' and 3' untranslated regions and 2) contained a Kozak sequence (GCC ACC) before the ATG start codon to facilitate efficient translation initiation. Rat β4 cloned into pAMV was provided by Cesar Labarca (California Institute of Technology, Pasadena, CA). pEYFP-N1 and pECFP-N1 (Clontech, Mountain View, CA) were used to construct fluorescent nAChR cDNAs. mGAT1 labeled with CFP was provided by Fraser Moss (California Institute of Technology). YFP-Syntaxin was provided by Wolfhard Almers (Vollum Institute, Oregon Health and Science University, Portland, OR). CFP-tau was provided by George Bloom (University of Virginia, Charlottesville, VA). A QuikChange (Stratagene, La Jolla, CA) kit was used to construct β3 (WT or XFP-modified) cDNAs containing a V13'S point mutation.
To design fluorescently labeled 6 and β3 subunits, we chose to insert the XFP moiety in the M3-M4 loop each subunit. We have previously found that this region is appropriate for insertion in nAChR 4 and β2 subunits (Nashmi et al., 2003), the nAChR subunit (data not shown), and GluCl and β subunits (Slimko et al., 2002). Similar to our previous studies, we inserted the XFP moiety in the M3-M4 loop at positions that avoided the conserved amphipathic -helix and putative cell sorting motifs and phosphorylation sites (Fig. 1, A and B). To construct nAChRs with XFP inserted into the M3-M4 loop, a two-step PCR protocol was used. First, YFP or CFP was amplified with PCR using oligonucleotides (sequences available upon request) designed to engineer 5' and 3' overhangs of 15 base pairs that were identical to the site where XFP was to be inserted, in frame, into the nAChR M3-M4 loop. A Gly-Ala-Gly flexible linker was engineered between the nAChR sequence and the sequence for YFP/CFP at both the 5' and 3' ends. In the second PCR step, 100 ng of the first PCR reaction was used as a primer pair in a modified QuikChange reaction using Pfu Ultra II (Stratagene, Cedar Creek, TX) polymerase and the appropriate nAChR cDNA as a template. All DNA constructs were confirmed with sequencing and, in some cases, restriction mapping.
Fig. 1. 6 and β3 nicotinic ACh receptor constructs used in this study. A, XFP insertion points in the 6 M3-M4 intracellular loop. The M3-M4 loop primary sequence of the mouse 6 nAChR subunit was analyzed for sequences predicted to be involved in forming -helices (light gray boxes), phosphorylation sites (white boxes), or intracellular trafficking motifs (dark gray boxes); these were specifically avoided. Arrows adjacent to XFP indicate insertion points. The inserted XFP (YFP or CFP) protein was modified 1) to have a flexible Gly-Ala-Gly linker flanking the XFP coding sequence and 2) to lack its STOP codon. B, XFP insertion points in β3 M3-M4 intracellular loop. Mouse β3-XFP fusion proteins were designed similarly to 6, as indicated in A. C, 6 and β3 nAChR constructs used in this study. In addition to WT, three 6-XFP and two β3-XFP fusions were constructed. A V13'S mutation on the WT and XFP background was introduced into β3 for characterization in X. laevis oocytes.
cRNA for injection and expression in X. laevis oocytes was prepared using a T7 or SP6 in vitro transcription kit (mMessage mMachine; Ambion, Foster City, CA) according to the manufacturer's instructions. RNA yield was quantified with absorbance at 260 nm. RNA quality was assessed by observing absorbance profiles across a range of wavelengths between 220 and 320 nm. Spectrophotometric analysis was performed using a ND-1000 spectrophotometer (Nano-Drop, Wilmington, DE).
Confocal Microscopy. N2a cells were plated on 35-mm glass-bottomed dishes, transfected with nAChR cDNAs, and they were imaged live 24-48 h after transfection. X. laevis oocytes were imaged 3 days after RNA injection. Oocytes were placed in an imaging chamber and allowed to settle for 20 min before imaging. To eliminate autofluorescence, growth medium was replaced with an extracellular solution containing the following components: 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM D-glucose, pH 7.4. Cells were imaged with a Nikon (Nikon Instruments, Melville, NY) C1 laser-scanning confocal microscope system equipped with spectral imaging capabilities and a Prior (Rockland, ME) remote-focus device. For oocytes, a Nikon Plan Apo 20 x 0.75 numerical aperture (NA) air objective was used, whereas a Nikon Plan Apo 60 x 1.40 NA oil objective was used for mammalian tissue culture cells. Pinhole diameter was 30-60 µm, and cells were imaged at 12-bit intensity resolution over 512 x 512 pixels at a pixel dwell time of 4 to 6 µs. CFP was excited using a 439.5-nm modulated diode laser, and YFP was excited with an argon laser at 514.5-nm. In most cases, imaging was carried out using the Nikon C1si DEES grating and spectral detector with 32 parallel photomultiplier tubes. This allowed us to collect spectral images ( stacks). In such images, each pixel of the X-Y image contains a list of emission intensity values across a range of wavelengths. We collected light between 450 and 600 nm at a bandwidth of 5 nm. The 515-nm channel was intentionally blocked while we used the 514.5-nm laser for YFP bleaching. Because the emission profile of YFP and CFP significantly overlap, we used the Nikon EZC1 linear unmixing algorithm to reconstruct YFP and CFP images. Experimental spectral images with both YFP- and CFP-labeled nAChR subunits were unmixed using reference spectra from images with only YFP- or CFP-labeled nAChR subunits. For each pixel of a spectral image, intensity of YFP and CFP was determined from fluorescence intensity values at the peak emission wavelength derived from the reference spectra.
Spectral FRET Analysis. To examine FRET between various nAChR subunits, the acceptor photobleaching method (Nashmi et al., 2003) was used with a modified fluorescence recovery after photobleaching macro built into the Nikon EZC1 imaging software. In this method, FRET was detected by recording CFP dequenching during incremental photodestruction of YFP. A spectral image was acquired once before YFP bleaching and at six time points every 10 s during YFP bleaching at 514.5 nm. Laser power during bleaching varied from cell to cell, but was between 25 and 50%. One bleach scan per cycle was used. This bleaching protocol was optimized to achieve 70 to 80% photodestruction of YFP while still enabling us to record incremental increases in CFP emission at each time point. In the confocal microscope, nAChRs labeled with XFP usually exhibit a uniform, intracellular distribution, regardless of the subunit being examined. This is consistent with our previous observations (Nashmi et al., 2003). To measure FRET, spectral images were unmixed into their CFP and YFP components as described above. We found little or no difference in FRET for various cellular structures or organelles in N2a cells, and we measured CFP and YFP mean intensity throughout the entire cell by selecting the cell perimeter as the boundary of a region of interest in Nikon's EZC1 software. CFP and YFP components were saved in Excel format, and fluorescence intensities were normalized to the prebleach time point (100%). FRET efficiency (E) was calculated as E = 1 - (IDA/ID), where IDA represents the normalized fluorescence intensity of CFP (100%) in the presence of both donor (CFP) and acceptor (YFP), and ID represents the normalized fluorescence intensity of CFP in the presence of donor only (complete photodestruction of YFP). The ID value was extrapolated from a scatter plot of the fractional increase of CFP versus the fractional decrease of YFP. The E values were averaged from several cells per condition (see Table 1 for n values). Data are reported as mean ± S.E.M.
TABLE 1 FRET efficiency calculations for nicotinic ACh receptors with various subunit compositions
Unless noted otherwise, all experiments with 6 and β3 subunits were performed with 6A405 and β3P379. Data are reported as mean FRET E ± S.E.M. n refers to the number of independently analyzed cells.
TIRF Microscopy. N2a cells cultured in glass-bottomed, polyethylenimine-coated imaging dishes were transfected with cDNA mixtures as described above. Cells, superfused with the same imaging solution used for confocal microscopy, were imaged 18 to 24 h after transfection to minimize overexpression artifacts. TIRF images were obtained with an inverted microscope (Olympus IX71; Olympus America, Inc., Center Valley, PA) equipped with a 488-nm air-cooled argon laser (P/N IMA101040ALS; Melles Griot, Carlsbad, CA). Laser output was controlled with a UNIBLITZ shutter system and drive unit (P/N VMM-D1; Vincent Associates, Rochester, NY) equipped with a Mitutoyo (Mitutoyo America, City of Industry, CA) micrometer to control TIRF evanescent field illumination. TIRF imaging was carried out with an Olympus PlanApo 100 x 1.45 NA oil objective, and images were captured with a 16-bit resolution Photometrics Cascade charge-coupled device camera (Photometrics, Tucson, AZ) controlled by SlideBook 4.0 imaging software (Intelligent Imaging Innovations, Santa Monica, CA).
Two-Electrode Voltage-Clamp Electrophysiology. Stage V to VI X. laevis oocytes were isolated as described previously (Quick and Lester, 1994). Stock RNAs were diluted into diethyl pyrocarbonate-treated water and injected 1 day after isolation. RNA was injected in a final volume of 50 nl per oocyte using a digital microdispenser (Drummond Scientific, Broomall, PA). After injection, oocytes were incubated in ND-96 solution (96 mM NaCl, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES/NaOH, pH 7.6) supplemented with 50 µg/ml gentamicin and 2.5 mM sodium pyruvate. After 1 to 4 days for nAChR expression, oocytes were used for recording or confocal microscopy.
Agonist-activated nicotinic receptor responses were measured by two-electrode voltage-clamp recording using a GeneClamp 500 (Molecular Devices, Sunnyvale, CA) voltage clamp. Electrodes were constructed from Kwik-Fil borosilicate glass capillary tubes (1B150F-4; WPI, Sarasota, FL) using a programmable microelectrode puller (P-87; Sutter Instrument Company, Novato, CA). The electrodes had tip resistances of 0.8 to 2.0 M after filling with 3 M KCl. During recording, oocytes were superfused with Ca2+-free ND-96 via bath-application and laminar-flow microperfusion using a computer-controlled application and washout system (SF-77B; Warner Instruments, Hamden, CT) (Drenan et al., 2005). The holding potential was -50 mV, and ACh was diluted in Ca2+-free ND-96 and applied to the oocyte for 2 to 10 s followed by rapid washout. Data were sampled at 200 Hz and low-pass filtered at 10 Hz using the GeneClamp 500 internal low-pass filter. Membrane currents from voltage-clamped oocytes were digitized (Digidata 1200 acquisition system; Molecular Devices) and stored on a PC running pCLAMP 9.2 software (Molecular Devices). Concentration-response curves were constructed by recording nicotinic responses to a range of agonist concentrations (six to nine doses) and for a minimum of six oocytes. EC50 and Hill coefficient values were obtained by fitting the concentration-response data to the Hill equation. All data are reported as mean ± S.E.M.
Whole-Cell Patch-Clamp Electrophysiology. N2a cells expressing YFP-labeled nicotinic receptors were visualized with an inverted microscope (Olympus IMT-2, DPlan 10 x 0.25 NA and MPlan 60 x 0.70 NA) under fluorescence illumination (mercury lamp). Patch electrodes (3-6 M) were filled with pipette solutions containing 88 mM KH2PO4, 4.5 mM MgCl2, 0.9 mM EGTA, 9 mM HEPES, 0.4 mM CaCl2, 14 mM creatine phosphate (Tris salt), 4 mM Mg-ATP, and 0.3 mM GTP (Tris salt), pH 7.4 with KOH. The extracellular solution was 150 mM NaCl, 4 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM D-glucose, pH 7.4. Standard whole-cell recordings were made using an Axopatch 1-D amplifier (Molecular Devices), low-pass filtered at 2 to 5 kHz, and digitized online at 20 kHz (pClamp 9.2; Molecular Devices). Series resistance was compensated 80%, and the membrane potential was held at -70 mV. Recorded potentials were corrected for junction potential.
ACh was delivered using a two-barrel glass -shaped tube (outer diameter 200 µm; pulled from 1.5-mm-diameter -shaped borosilicate tubing) connected to a piezoelectric translator (LSS-3100, Burleigh Instruments, Fishers, NY) as described previously (Nashmi et al., 2003). ACh was applied for 500 ms (triggered by pCLAMP 9.2), and solution exchange rates measured from open tip junction potential changes during application with 10% extracellular solution were typically 300 µs (10-90% peak time). Data are reported as mean ± S.E.M. for the peak current response to 1 µM ACh, and statistical significance was determined using a Wilcoxon signed rank test.
Design and Construction of 6 and β3 XFP Fusions. Based on previous work (Slimko et al., 2002; Nashmi et al., 2003), we chose to insert XFP fusions in the M3-M4 loop of mouse 6 and β3 nAChR subunits. Like all members of the Cys-loop family, 6 and β3 have predicted -helices at the N- and C-terminal ends of their M3-M4 loop (Fig. 1, A and B) that may be important in ion permeation (Miyazawa et al., 1999). In addition to avoiding these regions, we also avoided potential phosphorylation sites and trafficking motifs (Fig. 1, A and B). Our XFP fusion cassette also consisted of a Gly-Ala-Gly flexible linker flanking the XFP open reading frame on both the N- and C-terminal side. We built three independent XFP fusions for 6 and two XFP fusions for β3 (Fig. 1C). These were designated according to the residue immediately N-terminal to the beginning of the Gly-Ala-Gly linker (e.g., 6-YFPG366 denotes that the GAG-YFP-GAG cassette was inserted between G366 and V367). Unless otherwise noted, all experiments were conducted with 6-XFPA405 and β3-XFPP379.
Fig. 2. Fluorescently labeled β3 subunits are functional and expressed on the cell surface in X. laevis oocytes. A, X. laevis oocytes were injected with cRNA encoding WT (control) or YFP-labeled β3 (15 ng) along with 3(2 ng) and β4 (3 ng). The oocyte surface was imaged with direct fluorescence confocal microscopy. True YFP signal was acquired by linear unmixing of the background fluorescence spectra (untagged β3 with 3β4) and a YFP reference spectrum. Scale bar, 54 µm. B, fluorescently labeled β3 subunits are indistinguishable from WT subunits in their ability to attenuate nicotinic receptor responses. A representative voltage-clamped response from X. laevis oocytes expressing 3β4, 3β4β3, or 3β4β3-YFP is shown. Agonist (ACh; 200 µM) was applied and removed as indicated by the bar. C, reversal of β3-mediated suppression of nicotinic responses is identical in untagged and YFP-labeled hypersensitive β3. Representative voltage-clamped responses from oocytes expressing 3β4, 3β4β3V13S, or 3β4β3-YFPV13S are shown. Agonist application is identical to B.
Functional Expression of 6 and β3 Subunits. Despite exhaustive attempts to functionally reconstitute 6* nAChRs in X. laevis oocytes and mammalian tissue culture cells, we recorded no robust, reproducible responses from cells expressing 6, either with untagged subunits or the fluorescent subunits (Supplemental Data; Table 1). β3-YFP, however, was well expressed on the plasma membrane of X. laevis oocytes when coexpressed with 3 and β4 subunits to support functional expression (Fig. 2A). As a control for oocyte autofluorescence, we imaged oocytes expressing untagged β3 subunits (Fig. 2A). No fluorescence was detected in this case, indicating that our β3-YFP signal was specific.
β3 subunits do not drastically alter the EC50 for ACh or nicotine when incorporated into nAChRs (Boorman et al., 2003), but they do profoundly alter single-channel kinetics (Boorman et al., 2003). Channel burst duration was significantly shortened for nAChRs containing β3 versus those without it (Boorman et al., 2003), suggesting that β3 reduces the probability of channel opening, Popen. Consistent with this, macroscopic voltage-clamped responses from oocytes and mammalian cells expressing β3* receptors were significantly smaller than for non-β3* receptors (Broadbent et al., 2006). To assess the functionality of our β3-YFP construct, we compared the ability of untagged and YFP-labeled β3 subunits to attenuate nicotinic responses. β3 must be coexpressed with other and β subunits, so we chose to use 3β4 receptors for this purpose. We did so because β3 has been well characterized with this receptor combination (Boorman et al., 2003; Broadbent et al., 2006). When WT, untagged β3 was coexpressed with 3β4 receptors, we found a significant attenuation of the peak response to 200 µM ACh (Fig. 2B), consistent with previous findings (Broadbent et al., 2006). When β3-YFP was tested in this assay, it was also able to attenuate the maximal response in a manner identical to untagged β3 (Fig. 2B). It is possible that, although β3-WT attenuates responses via a gating mechanism on the plasma membrane, YFP-labeled β3 might do so via a different mechanism such as sequestering 3 or β4 subunits inside the cell. To further test the functionality of YFP-labeled β3, we took advantage of the fact that a gain-of-function TM2 mutation in β3 is able to reverse the attenuation of peak responses seen for β3-WT (Broadbent et al., 2006). We reasoned that if the YFP label in the M3-M4 loop is not disturbing the function of β3, we should detect the same gain-of-function response for unlabeled and YFP-labeled β3 when they are engineered to express a mutation of this sort. When a Val13' to Ser mutation (V13S) was introduced into unlabeled β3, we observed not only a reversal of this attenuation behavior, but a significant increase in the peak response to 200 µM ACh with 3β4 receptors (Fig. 2C). When β3-YFPV13S was tested in this assay, we observed an identical behavior. Taken together, these data suggest that β3-YFP is fully functional and incorporates into nAChRs in X. laevis oocytes.
To further characterize our β3-YFP construct, we constructed concentration-response curves for 3β4 receptors containing either β3WTor β3-YFP. Consistent with previous reports (Boorman et al., 2000), we measured an EC50 for ACh of 230 ± 22 µM for 3β4β3 receptors, which is slightly higher than for 3β4 (165 ± 9 µM) (Fig. 3A). When β3-YFP was substituted for WT β3, the EC50 was shifted slightly, but acceptably, to 109 ± 8 µM (Fig. 3A). We also noticed that the addition of β3 to 3β4 receptors increased the Hill coefficient from 1.5 ± 0.1 to 2.0 ± 0.3, and this effect was retained when β3-YFP was coexpressed with 3β4 receptors. Likewise, we also constructed concentration-response relationship curves for oocytes expressing β3V13S and β3-YFPV13S. Compared with the EC50 for 3β4β3 (230 ± 22 µM), we measured an EC50 for 3β4β3V13S of 28 ± 3 µM (Fig. 3B). This is consistent with others who have reported an approximate 6-fold reduction in EC50 for the inclusion of β3 with a similar hypersensitive mutation, Val9'Ser (Boorman et al., 2000). We reasoned that if β3-YFP retained the WT function of β3, then there should be a similar gain-of-function phenotype when it is coexpressed with 3β4. We measured an EC50 for 3β4β3-YFPV13S of 34 ± 3 µM (Fig. 3B), confirming that this construct behaves identically to β3-WT. Collectively, our work in X. laevis oocytes with YFP-labeled β3 subunits suggests that insertion of YFP into the M3-M4 loop does not significantly alter the assembly, subcellular trafficking, or function of this subunit.
Fig. 3. β3 nAChR subunit function is not affected by XFP insertion in M3-M4 loop. A, concentration-response relations for WT and fluorescently labeled β3-containing receptors are similar. X. laevis oocytes expressing the indicated receptor subunits were voltage-clamped during agonist application and washout. Peak responses to the indicated ACh concentration (molar) were normalized and the data were fitted to the Hill equation. B, concentration-response relation for β3 subunits with a hypersensitive mutation is not affected by the presence of YFP in the M3-M4 loop. X. laevis oocytes were assayed and data were analyzed as described in A. 3β4 data from A are shown for comparison. Error bars are ± S.E.M., and n = 6 for each condition.
Subcellular Localization and Trafficking of 6 and β3 Subunits. To probe the subcellular localization and trafficking of 6* and β3* receptors, we chose a mouse neuroblastoma cell line, N2a, to transiently express our fluorescent nicotinic receptor subunits. We prefer these cells over, for example, HEK293 cells, because they 1) are of mouse origin, the same species as our fluorescent constructs; 2) are of neuronal origin, suggesting that they will be a permissive environment for correct expression, subcellular localization, and assembly of our ectopic nAChR subunits; and 3) express only moderate quantities of transfected membrane protein. To study the subcellular localization of β3* receptors, we coexpressed β3-YFP with the previously described fluorescently labeled 4 and β2 subunits (Nashmi et al., 2003). β3 is able to assemble and function when coexpressed with 4β2 receptors (Broadbent et al., 2006). When coexpressed with fluorescent 4 or β2 receptors, β3-YFP was localized primarily in the endoplasmic reticulum of live N2a cells (Fig. 4A, i and B, i). We used CFP-labeled 4 (Fig. 4B, ii) or β2 (Fig. 4, A and C, ii) subunits along with a confocal microscope with spectral imaging capabilities to unambiguously assign YFP and CFP signals to each pixel for the spectral images of the cells. In these experiments, YFP was assigned green, CFP was assigned red, and yellow indicated pixels where β3-YFP was colocalized with either β2-CFP (Fig. 4A, iii) or 4-CFP (Fig. 4B, iii). We noted that β3-YFP was completely colocalized with either 4 or β2 in this experiment, suggesting that these subunits are assembled in the same pentameric receptors. To further define the extent of this colocalization, we plotted the β3-YFP and 4-CFP or β2-CFP pixel intensity across a two-dimensional region of interest transecting the cell (Fig. 4, A and B, iv). We noted that the YFP and CFP intensity profiles strongly resembled each other, suggesting that these subunits were indeed colocalized and coassembled in intracellular compartments of the cell. With respect to 4β2* receptors, this localization pattern is not an artifact of overexpression, because this is the same pattern we observed previously (Nashmi et al., 2003). This is also the expression pattern of endogenous, YFP-labeled 4* receptors in 4-YFP knockin mice (Nashmi et al., 2007). This indicates that 1) a large pool of intracellular receptors exists in neurons, and 2) YFP tag does not interfere with the delivery of receptors to the plasma membrane. Thus, the localization pattern we observe here for β3 subunits is the expected result if it is assembling with 4β2 receptors.
Fig. 4. β3-YFP and 6-YFP expression in neuronal cells. A, β3 and β2 nAChR subunits are localized similarly in N2a cells. N2a cells expressing 4β2-CFPβ3-YFP receptors were imaged live with spectral confocal microscopy. Spectral images were acquired and specific β3-YFP and β2-CFP signals were extracted with linear unmixing. Green (β3-YFP; i) and red (β2-CFP; ii) pseudocolor was assigned, and yellow (Merge; iii) indicates colocalized proteins. Pixel intensities for the YFP and CFP channel were plotted (iv) along a line (iii) transecting the imaged cell. B, β3 and 4 nAChR subunits are localized similarly in N2a cells. N2a cells expressing 4-CFPβ2β3-YFP receptors were imaged live as described in A. C, 6 and β2 nAChR subunits are localized similarly in N2a cells. N2a cells expressing 6-YFPβ2-CFP were imaged live as described in A and B. D, 4-YFP, β3-YFP, and 6-YFP are localized intracellularly and in processes in differentiated neurons. N2a cells were differentiated for 2 days (see Materials and Methods for details) to induce neurite outgrowth, followed by transfection with the indicated nAChR cDNA combinations plus soluble CFP to mark cellular morphology. One day after transfection, cells were imaged live as described in A to C. Scale bar, 10 µm.
We expressed 6-YFP along with β2-CFP in N2a cells, and we analyzed its localization pattern as described above for β3. We also found that 6 was localized in intracellular compartments in the cell (Fig. 4C, i), and that it was completely colocalized with β2 subunits (Fig. 4C, ii-iv). Although this is the first fluorescence imaging reported for 6* receptors, there is other evidence to corroborate our findings. Studies with [3H]epibatidine demonstrate that a significant portion of 6β2 and 6β2β3 receptors are intracellular (50 and 20%, respectively), although some are delivered to the surface (Kuryatov et al., 2000; Tumkosit et al., 2006).
Fig. 5. Localization of β3* and 6* receptors in primary neurons. A, β3-YFP and 6-YFP are localized in the cell soma and in dendrites in primary hippocampal neurons. E18 rat hippocampal neurons were plated and cultured for 14 days followed by transfection with the indicated nAChR cDNAs. One day after transfection, cells were imaged live. Right, higher magnification image of the boxed area in the left panel. B, β3* and 6* receptors are absent from axons. Neurons were transfected with the indicated nAChR cDNAs along with CFP-tau, followed by live confocal imaging as described in A. Scale bar, 10 µm.
To further investigate the subcellular localization and trafficking of 6 and β3 subunits, we imaged live, differentiated N2a cells and primary neurons. N2a cells can be induced to differentiate and undergo neurite outgrowth if serum is withdrawn and an activator of protein kinase A, dibutyryl-cAMP, is added (Fowler et al., 2001). In our previous work, 4β2 receptors were localized to dendrites, but not axons, when expressed in primary midbrain neurons (Nashmi et al., 2003). We were interested in whether our fluorescent nicotinic receptor subunits were localized to N2a cell processes in a manner analogous to dendrites in primary neurons. Furthermore, we wanted to address the question of whether β3 is localized with other subunits at distal sites such as dendrites. This is an unsolved question, as there is no high-affinity probe (pharmacological or immunological) that can reliably and unambiguously isolate β3* receptors. N2a cells were plated on glass-bottomed dishes, and they were then differentiated for 2 days (see Materials and Methods) followed by transfection with various combinations of YFP-labeled and unlabeled nAChR subunits. Cells were also co-transfected with an expression plasmid for soluble CFP to mark total cell morphology. We found that 4β2 receptors were indeed localized to neuronal processes in differentiated N2a cells (Fig. 4D, arrow), along with abundant expression in the cell soma. When β3-YFP was coexpressed with 4β2, we observed a very similar pattern. We found that β3 was present even at the most distant elements of neuronal processes (Fig. 4D, arrow). Because this pattern is identical to that of 4β2 in differentiated N2a cells, we conclude that β3 is likely assembling with 4β2 receptors and that the YFP label in the M3-M4 loop is not disrupting the normal cellular trafficking of 4β2β3 pentamers. To further characterize the localization of β3* receptors, we coexpressed β3-YFP with 4β2 receptors in primary rat hippocampal neurons (Nashmi et al., 2003). To minimize overexpression artifacts, cells were imaged live only 18 to 24 h after transfection. We found that β3* receptors were localized very similarly to 4β2 receptors in our previous studies with primary neurons; we noted uniform localization in the soma, suggestive of endoplasmic reticulum, and dendritic localization (Fig. 5A, arrow) and an absence of localization in axons. A high-magnification micrograph demonstrates the dendritic localization of these putative 4β2β3 receptors (Fig. 5A, right). In cells coexpressing 4/β2/β3Y with soluble CFP [to mark total cell morphology, similar to Nashmi et al. (2003)], β3 subunits did not traffic to a subregion of the cell interior likely to be axons (data not shown). To more directly determine whether β3* receptors could be localized to axons in these neurons, we coexpressed 4β2β3Y receptors with a CFP-labeled axonal marker, tau. The tau-CFP decorated axons in hippocampal neurons, with proximal (relative to the cell body) portions of the axon being labeled more strongly than distal portions (Fig. 5B). In all cells examined, we noted the presence of YFP-labeled β3 subunits in these proximal axons but not distal axons (Fig. 5B, arrow). These data in differentiated N2a cells and primary neurons suggest that β3 assembles efficiently with 4β2 receptors, and it is thus cotrafficked and targeted to distal sites in neurons.
Because 6-YFP* receptors do not function in our hands, we wanted to determine whether this is due to a subtle trafficking defect that could prevent the correct delivery of 6-YFP to the plasma membrane. Although we could readily detect 6 fluorescence in the cell body of undifferentiated N2a cells, we wanted to further probe the cellular trafficking of 6* receptors by expressing them in differentiated N2a cells that contain processes. To evaluate the subcellular localization of 6* receptors, we expressed 6-YFP with β2 in differentiated N2a cells. To our surprise, we found that 6β2 receptors were trafficked to neuronal processes in a manner analogous to 4β2 and 4β2β3 receptors (Fig. 4D, arrow). To further address this question, we expressed 6-YFPβ2 receptors in rat hippocampal neurons as described for 4β2β3-YFP. We observed a localization pattern for 6-YFP that was very similar to 4β2β3-YFP. These receptors were well expressed in the cell soma, but they were readily detectable in dendrites as well (Fig. 5A, arrow). In experiments with coexpressed soluble CFP and 6-YFPβ2 receptors, 6 subunits were not detected in putative axons (data not shown). In tau-CFP/6-YFPβ2 coexpression experiments, 6 subunits (similar to 4β2 but not 4β2β3 receptors) were not detected in tau-labeled axons (Fig. 5B, arrow). These data indicate that, although 6* receptors produce little or no agonist-induced conductance in mammalian tissue culture cells, they are expressed well and trafficked similarly compared with 4β2 and β3* receptors.
FRET Revealed Assembly of 6 and β3 Subunits into nAChR Pentamers. The fact that 4/β2/β3 and 6/β2 subunits are colocalized in the cell body and cotargeted to processes and dendrites in neurons suggests that they are assembled into pentameric receptors. The question of receptor assembly is often answered by simply measuring agonist-induced conductance increases in cells expressing a subunit combination of interest, or by applying a selective agonist or inhibitor to a pure receptor population of known pharmacological properties. This approach is not applicable, however, for 6* and β3* receptors. 6* receptors do not function well in heterologous expression systems, so it is not straightforward to determine the extent to which free 6 subunits assemble into pentameric receptors. Similarly for β3, although it is functional in oocytes (Fig. 2), there are no pharmacological probes that can be applied to β3* receptors to study their assembly or subunit composition. Others have indirectly measured receptor assembly of nicotinic subunits by using biochemical techniques such as immunoprecipitation (Zoli et al., 2002; Champtiaux et al., 2003) and centrifugation (Kuryatov et al., 2000) or by forcing subunits to assemble by using molecular concatamers (Tapia et al., 2007). To directly determine whether two nicotinic receptor subunits interact and, possibly, assemble to form pentameric receptors, we have used FRET coupled with our CFP- and YFP-tagged receptors. In the context of our nicotinic receptor subunits labeled with YFP or CFP in the M3-M4 loop, only subunits that interact will undergo FRET, because FRET occurs only when donors and acceptors are within 100 Å. Furthermore, we previously demonstrated that the efficiency of FRET directly correlates with the number of functional, plasma membrane-localized pentameric receptors (Nashmi et al., 2003). To measure FRET between subunits, we used the acceptor photobleaching method (Nashmi et al., 2003). In this method, we measure CFP dequenching during incremental photodestruction of YFP. CFP was excited at 439 nm, whereas YFP was bleached at 514 nm (Fig. 6A). Because the emission spectra for CFP and YFP overlap significantly, we imaged using a confocal microscope with spectral imaging capabilities along with a linear unmixing algorithm (described under Materials and Methods).
Fig. 6. FRET reveals assembly of β3 and 6 nAChR subunits with 4 and β2. A, Nicotinic receptor FRET schematic diagram. Gray cylinders indicate nAChR subunits, whereas cyan or yellow cylinders attached to nAChR subunits indicate CFP or YFP, respectively. FRET between nAChR subunits used the acceptor photobleaching method. When subunits are assembled, 439-nm excitation of CFP (donor) results in some emission of CFP at 485 nm, and some nonradiative transfer of energy (FRET) to YFP (acceptor) and resulting in emission at 535 nm. Acceptor photobleaching reveals FRET by measuring incremental dequenching of CFP during photodestruction of YFP with high-power 514-nm excitation. B, β3 assembles with β2 in the presence of 4. N2a cells expressing 4β2-CFPβ3-YFP receptors were imaged live for FRET. The 439-nm laser line was coupled to a confocal microscope equipped with spectral imaging capabilities; this instrument generated spectral images before and after photodestruction of YFP using the 514-nm laser. Specific CFP and YFP signals were generated with linear unmixing as described under Materials and Methods. YFP and CFP intensity throughout the cell before and after YFP photodestruction is shown using intensity scaling. C, plot of β3-YFP and β2-CFP intensity during incremental photodestruction of β3-YFP. Normalized data from a representative cell were fitted to an exponential decay. D, β3 assembles with 4 in the presence of β2. N2a cells expressing 4-CFPβ2β3-YFP were imaged live for FRET as described in B. E, plot of β3-YFP and 4-CFP intensity during incremental photodestruction of β3-YFP, similar to C. F, 6 assembles with β2. N2a cells expressing 6-YFPβ2-CFP were imaged live for FRET as described in B. G, plot of 6-YFP and β2-CFP intensity during incremental photodestruction of 6-YFP, similar to C. Scale bar, 10 µm.
Fluorescent 4 and β2 subunits are functional and undergo robust FRET in mammalian cells (Nashmi et al., 2003), so we used these subunits in our acceptor photobleaching assay with XFP-tagged β3 and 6. We expressed β3-YFP with untagged 4 and β2-CFP in N2a cells, followed by live cell FRET imaging. We recorded the whole-cell fluorescence intensity for β3-YFP and β2-CFP before and after photobleaching of YFP with the 514-nm laser, and we expressed with pseudocolor intensity scaling (Fig. 6B). In this experiment, β2-CFP was clearly dequenched after β3-YFP photodestruction (Fig. 6B), indicating that the two subunits had been undergoing FRET. In a similar experiment, we recorded multiple spectral images at several time points during YFP photodestruction. This revealed a corresponding increase in CFP intensity (Fig. 6C). A reciprocal experiment was also done, where β3-YFP was coexpressed with 4-CFP and untagged β2. We recorded a similar dequenching for 4-CFP after YFP photobleaching (Fig. 6, D and E), indicating FRET between these subunits as well. Both for β2/β3 and 4/β3 FRET, we found no difference between FRET inside the cell versus FRET at the cell periphery at or near the plasma membrane. These results directly demonstrate that β3 is able to assemble with 4β2 receptors in neuronal cells. This assembly likely occurs in the endoplasmic reticulum, which is consistent with previous findings (Nashmi et al., 2003).
There are many different putative 6* receptor subtypes in brain, including 6β2, 6β2β3, 64β2, and 64β2β3 (Salminen et al., 2007). To begin to study 6* receptor assembly, we measured FRET between 6-YFP and β2-CFP. In response to YFP bleaching, we recorded a robust dequenching of β2-CFP throughout the cell, indicating FRET between these subunits (Fig. 6, F and G). The pattern of localization and FRET pattern was identical to 4β2β3 receptors.
To further quantify FRET between 4/β2 subunits and β3 or 6, we measured FRET E values for various receptor subtypes. 4-CFPβ2-YFP, 4β2-CFPβ3-YFP, and 4-CFPβ2β3-YFP receptors were expressed in N2a cells followed by acceptor photobleaching FRET (Fig. 7A). We acquired spectral images with 439-nm laser excitation before and during incremental photobleaching of YFP-labeled subunits, followed by extraction of true CFP and YFP image data using linear unmixing (see Materials and Methods). A scatterplot of CFP intensity in response to YFP photobleaching reveals FRET between the subunits in question (Fig. 7B) when the slope of the linear regression line is >0. This slope was used to calculate FRET efficiency values, which were expressed as bar graphs (Fig. 7C) or listed (Table 1). As shown qualitatively in Fig. 6, significant FRET occurred in all nAChR pentamer conditions. We noted a higher FRET E for 4C/β2Y than for β3Y with either β2C or 4C (Y, YFP; C, CFP). To assess the specificity of this measurement, we also measured FRET between β3 and a non-nAChR, CFP-labeled protein, mGAT1. GAT1 is also a multipass transmembrane protein with a CFP-tag at its C terminus, which faces the cytoplasm. This protein is mainly localized to the endoplasmic reticulum (data not shown). These two points are important, because it was critical for a specificity probe to have 1) the same membrane topology as our labeled nicotinic receptors, with respect to the attached fluorophore; and 2) the same subcellular localization such that they are capable of interacting with each other. In N2a cells expressing β3-YFP and mGAT1-CFP, we could not detect any FRET between these proteins (Table 1; Fig. 7C). In an even more rigorous test, we assessed FRET between β3-YFP and another Cys-loop receptor labeled in the M3-M4 loop, the CFP-labeled GluCl β subunit (Slimko et al., 2002; Nashmi et al., 2003). FRET between β3 and the GluCl β subunit was significantly smaller (FRET E = 6 ± 4%) than for 4 or β2 nAChR subunits (Table 1; Fig. 7C). Thus, our FRET results between β3 and other labeled nAChR subunits cannot be explained by random collision or interaction with unassembled subunits. Finally, we were interested in whether subtle changes in the location of the fluorophore within the β3 M3-M4 loop could influence its ability to undergo FRET with another subunit. FRET E decreases strongly with the distance between fluorophores. We reasoned that changes in the insertion point of YFP in β3, while holding the position of CFP in β2 constant, might alter FRET between these two subunits. To address this, we compared the FRET E between β2-CFP and two different β3-YFP constructs, β3-YFPP379 and β3-YFPG367, which have different insertion points for YFP within the M3-M4 loop. To our surprise, there was no change in the FRET E for these two subunits (Table 1; Fig. 7D).
Fig. 7. β3 specifically assembles with 4β2 nAChRs. A, fluorescently labeled nicotinic receptor pentamers assayed for FRET in this experiment. N2a cells expressing the indicated receptor pentamers were assayed live for FRET using the acceptor photobleaching method. B, linear plots of donor (CFP) dequenching versus acceptor (YFP) photodestruction for nAChRs with the indicated fluorescent subunits. FRET efficiency was calculated by extrapolating linear regression plots to 100% YFP photodestruction as described under Materials and Methods. C, specific FRET signal detected between β3 and 4 or β2. The FRET efficiency for the given donor-acceptor pair was calculated from the linear plot shown in B as described under Materials and Methods. FRET between β3-YFP and mGAT1-CFP or GluCl β-CFP was measured as a specificity control. D, FRET efficiency for β3-YFP does not depend on the insertion site in the M3-M4 loop. Two β3-YFP constructs, β3-YFPP379 and β3-YFPG367, were compared for their ability to assemble with 4β2-CFP as measured by FRET. FRET efficiency was calculated as described in C. Error bars are ± S.E.M., and n = 5 to 15 cells for each condition. ***, p < 0.001; **, p < 0.01.
We quantitatively measured FRET between 6 and 4/β2 subunits as well. We expressed either 6Yβ2C or 6Y4Cβ2 in N2a cells to measure FRET (Fig. 8A). The latter receptor was studied because recent work indicates that nAChR receptors containing both 6 and 4 1) exist and are functional in mouse brain tissue (Salminen et al., 2007), and 2) are both necessary to form the nAChR subtype with the highest affinity for nicotine yet reported in a functional assay (Salminen et al., 2007). Acceptor photobleaching FRET experiments reveal robust CFP dequenching in response to YFP photobleach for both of these receptor subtypes, indicating FRET (Fig. 8B). Similar to β3-YFP* receptors, we measured FRET E values for these two subtypes, and we found a FRET E of 36.0 ± 2.4% for 6Yβ2C and 21.9 ± 1.1% for 6Y4Cβ2 (Table 1; Fig. 8C). We also assessed the specificity of our FRET measurements for 6 by measuring FRET between 6-YFP and mGAT1-CFP as described above for Fig. 7. Similar to β3 and mGAT1, we could record no significant FRET between 6 and mGAT1 (Table 1; Fig. 8C). FRET experiments between 6 and the GluCl β subunit, the most rigorous test conducted, yielded a small FRET signal (FRET E = 14 ± 2%) (Table 1; Fig. 8C). Because these subunits presumably do not form functional channels, there may be a small distortion of our 6 FRET signals that is due to partially assembled receptors. Because this signal is significantly smaller than for all other 6 combinations, FRET between subunits in pentameric receptors remains the most plausible explanation for the energy transfer we observe for 6. Finally, we studied FRET between β2-CFP and three 6-YFP constructs (6-YFPA405, 6-YFPG387, and 6-YFPG366) that differed only in their insertion point for YFP within the M3-M4 loop. Again, we were surprised to find no significant difference in FRET E between these three 6 constructs (Table 1; Fig. 8D).
Fig. 8. 6 specifically assembles with 4 and β2 nAChR subunits. A, fluorescently labeled nicotinic receptor pentamers assayed for FRET in this experiment. N2a cells expressing the indicated receptor pentamers were assayed live for FRET using the acceptor photobleaching method. B, linear plots of donor (CFP) dequenching versus acceptor (YFP) photodestruction for nAChRs with the indicated fluorescent subunits. FRET efficiency was calculated by extrapolating linear regression plots to 100% YFP photodestruction as described under Materials and Methods. C, specific FRET signal detected between 6 and 4 or β2. The FRET efficiency for the given donor-acceptor pair was calculated from the linear plot shown in B as described under Materials and Methods. FRET between 6-YFP and mGAT1-CFP or GluCl β-CFP was measured as a specificity control. D, FRET efficiency for 6-YFP does not depend on the insertion site in the M3-M4 loop. Three 6-YFP constructs, 6-YFPA405, 6-YFPG387, and 6-YFPG366, were compared for their ability to assemble with β2-CFP as measured by FRET. FRET efficiency was calculated as described in C. Error bars are ± S.E.M., and n = 5 to 15 cells for each condition. ***, p < 0.001; **, p < 0.01.
Several results described above gave us confidence that our XFP-labeled β3 and 6 constructs were performing as expected. After confirming that these subunits assemble and traffic normally when expressed independently of each other, we used these constructs together to study 6β2β3 nAChRs. This receptor represents a modest population of the total striatal nAChR pool, and it contributes to nicotine-stimulated dopamine release (Salminen et al., 2007). 6β2β3 receptors, where one subunit is untagged and the remaining subunits are either YFP- or CFP-tagged (6Yβ2Cβ3, 6Yβ2β3C, and 6β2Yβ3C), were expressed in N2a cells (Fig. 9A). We measured robust donor dequenching for all receptor subtypes (Fig. 9B), which was confirmed with FRET E measurements (Table 1; Fig. 9C). Thus, aside from 6 functional measurements, we conclude that XFP-labeled 6 and β3 subunits exhibit normal subcellular trafficking and assembly compared with our well characterized fluorescent 4 and β2 subunits.
Fig. 9. FRET reveals assembly of 6β2β3 nAChRs. A, fluorescently labeled nicotinic receptor pentamers assayed for FRET in this experiment. N2a cells expressing the indicated receptor pentamers were assayed live for FRET using the acceptor photobleaching method. B, linear plots of donor (CFP) dequenching versus acceptor (YFP) photodestruction for nAChRs with the indicated fluorescent subunits. FRET efficiency was calculated by extrapolating linear regression plots to 100% YFP photodestruction as described under Materials and Methods. C, specific FRET signal detected between 6-YFP and β2-CFP with β3 present, 6-YFP and β3-CFP with β2 present, and β2-YFP and β3-CFP with 6 present. The FRET efficiency for the given donor-acceptor pair was calculated from the linear plot shown in B as described under Materials and Methods. Error bars are ± S.E.M., and n = 10 to 15 cells for each condition.
6 and β3 Subunit Stoichiometry Probed with FRET. Having established that fluorescently labeled 6 and β3 are functional (β3 only), have a reasonable subcellular localization pattern, and assemble into nicotinic receptor pentamers with other subunits, we used these tools to probe an important question facing the nicotinic receptor field: subunit stoichiometry. A variety of creative approaches have been taken to understand subunit stoichiometry, including immunoprecipitation (Zoli et al., 2002; Champtiaux et al., 2003), density gradient centrifugation (Kuryatov et al., 2000), molecular concatamers/linked subunits (Tapia et al., 2007), reporter mutations (Boorman et al., 2000), and mouse genetic approaches (Gotti et al., 2005a; Salminen et al., 2007). We now use FRET to address the problem of subunit stoichiometry because FRET occurs only when subunits are directly interacting, and often assembled, with one another.
We have previously shown that FRET efficiency correlates directly with functional receptor pentamers (Nashmi et al., 2003). To examine the number of 6 and β3 subunits in a nicotinic receptor pentamer, we first used FRET to examine the stoichiometry of a well studied receptor, namely, 4β2 receptors. It is widely accepted that 4 and β2 subunits assemble to form both high-sensitivity (HS) and low-sensitivity (LS) receptors. Cells often produce a mixture of these two receptors (Buisson and Bertrand, 2001; Nashmi et al., 2003), although they can be induced to express a pure population of one or the other (Nelson et al., 2003; Briggs et al., 2006). The subunit stoichiometry of HS receptors is postulated to be (4)2(β2)3, whereas the LS receptors is thought to be (4)3(β2)2 (Nelson et al., 2003). Regardless of the fraction of HS and LS receptors, we took advantage of the fact that all 4β2 receptors presumably contain two or more 4 and two or more β2 subunits. We reasoned that when cells express 4-YFP and 4-CFP along with β2 (Fig. 10A), a fraction of the receptors will contain both YFP- and CFP-labeled 4 subunits, and they will therefore be detectable by FRET. Confirming this hypothesis, we did detect modest dequenching of 4-CFP upon incremental 4-YFP photobleaching (Fig. 10B). FRET E for 4Y4Cβ2 receptors was 22.2 ± 2.3% (Table 1; Fig. 10C). We also conducted a similar experiment with β2, and we found a modest FRET signal (FRET E = 16.3 ± 1.7%) between β2-YFP and β2-CFP within the same pentamer (Table 1; Fig. 10, B and C). We next used this assay to determine whether 6* and β3* receptors have one or more than one 6 or β3 subunit per pentamer. N2a cells expressing either 6Y6Cβ2 or 4β2β3Yβ3C receptors were analyzed for FRET (Fig. 10A). We measured a strong FRET signal between 6-YFP and 6-CFP in donor dequenching (Fig. 10B), corresponding to a robust FRET E of 27.8 ± 1.7% (Table 1; Fig. 10C). Thus, these data are the first to directly demonstrate that 6* receptors are capable of containing at least two 6 subunits, similar to other subunits such as 3 and 4. In contrast to 6, β3 is thought to be an "ancillary subunit", only able to incorporate into nAChRs with other and β subunits (Groot-Kormelink et al., 1998). We could detect little or no FRET between β3-YFP and β3-CFP (FRET E = 2.6 ± 1.3%) (Table 1; Fig. 10, B and C). This was a specific result, because β3-YFP and β3-CFP were able to FRET with other subunits (Figs. 7 and 9), thus ruling out the notion that one of these subunits is not able to undergo FRET. These data are the first to directly demonstrate that receptors containing β3 subunits are only able to incorporate a single copy of this subunit. We interpret this to mean that β3 incorporates into the "accessory" position in a nAChR pentamer (Tumkosit et al., 2006), and it likely does not contribute to either of the two :non- interfaces that form the ligand-binding sites.
Fig. 10. β3 and 6 subunit stoichiometry studied by FRET. A, fluorescently labeled nicotinic receptor pentamers assayed for FRET in this experiment. N2a cells expressing the indicated receptor pentamers were assayed live for FRET using the acceptor photobleaching method. B, linear plots of donor (CFP) dequenching versus acceptor (YFP) photodestruction for nAChRs with the indicated fluorescent subunits. FRET efficiency was calculated by extrapolating linear regression plots to 100% YFP photodestruction as described under Materials and Methods. C, 6-containing receptors have multiple 6 subunits whereas β3-containing receptors have only one β3 subunit. The FRET efficiency for the given donor-acceptor pair was calculated from the linear plot shown in B as described under Materials and Methods. D to F, linear plots of donor (CFP) dequenching versus acceptor (YFP) photodestruction for nAChRs with the indicated fluorescent subunits. FRET efficiency was calculated by extrapolating linear regression plots to 100% YFP photodestruction as described under Materials and Methods.G, β3 coexpression with 4β2 or 6β2 receptors reduces FRET between YFP- and CFP-labeled subunits. FRET E value for a given subunit combination was calculated from the linear plot in D, E, or F. Error bars are ± S.E.M., and n = 10 to 15 cells for each condition. ***, p < 0.001; *, p < 0.05.
After confirming via FRET that β3 incorporates into nAChRs at a frequency of one subunit per pentamer, we used β3 coexpression to further probe the subunit stoichiometry of 4* and 6* receptors. We coexpresssed β3-WT with 4-YFP, 4-CFP, and β2 such that β3 was in excess. In this experiment, β3 is incorporated into 4-XFPβ2 receptors and will displace either an 4 or β2 subunit. There was a significant decline in FRET for cells expressing 4Y4Cβ2β3 receptors versus those expressing 4Y4Cβ2 (Table 1; Fig. 10, D and G). We interpret this result to mean that β3 incorporation has fixed the subunit stoichiometry of FRET-competent receptors to (4Y)1(4C)1(β2)2(β3)1 versus the following mixture of FRET-competent receptors without β3: (4Y)2- (4C)1(β2)2,(4Y)1(4C)2(β2)2 and (4Y)1(4C)1(β2)3. A reduction in FRET for two XFP-labeled 4 subunits (YFP and CFP) versus three is reasonable and expected based on the work of others (Corry et al., 2005), and on our calculations that predict the relative FRET efficiencies in pentamers with XFP-labeled subunits (data not shown). Thus, β3 incorporation into nAChR pentamers likely displaces one subunit, and results in a decrease in 4 to 4 FRET for pentamers with a mixed subunit stoichiometry.
Fig. 11. Distinct plasma membrane localization for 4β2 versus β3* and 6* receptors. A, plasma membrane localization of 4β2 receptors. N2a cells plated on polyethylenimine, expressing 4-YFPβ2 receptors were imaged live under TIRF illumination. Arrows indicate 4β2 receptors in distal parts of membrane protrusions. Epifluorescence (non-TIRF) images and bright-field images are shown for reference. Scale bars, 10 µm. B, filopodia in N2
【参考文献】
Azam L, Chen Y, and Leslie FM (2007) Developmental regulation of nicotinic acetylcholine receptors within midbrain dopamine neurons. Neuroscience 144: 1347-1360.
Bezprozvanny I, Scheller RH, and Tsien RW (1995) Functional impact of syntaxin on gating of N-type and Q-type calcium channels. Nature 378: 623-626.
Bierut LJ, Madden PA, Breslau N, Johnson EO, Hatsukami D, Pomerleau OF, Swan GE, Rutter J, Bertelsen S, Fox L, et al. (2007) Novel genes identified in a high-density genome wide association study for nicotine dependence. Hum Mol Genet 16: 24-35.[Abstract/Free Full Text]
Boorman JP, Beato M, Groot-Kormelink PJ, Broadbent SD, and Sivilotti LG (2003) The effects of β3 subunit incorporation on the pharmacology and single channel properties of oocyte-expressed human 3β4 neuronal nicotinic receptors. J Biol Chem 278: 44033-44040.[Abstract/Free Full Text]
Boorman JP, Groot-Kormelink PJ, and Sivilotti LG (2000) Stoichiometry of human recombinant neuronal nicotinic receptors containing the β3 subunit expressed in Xenopus oocytes. J Physiol 529: 565-577.[Abstract/Free Full Text]
Briggs CA, Gubbins EJ, Marks MJ, Putman CB, Thimmapaya R, Meyer MD, and Surowy CS (2006) Untranslated region-dependent exclusive expression of high-sensitivity subforms of 4β2 and 3β2 nicotinic acetylcholine receptors. Mol Pharmacol 70: 227-240.[Abstract/Free Full Text]
Broadbent S, Groot-Kormelink PJ, Krashia PA, Harkness PC, Millar NS, Beato M, and Sivilotti LG (2006) Incorporation of the β3 subunit has a dominant-negative effect on the function of recombinant central-type neuronal nicotinic receptors. Mol Pharmacol 70: 1350-1357.[Abstract/Free Full Text]
Buisson B and Bertrand D (2001) Chronic exposure to nicotine upregulates the human 4β2 nicotinic acetylcholine receptor function. J Neurosci 21: 1819-1829.[Abstract/Free Full Text]
Champtiaux N, Gotti C, Cordero-Erausquin M, David DJ, Przybylski C, Lena C, Clementi F, Moretti M, Rossi FM, Le Novere N, et al. (2003) Subunit composition of functional nicotinic receptors in dopaminergic neurons investigated with knockout mice. J Neurosci 23: 7820-7829.[Abstract/Free Full Text]
Champtiaux N, Han ZY, Bessis A, Rossi FM, Zoli M, Marubio L, McIntosh JM, and Changeux JP (2002) Distribution and pharmacology of 6-containing nicotinic acetylcholine receptors analyzed with mutant mice. J Neurosci 22: 1208-1217.[Abstract/Free Full Text]
Corry B, Jayatilaka D, and Rigby P (2005) A flexible approach to the calculation of resonance energy transfer efficiency between multiple donors and acceptors in complex geometries. Biophys J 89: 3822-3836.
Cui C, Booker TK, Allen RS, Grady SR, Whiteaker P, Marks MJ, Salminen O, Tritto T, Butt CM, Allen WR, et al. (2003) The β3 nicotinic receptor subunit: a component of -conotoxin MII-binding nicotinic acetylcholine receptors that modulate dopamine release and related behaviors. J Neurosci 23: 11045-11053.[Abstract/Free Full Text]
Drenan RM, Doupnik CA, Boyle MP, Muglia LJ, Huettner JE, Linder ME, and Blumer KJ (2005) Palmitoylation regulates plasma membrane-nuclear shuttling of R7BP, a novel membrane anchor for the RGS7 family. J Cell Biol 169: 623-633.[Abstract/Free Full Text]
Fowler MJ, Flaskos J, McLean WG, and Hargreaves AJ (2001) Effects of neuropathic and non-neuropathic isomers of tricresyl phosphate and their microsomal activation on the production of axon-like processes by differentiating mouse N2a neuroblastoma cells. J Neurochem 76: 671-678.
Gotti C, Moretti M, Clementi F, Riganti L, McIntosh JM, Collins AC, Marks MJ, and Whiteaker P (2005a) Expression of nigrostriatal 6-containing nicotinic acetylcholine receptors is selectively reduced, but not eliminated, by β3 subunit gene deletion. Mol Pharmacol 67: 2007-2015.[Abstract/Free Full Text]
Gotti C, Moretti M, Zanardi A, Gaimarri A, Champtiaux N, Changeux JP, Whiteaker P, Marks MJ, Clementi F, and Zoli M (2005b) Heterogeneity and selective targeting of neuronal nicotinic acetylcholine receptor (nAChR) subtypes expressed on retinal afferents of the superior colliculus and lateral geniculate nucleus: identification of a new native nAChR subtype 3β2(5 or β3) enriched in retinocollicular afferents. Mol Pharmacol 68: 1162-1171.[Abstract/Free Full Text]
Grady SR, Murphy KL, Cao J, Marks MJ, McIntosh JM, and Collins AC (2002) Characterization of nicotinic agonist-induced [3H]dopamine release from synaptosomes prepared from four mouse brain regions. J Pharmacol Exp Ther 301: 651-660.[Abstract/Free Full Text]
Graham AJ, Martin-Ruiz CM, Teaktong T, Ray MA, and Court JA (2002) Human brain nicotinic receptors, their distribution and participation in neuropsychiatric disorders. Curr Drug Targets CNS Neurol Disord 1: 387-397.
Grailhe R, de Carvalho LP, Paas Y, Le Poupon C, Soudant M, Bregestovski P, Changeux JP, and Corringer PJ (2004) Distinct subcellular targeting of fluorescent nicotinic 3β4 and serotoninergic 5-HT3A receptors in hippocampal neurons. Eur J Neurosci 19: 855-862.
Groot-Kormelink PJ, Luyten WH, Colquhoun D, and Sivilotti LG (1998) A reporter mutation approach shows incorporation of the "orphan" subunit β3 into a functional nicotinic receptor. J Biol Chem 273: 15317-15320.[Abstract/Free Full Text]
Kuryatov A, Olale F, Cooper J, Choi C, and Lindstrom J (2000) Human 6 AChR subtypes: subunit composition, assembly, and pharmacological responses. Neuropharmacology 39: 2570-2590.
Léna C, de Kerchove D'Exaerde A, Cordero-Erausquin M, Le Novere N, del Mar Arroyo-Jimenez M, and Changeux JP (1999) Diversity and distribution of nicotinic acetylcholine receptors in the locus ceruleus neurons. Proc Natl Acad Sci U S A 96: 12126-12131.[Abstract/Free Full Text]
Marks MJ, Burch JB, and Collins AC (1983) Effects of chronic nicotine infusion on tolerance development and nicotinic receptors. J Pharmacol Exp Ther 226: 817-825.[Free Full Text]
Miwa JM, Ibanez-Tallon I, Crabtree GW, Sanchez R, Sali A, Role LW, and Heintz N (1999) lynx1, an endogenous toxin-like modulator of nicotinic acetylcholine receptors in the mammalian CNS. Neuron 23: 105-114.
Miyazawa A, Fujiyoshi Y, Stowell M, and Unwin N (1999) Nicotinic acetylcholine receptor at 4.6 angstrom resolution: transverse tunnels in the channel wall. J Mol Biol 288: 765-786.
Nashmi R, Dickinson ME, McKinney S, Jareb M, Labarca C, Fraser SE, and Lester HA (2003) Assembly of 4β2 nicotinic acetylcholine receptors assessed with functional fluorescently labeled subunits: effects of localization, trafficking, and nicotine-induced upregulation in clonal mammalian cells and in cultured midbrain neurons. J Neurosci 23: 11554-11567.[Abstract/Free Full Text]
Nashmi R, Xiao C, Deshpande P, McKinney S, Grady SR, Whiteaker P, Huang Q, McClure-Begley T, Lindstrom JM, Labarca C, et al. (2007) Chronic nicotine cell specifically upregulates functional alpha4* nicotinic receptors: basis for both tolerance in midbrain and enhanced long-term potentiation in perforant path. J Neurosci 27: 8202-8218.[Abstract/Free Full Text]
Nelson ME, Kuryatov A, Choi CH, Zhou Y, and Lindstrom J (2003) Alternate stoichiometries of 4β2 nicotinic acetylcholine receptors. Mol Pharmacol 63: 332-341.[Abstract/Free Full Text]
Ohara-Imaizumi M, Nishiwaki C, Nakamichi Y, Kikuta T, Nagai S, and Nagamatsu S (2004) Correlation of syntaxin-1 and SNAP-25 clusters with docking and fusion of insulin granules analysed by total internal reflection fluorescence microscopy. Diabetologia 47: 2200-2207.
Quick M and Lester HA (1994) Methods for expression of excitability proteins in Xenopus oocytes, in Ion Channels of Excitable Cells (Narahashi T ed) pp 261-279, Academic Press, San Diego, CA.
Quik M and McIntosh JM (2006) Striatal 6* nicotinic acetylcholine receptors: potential targets for Parkinson's disease therapy. J Pharmacol Exp Ther 316: 481-489.[Abstract/Free Full Text]
Rowell P (2002) Effects of nicotine on dopaminergic neurotransmission, in Nicotinic Receptors in the Nervous System (Levin ED ed) pp 51-80, CRC, Boca Raton, FL.
Salminen O, Drapeau JA, McIntosh JM, Collins AC, Marks MJ, and Grady SR (2007) Pharmacology of -Conotoxin MII-Sensitive Subtypes of Nicotinic Acetylcholine Receptors Isolated by breeding of null mutant mice. Mol Pharmacol 71: 1563-1571.[Abstract/Free Full Text]
Slimko EM, McKinney S, Anderson DJ, Davidson N, and Lester HA (2002) Selective electrical silencing of mammalian neurons in vitro by the use of invertebrate ligand-gated chloride channels. J Neurosci 22: 7373-7379.[Abstract/Free Full Text]
Tapia L, Kuryatov A, and Lindstrom J (2007) Ca2+ permeability of the (4)3(β2)2 stoichiometry greatly exceeds that of (4)2(β2)3 human acetylcholine receptors. Mol Pharmacol 71: 769-776.[Abstract/Free Full Text]
Tumkosit P, Kuryatov A, Luo J, and Lindstrom J (2006) β3 subunits promote expression and nicotine-induced up-regulation of human nicotinic 6* nicotinic acetylcholine receptors expressed in transfected cell lines. Mol Pharmacol 70: 1358-1368.[Abstract/Free Full Text]
Whiteaker P, McIntosh JM, Luo S, Collins AC, and Marks MJ (2000) [125I]--Conotoxin MII identifies a novel nicotinic acetylcholine receptor population in mouse brain. Mol Pharmacol 57: 913-925.[Abstract/Free Full Text]
Xu J, Zhu Y, and Heinemann SF (2006) Identification of sequence motifs that target neuronal nicotinic receptors to dendrites and axons. J Neurosci 26: 9780-9793.[Abstract/Free Full Text]
Zheng JQ, Felder M, Connor JA, and Poo MM (1994) Turning of nerve growth cones induced by neurotransmitters. Nature 368: 140-144.
Zoli M, Moretti M, Zanardi A, McIntosh JM, Clementi F, and Gotti C (2002) Identification of the nicotinic receptor subtypes expressed on dopaminergic terminals in the rat striatum. J Neurosci 22: 8785-8789.[Abstract/Free Full Text]
作者单位：Division of Biology, California Institute of Technology, Pasadena, California