EXOCYTIC TRAFFICKING IS REQUIRED FOR NICOTINE-INDUCEDUPREGULATION OF alpha4beta2 NICOTINIC ACETYLCHOLINE

RECEPTORSTamara Darsow, T.K. Booker, Juan Carlos Piña-Crespo, and Stephen F. Heinemann

From Molecular Neurobiology Laboratories, The Salk Institute for Biological Studies, LaJolla, California 92037

Running Title: nAChR upregulation requires exocytic traffickingAddress correspondence to: Tamara Darsow, Molecular Neurobiology Laboratories, The SalkInstitute for Biological Studies, La Jolla, CA 92037, Tel. (858) 453-4100 x1172; Fax (858) 453-0852; Email: darsow@salk.edu

The primary target for nicotine in thebrain is the neuronal nicotinic acetylcholinereceptor (nAChR). It has been welldocumented that nAChRs respond to chronicnicotine exposure by upregulation of receptornumbers, which may underlie some aspects ofnicotine addiction. In order to investigate themechanism of nicotine-induced nAChRupregulation, we have developed a cell culturesystem to assess membrane trafficking andnicotine-induced upregulation of surfaceexpressed alpha4beta2 nAChRs. Previousreports have implicated stabilization of thenAChRs at the plasma membrane as thepotential mechanism of upregulation. We havefound that while nicotine exposure results inupregulation of surface receptors in our system,it does not alter surface receptor internalizationfrom the plasma membrane, post-endocytictrafficking, or lysosomal degradation. Instead,we find that transport of nAChRs through thesecretory pathway to the plasma membrane isrequired for nicotine-induced upregulation ofsurface receptors. Therefore, nicotine appearsto regulate surface receptor levels at a stepprior to initial insertion in the plasmamembrane, rather than by altering theirendocytic trafficking or degradation rates ashad been previously suggested.

INTRODUCTION

Neuronal nicotinic acetylcholine receptors(nAChRs) in mammalian brain compose a familyof proteins encoded by eleven genes, ( alpha2-7, 9-10, and beta2-4) that assemble into pentamericligand-gated ion channels (1-4). Although allcombinations of subunits that form functionalnicotinic receptors can bind nicotine, alpha4 andbeta2 containing receptors form the high affinitynicotine binding site (5-9) and are thereforethought to be the primary nAChR subtype affected

by the relatively low nicotine concentrations (100-500 nM) found in the blood of smokers (10).

Chronic exposure of alpha4beta2 receptorsto nicotine, as occurs in smokers, results initiallyin receptor desensitization, followed bysubsequent upregulation of high affinity nicotinebinding sites (11). Upon nicotine withdrawal, theincreased number of nAChRs recover fromdesensitization, resulting in excess activity in thenAChR system. It has been proposed that thiscycle of nicotine-induced receptor upregulationmay contribute to the negative symptomsassociated with nicotine withdrawal, resulting incontinued tobacco consumption and eventually,nicotine dependence (12).

The mechanisms and cellular machineryrequired for nicotine induced upregulation remainunknown. However a large body of researchsupports a consensus on several relevant points.First, upregulation is observed in the brains ofhuman smokers (13) as well as in chronicallynicotine-treated animal models (8,14) and incultured cells heterologously expressing nAChRs(15-18), suggesting that upregulation requiresbasic, conserved cellular processes. It has beenconvincingly shown that increased nicotinebinding reflects an increase in receptor numberrather than receptor affinity (15,19-21) implyingthat upregulation involves receptor proteindynamics rather than intrinsic changes in theexisting receptors. Finally, chronic nicotinetreatment does not increase nAChR subunitmRNA levels (14), making transcriptionalregulation unlikely.

The functionally relevant pool of nAChRsin neuronal cells exists at the plasma membrane,where exposure to neurotransmitter regulatesmembrane excitability. In many cases, cellsurface receptor activity is regulated by insertionand removal of receptors at the plasma membrane(22). For example, it is becoming increasinglyclear that modulation of neuronal synaptictransmission is regulated at least in part by the

JBC Papers in Press. Published on March 1, 2005 as Manuscript M501157200

Copyright 2005 by The American Society for Biochemistry and Molecular Biology, Inc.

dynamic localization and turnover ofneurotransmitter receptors (23,24). Furthermore,ligand-induced opiate receptor endocytosis hasbeen shown to be a determinant of the addictivepotential of opiate agonists (25). Therefore,mechanisms dictating the number of surfacereceptors at the plasma membrane may also beinvolved in nicotine-induced upregulation.

To directly test this hypothesis, weanalyzed the upregulation and trafficking ofsurface alpha4beta2 nAChRs using a cell culturemodel that expresses functional surface localizedalpha4beta2 receptors. We show that in thissystem, chronic nicotine treatment induces theupregulation of surface alpha4beta2 nAChRs in amanner that is similar to nAChR upregulationobserved in the brains of human smokers and inanimal models chronically exposed to nicotine.Our analysis of nicotine dependent nAChRtrafficking suggests that upregulation is regulatednot via modulation of endocytic trafficking, but ata biosynthetic step prior to insertion ofalpha4beta2 nAChRs into the plasma membrane.

Materials and Methods

Cell Culture, Transfection and DNAConstructs: Human embryonic kidney cells(HEK293T) were maintained at 37˚C in 5% CO2and passaged in HEK Media (Dulbecco’s modifiedEagles medium (DMEM) (Specialty Media,Phillipsburg, NJ) supplemented with 10% heatinactivated fetal bovine serum (Gibco, GrandIsland, NY) , 2mM L-Glutamine (Specialty Media,Phillipsburg, NJ) and 50 U/ml penicillin andstreptomycin (Gibco, Grand Island, NY)).Transient transfection of cDNA constructs intoHEK293T cells was accomplished by calciumphosphate precipitation (26). Mouse cDNAsencoding the alpha4 and beta2 nAChR subunitswere a generous gift from Dr. Jerry Stitzel(University of Colorado). HA epitope taggedmouse SKD1 and SKD1(E235Q) clones were agift from Dr. Scott Emr (University of California,San Diego) and Dr. Markus Babst (University ofUtah).Electrophysiology: HEK293T cells were co-transfected by calcium phosphate method withseparate plasmids containing alpha4,beta2 andEGFP at a 1:1:0.05 ratio. Whole-cell recordingswere performed at room temperature (RT= 21°C)from EGFP expressing cells 24 to 48 hours aftertransfection. Coverslips were transferred from the

incubator (37 °C, 8% CO2) to a recording chamber(RC-26G; Warner Instruments) fitted to the stageof an upright microscope (Axioscope, Zeiss).Cells were continuously superfused with aHEPES-buffered extracellular solution composedof (mM): 146 NaCl; 2.5 KCl, 1.2 CaCl2, 1 MgCl2,20 mM sucrose, 20 mM glucose, 10 mM Hepes, 1µM atropine, pH 7.4 (adjusted with NaOH), 310mOsm. Patch pipettes were pulled on amicropipette puller (P83, Sutter Instruments) fromborosilicate glass capillaries (GC150F-10, I.D.:0.86, O.D.: 1.50 mm, Harvard Apparatus, UK) andfire polished on a microforge (MF-83, Narishige,Japan) to a final open tip resistance of 3-5 MW.Patch pipettes were filled with a CsCl-basedinternal solution composed of (mM): 140 CsCl, 4NaCl, 0.5 CaCl2, 5 EGTA, 10 HEPES, 0.5 NaGTPand 2 Mg-ATP, 290 mOsm, pH 7.33 (adjustedwith CsOH).

Brief pulses (300 ms) of acetylcholine (1mM) were applied onto the cells every 10 minutesfor up to 60 minutes. ACh-evoked whole-cellcurrents were recorded using a patch-clampamplifier (Axopatch 200A, Union City, CA) in thewhole-cell voltage-clamp configuration. Currentswere filtered on line at 2 kHz using a 8-poleBessel low pass filter (902LPF, FrequencyDevices, Haverhill, MA), digitized at 1kHz usingan analog-to-digital converter (Digidata 1322A,Axon Instruments, Union City, CA) and stored onthe hard drive of a personal computer (DimensionXPS T600, Dell) using pClamp8 (Axoninstruments, Union City, CA). Data analysis wascarried out using Clampfit8 (Axon Instruments,Union City, CA). Drugs and chemicals wereobtained from Sigma-Aldrich.[3H]Epibatidine binding assays: Cells weretransfected with plasmids containing alpha4 andbeta2 subunit genes either singly or incombination and grown for 24 hours followingtransfection. Cells were washed once in Ringer’sBinding Buffer (140mM NaCl, 1.5 mM KCl, 2mMCaCl2, 1mM Mg(SO)4-(7H20), 25mM HEPES,pH7.5) resuspended from culture dishes inRinger’s Binding Buffer and harvested bycentrifugation at 1000xg for 2 minutes. Cells wereresuspended in 5mM HEPES pH 7.5 andhomogenized by passing through a 27 gaugeneedle 15 times. Lysates were cleared by a briefcentrifugation step at 1000xg for 2 minutes. Thetotal membranes were fractionated from thesupernatant fractions by centrifugation at 35,000rpm in a TLA100 ultracentrifuge and washed atotal of three times in Ringer’s Binding Buffer

before resuspension in water. Membranes werethen directly assayed for binding activity. Thetotal number of nicotinic binding sites wasdetermined by equilibrium binding of[3H]epibatidine (Amersham, Piscataway, NJ)similar to previously published protocols (27).Blanks were determined by inclusion of 1 mM (-)-nicotine in the incubation. Total binding wascalculated as fmol/mg protein and bindingparameters were compared using a one-wayANOVA followed by LSD post hoc test.Immunofluorescence and Microscopy:

Surface staining of live cells wasperformed on cells grown on glass coverslips andtransfected with plasmids containing alpha4 andbeta2 subunit genes. At 24 hours post-transfection,cells were transferred to 4˚C for 10 minutes toinhibit membrane trafficking. Growth medium wasreplaced with fresh chilled medium containingprimary antibodies, mAb299 or mAb270(Covance, Richmond, CA) diluted at 1:500 againsteither the a4 or b2 nAChR subunits respectivelyand incubated for 1 hour at 4˚C. Cells werewashed twice with chilled phosphate bufferedsaline (PBS) to remove unbound antibodies andthen fixed first with 2% paraformaldehyde for 10minutes and then with 4% paraformaldehyde foran additional 30 minutes. Fixed cells were washedtwice in PBS and then blocked and permeabilizedin IF buffer (PBS containing 1% bovine serumalbumin (BSA) and 0.1% saponin) plus 4% fetalbovine serum (FBS) for 30 minutes. Followingthe blocking step, the specimens were incubatedwith Cy3 goat anti-rat secondary antibodies(Chemicon, Temecula, CA) diluted at 1:1000 in IFbuffer for 1 hour, washed and mounted in AquaPolymount (VWR, West Chester, PA) on glassslides for microscopy. The specimens were thenobserved on an LSM 510 Zeiss laser scanningconfocal microscope.

To examine internalization surfacereceptors were labeled as described above, exceptfollowing incubation with primary antibodies, thecells were washed with PBS, returned to pre-warmed growth media and incubated at 37˚C forthe indicated chase times to allow internalizationof labeled surface receptors. Following chase, thespecimens were fixed, blocked, permeabilized andincubated with secondary antibodies and viewedby confocal microscopy as described above.

To examine co-localization of internalizedalpha4beta2 receptors, pulse-chase of labeledsurface receptors was performed as describedabove except protease inhibitors (100 µg/ml

leupeptin, 200 µM chloroquine) were included inthe media to inhibit lysosomal proteases in orderto maintain protein integrity in the lysosome.Following fixation, the specimens were blockedand permeabilized in IF buffer plus 4% FBS for 30minutes. The specimens were then incubated forone hour in primary antibodies against LampI (BDBiosciences Pharmingen, San Diego, CA) dilutedat 1:500, or HA (Covance, Richmond, CA) dilutedat 1:500, in IF buffer. Following antibodyincubations, cells were washed twice and blockeda second time in IF buffer plus 4% normal goatserum (NGS) for 30 minutes. Finally thespecimens were incubated for 30 minutes withgoat anti-rat FITC or goat anti-rat Cy3 secondaryantibodies (Chemicon, Temecula, CA) diluted at1:1000 in IF buffer against mAb299 and goat anti-rabbit Cy3 or goat anti-mouse FITC (JacksonImmunoResearch, West Grove, PA) against HAand Lamp1 respectively. Following secondaryantibody incubations, cells were washed andmounted for confocal microscopy as describedabove. Co-localization experiments withtransferrin receptor and SKD1 mutants andcalnexin and the beta2 receptor subunit wereperformed as described above except cells werefixed first and then labeled with primaryantibodies at 1:500 each against transferrinreceptor (Zymed, San Francisco, CA) and HAconcurrently or calnexin (Stressgen, San Diego,CA ) and beta2 (Covance, Richmond, CA)concurrently. Labeled samples were observedwith a Zeiss confocal microscope. Fluorescencebleed through between channels was not detectedin any double labeling experiments,Analysis of Receptor Stability: Transfected cellsexpressing alpha4beta2 receptors were grown toconfluence and washed twice with roomtemperature borate buffer (10 mM borate, 100 mMNaCl). Cell surface proteins were biotinylatedwith 6 mg/ml sulfo-NHS-biotin for 10 minutes inborate buffer. Biotinylation reagent was removedand remaining reactive biotin was quenched bysuccessive washes first in TS buffer (50 mM TrispH7.5, 100 mM NaCl) containing 0.1Mammonium chloride and then with TS buffercontaining 10 mM glycine and finally in TS bufferalone. The cells were then transferred back intogrowth media with or without 100 µM nicotine at37˚C for the designated chase times. Followingchase, the cells were washed twice in ice cold TSbuffer and then harvested in lysis buffer (50 mMHEPES pH 7.4, 150 mM NaCl, 1% TritonX-100)containing protease inhibitor cocktail tablets

(Roche Molecular). The cells were extracted for10 minutes on ice and then clarified bycentrifugation at 14,000xg for 10 minutes at 4˚C.Clarified extracts were incubated with streptavidinagarose for 1-2 hours at 4˚C to precipitatebiotinylated proteins. Streptavidin agaroseprecipitates were washed twice in SWB (100 mMNaCl, 100 mM Tris pH 8.0, 10 mM EDTA)containing 1% TritonX-100 and once in SWBcontaining 0.1% TritonX-100. Proteins wereeluted from streptavidin agarose in SDS samplebuffer (125 mM Tris pH 6.8, 20% (wt/vol)glycerol, 6% SDS, 0.005% bromphenol blue,0.002% beta-mercaptoethanol), separated by SDS-PAGE, transferred to PVDF membranes andanalyzed by immunoblotting with antibodiesagainst alpha4 (mAb299).Quantitation of Surface Expression andU p r e g u l a t i o n : For experiments withagonists/antagonists and/or other pharmacologicalagents, the compounds were added to the cells atleast 24 hours post-transfection and then incubatedfor the indicated times prior to the start of theexperiment. Once cells were harvested, they weremaintained at 4˚C at all times and allcentrifugation steps were done at 1000xg.

Transfected cells expressing alpha4beta2receptors were grown to confluence (48 hourspost-transfection) and suspended by gentlypipetting in ice-cold PBS. The cells wereharvested and resuspended in HEK mediacontaining an additional 10% FBS and incubatedat 4˚C with rotating for 30 minutes. Followingblocking, cells were harvested and thenresuspended and incubated in mAb299 antibodiesagainst surface alpha4beta2 receptors diluted inPBS/BSA (containing 1% bovine serum albumin)for 1 hour. Cells were then washed three times incold HEK media and resuspended in biotinylatedanti-rat secondary antibodies (Chemicon,Temecula, CA) and incubated 30 minutes at 4˚C.Following secondary antibody incubations, thecells were again washed 3 times in HEK mediaand resuspended in PBS/BSA containingfluorescein conjugated to streptavidin (Vector,Burlingame, CA) and incubated for 30 minutes at4˚C. Following the streptavidin incubation, thecells were washed twice in PBS/BSA andresuspended in PBS containing 1 µg/ml propidiumiodide (Sigma, St. Louis, MO) to differentiatebetween live and dead cells. The fluorescenceintensity of 30,000 single, live cells was collectedfor each sample using a Becton-DickinsonFACScan flow cytometer. Each experimental

value reported in figures is the average from atleast 3 independent experiments.Quantitation of Surface Receptor Clearance:Cells were prepared and treated exactly asdescribed above for surface expression analysisexcept following incubation with primaryantibodies, the cells were washed three times andresuspended in warmed HEK media and incubatedwith rocking at 37˚C for the indicated time pointsto allow internalization of surface labeledreceptors. The cells were then harvested in 1 mlaliquots and immediately transferred to ice for 5minutes to inhibit membrane trafficking. For theduration of the experiment the cells were labeledwith secondary and tertiary antibodies andanalyzed by FACS as described above. Eachexperimental value reported in figures is theaverage from at least 3 independent experiments.

RESULTS

Functional alpha4beta2 nAChRs are surfaceexpressed in HEK293T cells. Many previousstudies of nicotine-induced nAChR upregulationhave used membrane permeable ligands to assessthe total number of cellular receptor binding sites(16-18). These studies have shown in at least onecell culture model, that a large percentage ofligand binding sites are localized intracellularly(17). Since under physiological conditions, therelevant receptors that contribute to the functionand excitability of neuronal cells reside at the cellsurface, we wanted to focus specifically on thispool of receptors. Up to this point, it has beenproblematic to examine the effects of nicotine onthe surface expressed alpha4beta2 nAChR proteinin living cells, in part due to the difficulties inachieving significant surface expression incultured cells (28). In addition, it remainstechnically impracticable to perform studies ofspecific native receptor subtypes in neuronal cellmodels due to the complex expression patterns ofthe nAChR subunits, the lack of antibodies able todifferentiate between individual subunits, and thelack of quantitative methods for measuring surfaceexpression in neurons. Therefore, to find anappropriate and tractable model system to studyupregulation and trafficking of surfacealpha4beta2 nACh receptors, we examined surfaceexpression of both rat and mouse alpha4beta2nACh receptors in various cultured cell types.

To assess surface expression, weperformed immunofluorescence in cultured cells

transiently transfected with alpha4 and beta2subunit cDNAs. At 24 hours post-transfection,live cells were labeled with antibodies directedagainst the extracellular N-terminal domain of thealpha4 subunit to specifically label surfaceexpressed receptors. Since neither alpha4 norbeta2 are capable of assembling into functionalhomo-oligomeric receptors (29), no surfacelabeling was observed when cells were mocktransfected or transfected with either alpha4 orbeta2 subunits alone (data not shown). However,when transfected together, mouse alpha4 andbeta2 subunits formed receptors that were detectedat the surface of HEK293T cells byimmunofluorescence with antibodies against thealpha4 (mAb299) subunit (Figure 1A).

To determine whether the surfaceexpressed alpha4beta2 nAChRs detected byimmunofluorescence corresponded to functionalreceptors, transfected HEK293T cells wereassayed 24-48 hours post-transfection usingwhole-cell patch clamp recordings. Although wecould not detect nicotine evoked currents inuntransfected cells or cells expressing alpha4alone (data not shown), cells co-expressing thealpha4 and beta2 subunits responded in aconcentration-dependent manner to pulses ofacetylcholine (ACh, 300 ms) at concentrationsranging from 0.1 to 1000 m M (Figure 1B).Furthermore, when whole-cell currents wererecorded from alpha4beta2 transfected cells atmembrane potentials ranging from -100 to +80mV, we observed strong inward rectification atpositive membrane potentials (data not shown),consistent with what has been previously reportedfor the alpha4beta2 nAChR subtype (30). Sincemouse alpha4beta2 receptors were functionallyexpressed in HEK293T cells, we then wanted todetermine if nicotine could induce upregulation inthis transfected cell system.

Total and surface nAChRs are upregulatedunder chronic nicotine treatment conditions. Toexamine nicotine-induced upregulation ofnAChRs, we first assayed the total number ofbinding sites of the nicotinic ligand[3H]epibatidine in the presence and absence ofchronic nicotine treatment. HEK293T cellstransfected with both alpha4 and beta2 subunitgenes were grown for 24 hours followingtransfection and then either maintained undernormal growth conditions or exposed to 500 nMnicotine for 12 hours. The concentration of 500nM nicotine was chosen because it is in the upper

range of nicotine levels found in the blood ofsmokers (10) and therefore is a physiologicallyrelevant concentration. Total membranes wereprepared from both control and nicotine exposedcells and [3H]epibatidine binding assays wereperformed. Transfected cells grown under normalconditions exhibited significant [3H]epibatidinebinding activity (50 ± 4.13 fmol/mg) (Figure 1C)as compared to untransfected controls (0.54 ±0.083 fmol/mg) or singly transfected alpha4 (0.55± 0.054 fmol/mg) or beta2 (0.61 ± 0.117fmol/mg) controls. Importantly, following 12hours of nicotine exposure, total binding sites wereupregulated (118 ± 5.3 fmol/mg) more than 2 foldover paired untreated controls (Figure 1C),indicating that nicotine is capable of inducingsignificant upregulation of total binding sites inthis cultured cell system.

While total [3H]epibatidine binding siteswere upregulated significantly, we were primarilyinterested in the upregulation of the surfaceexpressed receptors. To assess the ability ofnicotine to drive upregulation specifically of thesurface pool of nAChRs, we performedquantitative cell surface labeling of alpha4beta2receptors using a flow cytometry/ fluorescenceactivated cell sorting (FACS) based assay. Thisassay was designed to overcome several problemsassociated with quantitation of surface proteinexpression levels in cultured cells. First, the assaywas performed in living cells, as defined bypropidium iodide staining, to assure that thelabeling was representative of surface protein inlive cells. Second, cells were selected based onshape and size to measure only fluorescence ofsingle cells, and not clusters of cells. Finally, eachdata point represents the average fluorescenceintensity of 30,000 randomly selected cells, whichallows normalization of the total population andmore accurately represents the expression levelsthan analysis of individual cells.

Using this assay, we determined both theconcentration and time dependence of surfacenAChR upregulation. Transfected HEK293Tcells were exposed to nicotine for 12 hours atconcentrations ranging from 10 nM to 100 µM for12 hours. The cells were then harvested andlabeled with antibodies against the alpha4 subunit(mAb299) at 4˚C. The cells were extensivelywashed and then labeled at 4˚C first withbiotinylated secondary antibodies and finally withFITC labeled avidin. The cells were then brieflytreated with propidium iodide and fluorescenceintensity of live cells was assessed by FACS

analysis. As was previously seen with totalreceptor binding assays, incubation of cells withnicotine over a 12 hour period increased cellsurface expression of alpha4beta2 receptors in aconcentration dependent manner (Figure 2A) withmaximal upregulation of approximately 2-foldover untreated controls achieved between 10 and100 µM nicotine.

Next, similar experiments were performedto establish a time course for surface receptorupregulation. Transfected cells were exposed to500 nM nicotine for varying time periods rangingfrom 2 to 24 hours prior to antibody labeling andFACS analysis. At 500 nM nicotineconcentration, maximum upregulation of surfacealpha4beta2 receptors of more than 2-fold overuntreated controls was observed following 20-24hours of nicotine exposure (Figure 2B).Therefore, upregulation of alpha4beta2 surfacenAChRs is both time and concentration dependentand the maximal extent of upregulation isapproximately 2-2.5 fold over untreated controls.

Since nicotine is membrane permeable, wewanted to determine if agonist exposure to onlythe surface alpha4beta2 nAChRs could also inducesurface receptor upregulation. Just as we observedupregulation of the receptors with membranepermeant nicotine, the membrane impermeantagonist tetramethylammonium (TMA) producedsignificant upregulation (Figure 2C) of surfacealpha4beta2 nAChRs. Therefore, nicotine actionon intracellular binding sites is not required for theinduction of upregulation. To address whetherion-channel function was required forupregulation, we tested several antagonists whichinhibit alpha4beta2 nAChR activity by distinctmechanisms. First, exposure to dihydro-berythroidine (DHbE), a competitive antagonist,was capable of inducing significant upregulationin the absence of nicotine (Figure 2C). Howevermecamylamine (MAA), an antagonist which actsas an open channel blocker, did not induceupregulation of surface alpha4beta2 nAChRs(Figure 2C). Together these data suggest thatoccupancy of the binding site, but not ion-channelactivity, are required for surface receptorupregulation. Consistent with this observation, thecombination of mecamylamine, an open channelblocker, and nicotine together inducedupregulation to the same extent as nicotine alone.Therefore, nicotine is capable of inducingupregulation of surface alpha4beta2 receptorseven in the absence of channel activity (Figure2C).

Together, the characteristics of surfacenAChR upregulation that we observed intransfected HEK293T cells are similar toupregulation of total nAChR populations observedin human smokers, animal models and othercultured cell systems (11,13,15-17,20,31).Furthermore, since HEK293T cells are tractablefor cell biological studies and allow specific andquantitative analysis of the surface expressed poolof receptors, they are a useful model system toexamine the contribution of receptor trafficking tonicotine induced upregulation.

Nicotine exposure does not influence theinternalization rates of surface nAChRs. It hasbeen shown for opiate receptors that interactionwith various agonists and antagonists differentiallyaffects their endocytosis (25). In a similar manner,ligand dependant changes in endocytosis ofnAChR could also result in stabilization ofreceptor protein and upregulation. We thereforewanted to determine whether endocytosis of thealpha4beta2 nAChRs from the plasma membranewas affected by nicotine. Since the traffickingpatterns of alpha4beta2 nAChRs have not beenextensively characterized, we first analyzedinternalization of surface nAChRs under normalconditions by immunofluorescence. Surfacereceptors in transfected cells were labeled at 4˚Cfor 1 hour with an antibody (mAb299) against theextracellular domain of the alpha4 subunit. Thecells were washed and incubated at either 4˚C orat 37˚C in growth media for 30 minutes, thenfixed, permeabilized, labeled with secondaryantibodies and prepared for immunofluorescence.As expected, when surface labeled cells wereretained at 4˚C (0 minute chase), the labeledreceptors remained at the surface (Figure 3A).However, when labeled cells were shifted to 37˚Cfor 30 minutes, the surface receptors weretranslocated in a time- and temperature- dependentmanner to intracellular punctate structures,consistent with efficient internalization throughthe endocytic pathway (Figure 3A). To assure thatthe translocation we observed these assays wasdue to endocytosis of the surface receptors, weperformed internalization experiments in thepresence of sucrose, which is known to inhibitendocytosis, and found that the labeled nAChRswere retained at the surface throughout the chaseperiod (data not shown). In addition, weperformed internalization experiments with Fabantibody fragments generated from mAb299. Fabfragments do not cluster receptors and therefore,

generally do not initiate artificial endocytosiswhen bound to surface receptors. Using Fabfragments, we observed identical translocation ofsurface receptors as with the intact antibody.These results indicate that the internalization weobserved was unlikely to be the result of antibodyinterference (data not shown).

Once we were satisfied that our assay wasmonitoring endocytic events, we examined theeffect of acute nicotine treatment oninternalization. Cells were treated in the samemanner as in the constitutive internalizationassays, except that 100 µM nicotine was added tothe cells during the chase period. Just as in theuntreated controls, nAChRs were translocated intointernal structures within the 30 minute chaseperiod in the presence of nicotine (Figure 3A),suggesting that acute exposure to nicotine does notdramatically change the initial internalization ofthese receptors from the plasma membrane.Relatively high concentrations of nicotine wereused in these assays to ensure that any effectswould be as dramatic and apparent as possible.However, at both high (100µM) and low (500nM,data not shown) concentrations, no differences ininternalization could be discerned.

To confirm the immunofluorescenceresults by an independent method, we utilized aquantitative FACS based assay to examine thekinetics of internalization following both acute andchronic nicotine exposure. The surfacealpha4beta2 nAChRs were first labeled withprimary antibodies against the alpha4 subunit, andthen returned to standard growth medium at 37˚Cfor the indicated time points. The cells were thencooled to 4˚C and remaining labeled surfacenAChRs were labeled with secondary and tertiaryantibodies. Under these conditions, only thosereceptors that were labeled by the primaryantibody and remained at the cell surface duringthe chase period were accessible to secondaryantibodies at any particular time point. Thus, theloss of a population of pulse labeled receptorsfrom the surface over time could be determined.To examine the efficacy of this assay, weperformed internalization experiments usingmAb299 to label surface receptors. The labeledsurface receptors were cleared from the plasmamembrane efficiently during the chase period,with only approximately 40% of labeled receptorsmaintained at the surface following the 30 minutechase period (Figure 3B). Furthermore, weobtained identical internalization rates using eitherfull length mAb299 or Fab fragments to label the

surface receptors and observed that maintainingthe cells at either 4˚C or under glucose-deprivationconditions dramatically inhibited internalizationover the course of the chase period (data notshown). Therefore, this assay monitors atemperature and glucose dependent internalizationprocess.

We then examined the effect of nicotinetreatment on internalization of surface alpha4beta2nAChRs using this assay. Again, in agreementwith the immunofluorescence experiments, whennicotine was added to labeled cells during the 30minute internalization period, only approximately35% of surface receptors remained following the30 minute chase and the rate of clearance from theplasma membrane (0.0366 min-1) was notstatistically different from that of untreated cells(0.0364 min-1) (Figure 3B).

Although acute nicotine treatment did notappear to influence the internalization of thenAChRs, it was possible that chronic incubationwith nicotine sufficient to induce upregulationmay eventually result in an observable change inthe rate of internalization. To address thispossibility, we examined internalization ofnAChRs following a 12 hour treatment with 500nM nicotine. As expected, the nicotine treatedcells exhibited a 40% increase in surfaceexpression as compared to untreated controls(Figure 3B). However, when surface receptorclearance was monitored, the rate of receptordisappearance was not significantly differentbetween cells chronically treated with nicotine(0.0369 min-1) and untreated cells (0.0364 min-1).Similar results were obtained using increasingconcentrations of nicotine up to 100 µM (data notshown). Therefore, although more alpha4beta2receptor protein is expressed at the surface of thechronically nicotine-treated cells, the rate at whichthe protein is internalized from the plasmamembrane is similar to that of untreated cells.Therefore, neither acute nor chronic exposure tonicotine changes the rate of nAChR proteinendocytosis from the cell surface.

Nicotine does not change post-endocyticdegradation rates of surface receptors.Previous studies of upregulation suggested thatnicotine may act by changing the rate of nAChreceptor turnover, stabilizing alpha4beta2 nAChRprotein and subsequently resulting in upregulation.To directly test this hypothesis, we wanted todetermine whether trafficking of the alpha4beta2nAChRs from the plasma membrane to the

lysosome was affected by nicotine. First, toidentify the intracellular compartment that thesurface receptors are directed to from the plasmamembrane, we performed co-localizationexperiments with internalized alpha4beta2receptors and Lamp1, a protein marker oflysosomal membranes. At 4˚C, labeledalpha4beta2 nAChRs were seen at the plasmamembrane, distinctly localized from the internalpunctate Lamp1 compartments (Figure 4A, 0 min.chase). However, following 30 minutes of chase,the internalized alpha4beta2 receptors exhibitedsignificant co-localization with Lamp1 positivecompartments (Figure 4A). Interestingly, at thissame time point, we saw no significant co-localization of internalized nAChRs with eithertransferrin receptor or EEA1(data not shown), twomarkers of early endosomal compartments fromwhich proteins are recycled to the plasmamembrane. Therefore, it appears that undernormal growth conditions, surface alpha4beta2receptors are internalized and rapidly directed tolysosomal compartments.

If nicotine changed the rate of transport tothe lysosome, we would expect to observedramatic changes in the degradation rate of thesurface receptors. Therefore, we examined thestability of the surface expressed pool of receptorsusing a surface biotinylation degradation assay ineither the presence or absence of nicotine. Intacttransfected cells were reacted with NHS-SS-biotin,a membrane impermeant biotinylation reagentwhich exclusively labels surface proteins. Thefate of the biotinylated proteins was monitoredover time by streptavidin immunoprecipitation andsubsequent detection with mAb299 directedagainst the alpha4 nAChR subunit. In the absenceof nicotine, the surface nAChR signal wasdramatically reduced following 30 minutes at 37˚C(Figure 4B). This experiment suggests thatinternalized receptor is not recycled to the plasmamembrane but is directly routed to the lysosomeand degraded. These results are consistent with ourimmunofluorescence and FACS data, and provideadditional evidence that the internalization that weobserve using antibodies to label surface receptorsis not likely to be due to antibody inducedinternalization.

Interestingly, the addition of nicotineduring the chase period did not dramaticallystabilize the surface nAChRs (Figure 4B) ascompared to the untreated control cells. Together,these data suggest that exposure to nicotine does

not affect the degradation of surface expressedalpha4beta2 nAChRs in the lysosome.

Upregulation of nAChRs is independent ofrecycling from the endosome. Although we didnot observe significant changes in internalizationor degradation of surface alpha4beta2 nAChRs i nthe presence of nicotine, the possibility remainedthat small changes in post-endocytic traffickingover extended periods of time may contribute toupregulation. As another means to assess thecontribution of endocytic trafficking toupregulation, we utilized a dominant negativemutant of SKD1, an endosomal associated AAA-ATPase previously shown to be required forefficient recycling of plasma membrane proteinsfrom endosomal compartments (32). Undernormal conditions, the majority of SKD1 issoluble and distributed throughout the cytoplasm.However, a dominant negative mutation in theATPase domain (E235Q) of SKD1 (SKD1(EQ))results in the accumulation of the SKD1(EQ)protein itself, as well as endocytic cargoes, in largeendosomal structures. Endocytic cargoes areunable to traffic out of these aberrantcompartments, resulting in an intracellularaccumulation of internalized surface proteins (33).If changes in the post-endocytic trafficking ofnAChR proteins were required for upregulation,disruption of this pathway with SKD1(EQ) shouldprevent upregulation.

To establish the SKD1 phenotype in theHEK293T cell line, we first examined thedistribution of transferrin receptor, a normallyrecycled protein, in cells transfected with HAepitope tagged versions of either wild-type SKD1(SKD1-HA) or dominant negative SKD1(SKD1(EQ)-HA). In cells expressing wild-typeSKD1-HA, transferrin receptor was distributedthroughout the cell in small punctate structures asexpected (Figure 5A). However, in cellsexpressing the mutant SKD1(EQ)-HA, transferrinreceptor accumulated with SKD1(EQ)-HA proteinin large aberrant endosomal compartments(Figure 5A), confirming that the SKD1(EQ)mutation disrupts endosomal recycling in theHEK293T cell line, as has been previously shownin other cultured cell lines (33).

When co-transfection experiments wereperformed with both alpha4beta2 and SKD1(EQ)-HA, internalized alpha4beta2 receptors weredetected in SKD1(EQ)-HA positive compartments(Figure 5B) suggesting that alpha4beta2 nAChRtrafficking to the lysosome normally proceeds via

the SKD1 compartment. Importantly, theaccumulation of alpha4beta2 was only observed incells co-expressing alpha4beta2 and SKD1(EQ)-HA. Therefore, the SKD1(EQ) mutant is defectivein post-endocytic trafficking of alpha4beta2nAChRs to the lysosome.

We then examined surface expression ofalpha4beta2 receptors in cells co-expressingalpha4beta2 with either vector alone, SKD1-HA,or the dominant negative SKD1(EQ)-HA mutant.As is usually observed in co-transfectionexperiments, virtually all cells expressingalpha4beta2 also expressed either SKD1-HA orSKD1(EQ)-HA (Figure 5B) assuring thatphenotypic differences that we observed werefrom cells expressing both constructs together. Asexpected, in cells expressing alpha4beta2 alone oralpha4beta2 together with wild type SKD1-HA,we observed nicotine induced upregulationfollowing a 12 hour incubation with nicotine(Figure 5C). Interestingly, however, in cellsexpressing both alpha4beta2 and SKD1(EQ)-HA,even though trafficking of nAChRs is disrupted(Figure 5B), we still observed upregulation ofalpha4beta2 nAChRs (Figure 5C). These resultsare consistent with the lack of nicotine effects oninternalization and degradation rates of surfacereceptors and further suggest that upregulation isindependent of post-endocytic protein sorting.

Transport through the secretory pathway fromthe Endoplasmic Reticulum is required fornAChR upregulation. Together, our data indicatethat nei ther in ternal iza t ion of thealpha4beta2 nAChRs from the plasma membrane,nor trafficking in the post-endocytic pathway isinvolved in nicotine-induced upregulation ofsurface alpha4beta2 nAChRs. We thereforewanted to examine the contribution of receptorbiosynthesis and/or insertion of receptors into theplasma membrane to upregulation. It has beenwell documented that nicotine-inducedupregulation of high affinity nicotine binding sitesdoes not result from an increase in mRNA levels,which suggests that upregulation is nottranscriptionally regulated. Furthermore, it hasbeen reported that upregulation of nicotine bindingsites can proceed in the presence of cycloheximide(15), suggesting that de novo protein synthesis isalso not required for upregulation. In our systemas well, we have found that the translationalinhibitors cycloheximide and emetine do not blockupregulation of surface nAChRs (data not shown).

Following translation, membrane proteinsare transported through the secretory pathway tothe plasma membrane. If trafficking of proteinthrough the secretory pathway were required forupregulation, disruption of secretory pathwayfunction should disrupt nicotine-inducedupregulation. Brefeldin A (BFA) is a fungalmetabolite that disrupts transport of secretedproteins from the Endoplasmic Reticulum (ER) toGolgi (34,35). To assess the contribution ofsecretory traffic to the plasma membrane inupregulation, we performed surface labelingexperiments in the presence of BFA. BFA waseither added individually to transfected cells or co-administered with nicotine for 10 hours prior tolabeling for surface expression analysis.Following exposure to nicotine alone, transfectedcells displayed the typical ~40% upregulation ofsurface nAChRs when compared to untreatedcontrols (Figure 6A). Cells exposed to BFA aloneshowed a marked decrease in basal surfaceexpression, which is indicative of the effectivenessof BFA in disrupting overall secretory pathwayfunction (data not shown). However, when BFAtreatment was combined with nicotine, we couldnot detect nicotine-dependent upregulation ofsurface expression. Instead, nAChR surfaceexpression was slightly, but not significantly moredepressed than with BFA alone (Figure 6A)indicating that secretory pathway function isnecessary for nicotine-induced upregulation.

To determine whether BFA acts directly todisrupt surface upregulation, or instead thatupregulation still occurs in the presence of BFA,but BFA blocks the transport of an internal pool ofupregulated receptors to the plasma membrane, weperformed [3H]epibatidine binding assays to assessnicotine-induced upregulation of the total pool ofnAChRs in the presence of BFA. As expected,exposure of alpha4beta2 transfected cells tonicotine for 8 hours induced significantupregulation of total [3H]epibatidine binding sitesto approximately 40% above untreated controls(Figure 6B). Interestingly, the addition of BFAduring the nicotine exposure did not eliminate theupregulation of total [3H]epibatidine binding sites(Figure 6B). These data indicate that themechanism underlying upregulation is intact inBFA treated cells, but that the transport ofupregulated receptors to the plasma membrane isdisrupted. This further suggests that the site ofupregulation is at or prior to the ER, the site ofBFA action in the cell.

If upregulation is regulated at the ER, aninternal pool of pre-existing nAChR subunitsrequired for upregulation most likely exists in theER. To determine which compartment internalalpha4beta2 protein is localized, we performed co-localization experiments with calnexin, amembrane bound ER resident protein. HEK293Tcells were transfected with alpha4 and beta2subunits and grown for 24 hours prior to fixationand permeabilization. Permeabilized cells werelabeled with antibodies against beta2 and calnexinto examine the distribution of the total pools ofboth proteins. As expected, calnexin was detectedin a disperse perinuclear compartment, consistentwith ER localization (Figure 7D). In addition, thebeta2 protein labeling was predominantlycoincident with calnexin (Figure 7D), suggestingthat the majority of the internal beta2 protein poolis in the ER, providing an available source ofnAChR subunits that can be converted to ligandbinding competent nAChRs by nicotine exposure.

DISCUSSION

The phenomenon of nicotine-inducednAChR upregulation is contrary to conventionalmodels of ligand-receptor interaction. Typically,exposure to ligand results in surface receptordownregulation to attenuate signaling. However,in the case of nAChRs, chronic exposure toagonist results in upregulation, which has beenproposed to be a compensatory mechanism toreplace desensitized receptors at the cell surface(12). Therefore, nicotine-induced upregulationseems to be novel means to control the activity ofcell surface receptors.

We have utilized a culture cell system anda quantitative FACS based surface expressionassay to characterize the upregulation andtrafficking patterns of surface expressedalpha4beta2 nAChRs in living cells. We havechosen to concentrate specifically on the surfaceexpressed proteins, which in vivo are thefunctionally relevant receptors required formembrane excitability. In our cultured cellsystem, we achieve robust and reproduciblesurface expression of functional mousealpha4beta2 nAChRs (Figure 1) and observenicotine-induced upregulation of both total andsurface alpha4beta2 nAChRs (Figures 1 and 2).The upregulation that we observe is similar topreviously reported high-affinity nicotine bindingsite upregulation in both cultured cells and animalmodels (15-17,31). This indicates that

upregulation can be achieved using basic,fundamental cellular machinery that is intact innon-neuronal cells. Therefore, understanding theprocesses responsible for upregulation of nAChRsmay not only be important with respect tonicotine response, but potentially for theregulation of other cell surface receptors as well.In addition, mechanisms and components that canbe identified using this reduced system may alsobe involved in regulation of these receptors inneuronal cells and once identified, can be directlytested in the more complicated context of neuronalcells or whole animals.

Ligand-dependent trafficking and upregulationOur examination of alpha4beta2 nAChR endocytictrafficking has shown that surface expressednAChRs are constitutively and rapidlyendocytosed from the plasma membrane anddirected into a lysosomal degradation pathway(Figures 3 and 4). Therefore, under normalconditions, surface nAChRs are constantlyreplaced by new receptors from the secretorypathway. Chronic exposure of surface nAChRs tonicotinic agonists is sufficient for induction ofsurface receptor upregulation, but does not alterthe endocytic trafficking of these receptors (Figure3). One mechanism that has been proposed forupregulation is that nicotine modulates stability ofthe nAChRs, redirecting internalized receptorsfrom a degradative pathway to increase theirsurface residence and stability (15). However, ourdata clearly demonstrates that neither acute orchronic exposure to nicotine has any effect on theclearance of the surface nAChRs from the plasmamembrane or their degradation in the lysosome(Figure 3 and Figure 4). Furthermore, disruptingpost-endocytic traffic with a dominant negativeSKD1 mutant does not prevent upregulation ofnAChRs (Figure 5). Together, these data stronglysuggest that nicotine dependent regulation ofnAChR surface expression is not via changes inendocytic trafficking or the lysosomal degradationrate of the surface nAChRs, a model that has beenpreviously invoked as a post-translationalmechanism for upregulation (15).

In contrast to these previous studies, ourdegradation and internalization experiments wereconducted by pulse-chase analysis of nAChRs insitu, on much shorter time courses and withoutadditional drug treatments. In addition, ourexperiments specifically analyzed the fate ofsurface receptors, and therefore, did not directlyaddress the possibility of changes in the

degradation of internal nAChRs through a non-lysosomal pathway. It is possible that the morelong term changes that were observed by Peng etal. could be explained by an inhibition ofproteasomal degradation of internal receptorprotein as a result extended drug exposure.However, our results clearly demonstrate a lack ofa direct effect of nicotine on the lysosomaldegradation pathway.

Although post-endocytic trafficking doesnot contribute significantly to upregulation, theinsertion of nAChRs into the plasma membranethrough the secretory pathway appears to berequired for nicotine-induced upregulation ofsurface receptors. Inhibition of secretory pathwayfunction with BFA results in complete loss ofnicotine-induced surface receptor upregulation(Figure 6). Interestingly however, de novo proteinsynthesis does not appear to be required forupregulation since translational inhibitors do notinhibit upregulation (15). There have beencontradictory interpretations of the effects oftranslational inhibitors on upregulation (15,16).However, the data in these studies each show anincrease of binding sites in the presence ofcycloheximide and nicotine when compared tocycloheximide treatment alone, suggesting thatsecondary effects following prolonged exposuretranslational inhibitors may result in changes inprotein expression, but that nicotine inducedupregulation still occurs. Furthermore, we haverepeated translational inhibitor experiments in ourcultured cell system and found no effect ofcycloheximide or emetine, two inhibitors that actat distinct points in the protein translationpathway, on upregulation (unpublishedobservations). Together these data indicate that apre-existing intracellular pool of protein issufficient for nicotine-induced upregulation. Inagreement with this, binding experiments done inthe presence of BFA indicate that although surfacereceptor upregulation is inhibited by disruption ofsecretory pathway traffic to the plasma membrane,the total pool of receptors are still upregulated bynicotine exposure. This suggests that theupregulation event is at or prior to the formation ofligand binding competent receptors and requirestheir subsequent transport through the secretorypathway.

Model for nicotine-induced upregulation Takentogether, our data suggests that an internal pool ofnAChRs relevant to upregulation may reside in theendoplasmic reticulum. First, we find that BFA

blocks nicotine-induced upregulation of surfacenAChRs, but not the upregulation of total bindingsites. Furthermore, the majority of internal beta2protein co-localizes with calnexin, an ER residentprotein. This is consistent with previous studies ofmuscle nAChR, where it has been shown that thelimiting step of surface expression appears to be atthe ER (36,37). At the ER, there are severalpotential steps in receptor biosynthesis that couldbe regulated by nicotine to affect expressionlevels. For multi-subunit receptors, includingnAChRs, folding and assembly into functionalreceptors is a prerequisite for packaging intosecretory vesicles and exit from the ER (38). Infact, recent studies using FRET as a means tomonitor assembly of nAChRs have shown thatnicotine increases FRET between alpha4 and beta2subunits which may be indicative of increasedassembly into functional receptors (39). Prior toassembly, in order to accomplish appropriatefolding, ER localized chaperone proteins act toprevent proteasomal degradation and promotefolding of nascent polypeptide chains (40).Muscle nAChR expression appears to be effectedby alterations in the ubiquitin-proteasome system(41) and this quality control step could beinfluenced by nicotine to affect upregulation.Finally, once properly folded, many receptorsubunits contain exposed ER retention signals thatact to keep unassembled components in the ERuntil assembly is complete, at which time theretention signals are masked and packaging intoforward transport vesicles can proceed. Bothmuscle and neuronal nicotinic receptor familymembers have been shown to interact withcalnexin, an ER chaperone protein, and they alsocontain putative ER retention signals (37,42,43).

Finally, although nicotine is membranepermeant and binds to intracellular sites, we andothers have shown that ligand interaction withsurface receptors alone is sufficient to induceupregulation, since exposure to membraneimpermeant ligands also induces upregulation(Figure 2 and (17)). Therefore, it is necessary toinvoke a signal initiated by the surface receptors inresponse to nicotine, to activate a secondmessenger that acts at a distinct internal site todrive upregulation. This pathway is unlikely to bedependent upon nAChR channel activity, sincechronic treatment of cells with competitiveantagonists, or the combination of nicotine andchannel blocking antagonist, both of which inhibitthe ion-channel activity of surface receptors, aresufficient to drive upregulation independent of

function (Figure 2) (15,16). Together, theseconsiderations suggests a revised model fornicotine-induced upregulation where theinteraction of nAChR with ligand at the cellsurface induces a second messenger signalingsystem. This results in an increased number ofnAChRs transiting from the ER to the surface,mediating the observed upregulation of surfacenicotinic receptors. The challenge now will be todefine the nicotine regulated molecules involvedin receptor dynamics at the ER and once thesemolecules are in hand, to move into neuronalsystems and whole animal models and directly testtheir involvement in nicotinic receptor traffickingand nicotine-induced behaviors.

AKNOWLEDGEMENTS

We thank Dr. Markus Babst, Dr. ChristineSchulteis and Dr. Nathalie Kayadjanian for criticalreading of the manuscript and many helpfuldiscussions. We also thank Dr. Jerry Stitzel formouse nicotinic acetylcholine receptor constructsand Dr. Markus Babst and Dr. Scott Emr forSKD1 constructs. T. D. is a Damon RunyonPostdoctoral Fellow supported by the DamonRunyon Cancer Research Foundation (DRG-#1661). This work was also supported by a grant(AA13018) from the National Institutes of Health(NIAAA) to S.F. Heinemann.

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FIGURE LEGENDS

Figure 1. Functional alpha4beta2 nAChRs expressed at the surface of HEK293T cells are upregulated bynicotine treatment. (A) To demonstrate surface localization of alpha4beta2 receptor protein, liveHEK293T cells transfected with alpha4 and beta2 subunit cDNAs were labeled at 4˚C with antibodiesagainst alpha4 (mAb299), followed by fixation and labeling with Cy3 conjugated secondary antibodies.(B) Characteristic whole-cell inward currents evoked by application of increasing concentrations ofacetylcholine (ACh) in HEK293T cells transfected with alpha4 and beta2 subunit genes. Cells werechallenged with 300 ms applications of ACh every 5 minutes at a holding potential of -60 mV. (C) Totalmembranes were prepared from alpha4 and beta2 transfected HEK293T cells grown in the absence orpresence of 500 nM nicotine for 12 hours. The number of specific [3H]epibatidine binding sites wasdetermined. Nicotine induced a significant (***=p≤0.001) increase in [3H]epibatidine binding activity ascompared to untreated controls

Figure 2. Nicotine effects on alpha4beta2 nAChR endocytosis (A) Live HEK293T cells expressingalpha4beta2 nAChRs were labeled at 4˚C with antibodies against alpha4 (mAb299) to examine surfaceexpression levels by FACS before and after treatment with the indicated increasing concentrations ofnicotine for 12 hours. (B) Live HEK293T cells expressing alpha4beta2 nAChRs were labeled at 4˚C withmAb299 antibodies against alpha4 to examine surface expression levels by FACS following incubationwith 500 nM nicotine for the indicated times. nAChR surface expression increases in a concentration and

time dependent manner. (C) alpha4beta2 expressing cells were incubated for 12 hours with indicatednicotinic agonists and antagonists. Surface receptors were labeled with mAb299 and FACS analysis wasperformed to examine upregulation efficacy of the various compounds (TMA= tetramethyl ammonium,DHßE= dihydro-ß-erythroidine, MAA= mecamylamine). Significant upregulation was seen under alltreatment conditions (***=p≤0.001,**=p<0.005) except with MAA treatment, in which no significantchange in surface expression was observed. In (A), (B) and (C), the reported values are normalized tountreated alpha4beta2 controls.

Figure 3. Quantitative internalization rates of alpha4beta2 nAChRs under nicotine treatment conditions.A) Surface alpha4beta2 receptors in live transfected HEK293T cells were labeled with mAb299 againstthe extracellular domain of the alpha4 subunit at 4˚C for 1 hour. Following labeling, the cells wereincubated for the indicated times at 37˚C in media with or without 100 µM nicotine. Followinginternalization cells were permeabilized and labeled with Cy3 conjugated secondary antibodies. Timedependent translocation of the labeled surface receptors from the plasma membrane to internal punctatestructures was monitored by immunofluorescence and confocal microscopy. (B) FACS basedinternalization assays were performed on HEK293T cells expressing alpha4beta2 nAChRs. Surfacereceptors were labeled with mAb299 at 4˚C and then allowed to internalize at 37˚C in growth medium forthe indicated time points. Cells were treated with ( grey triangles) or without 500 nM nicotine (blacksquares) during the chase period (acute treatment), or for 12 hours (grey circles, chronic treatment) priorto surface labeling with mAb299. The data are expressed as percent of untreated control to demonstrateupregulation of nAChRs in the chronically nicotine treated cells. The rate constants are 0.0364 min-1 forthe untreated control, 0.0366 min-1 for acute and 0.0369 for chronic nicotine treatment conditions. Thereis no statistical difference between the rate constants of internalization for either acute (p=0.884) orchronic (p=0.609) nicotine treatment as compared to untreated controls. Each data point is the average of3 independent experiments.

Figure 4. Lysosomal trafficking and degradation of surface alpha4beta2 nAChRs. (A) Live HEK293Tcells expressing alpha4beta2 receptors were labeled with mAb299 and then incubated at either 4˚C (0minute chase) or 37˚C for 30 minutes. Cells were then permeabilized and co-labeled with primaryantibodies against LampI. Co-localization of internalized receptors following 30 minutes ofinternalization at 37˚C with LampI positive late endosomal/lysosomal compartments was monitored byconfocal microscopy. (B) HEK293T cells transfected with alpha4beta2 nAChR subunits were surfacebiotinylated and returned to growth media with or without 100 µM nicotine for the indicated times.Biotinylated alpha4beta2 receptors were precipitated from cell lysates with streptavidin agarose and thestability of the labeled receptors was examined by SDS-PAGE and immunoblotting with mAb299.

Figure 5. Trafficking and upregulation of alpha4beta2 receptors in SKD1(EQ) mutant. (A) Thephenotype of SKD1(EQ) mutant is demonstrated by co-localization of SKD1(EQ)-HA mutant proteinwith transferrin receptor. SKD1-HA and transferrin receptor proteins were labeled with primaryantibodies, against either the HA epitope or transferrin receptor respectively, in fixed cells either mocktransfected, expressing SKD1-HA or SKD1(EQ)-HA. Expression of SKD1(EQ)-HA, but not SKD1-HAwild-type, disrupts endocytic membrane trafficking and causes accumulation of transferrin receptor andSKD1 protein in large aberrant endocytic compartments by immunofluorescence. (B) Surfacealpha4beta2 receptors were labeled with mAb299 against the alpha4 subunit and allowed to internalize for30 minutes. Cells were fixed and labeled first with primary antibodies against HA to detect SKD1(EQ)-HA and then with distinct secondary antibodies against both SKD1(EQ)-HA and internalized alpha4beta2nAChRs. Internalized alpha4beta2 receptors co-localize with SKD1(EQ)-HA in aberrant endocyticcompartments induced by SKD1(EQ)-HA expression in HEK293T cells. (C) Surface nAChRs in cellsco-expressing alpha4beta2 together with either empty plasmid, SKD1-HA or SKD1(EQ)-HA werelabeled with mAb299 and upregulation was examined by FACS. Statistically significant nicotine inducedupregulation (***=p£0.001) was observed in each condition. Each reported value is the average of threeindependent experiments.

Figure 6. Exocytic trafficking of alpha4beta2 nAChRs from the ER is required for upregulation (A)Cells expressing alpha4beta2 receptor subunits were treated with nicotine, BFA or nicotine incombination with BFA for 10 hours. Surface receptors were labeled with mAb299 and surface expressionin the presence of these compounds was quantitated by FACS. The values in the figure are the average ofthree independent experiments. Nicotine alone induced significant upregulation (***=p≤0.005) but BFAprevented nicotine-induced upregulation. (B) Total membranes were prepared from HEK293T cellsexpressing alpha4beta2 receptor subunits which were exposed for 8 hours prior to the experiment with orwithout nicotine, BFA, or a combination of nicotine and BFA. [3H]epibatidine binding was measured inthe membrane preps to determine upregulation of total binding sites. Significant upregulation wasobserved in cells treated with nicotine alone as compared to untreated controls and with the combinationof BFA and nicotine as compared to controls treated only with BFA (***=p≤0.001). The reported valuesare expressed as percent of untreated controls (without nicotine) and represent the average of threeindependent experiments. (C) Cells expressing alpha4beta2 receptor subunits were co-labeled withantibodies against both calnexin and the beta2 nAChR subunit. Co-localization between internal beta2protein (left panel) and calnexin (middle panel) is observed in merged images (right panel).

C

To

tal[

3 H]

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ine

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alpha4beta2anti-alpha4 (mAb299)

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µMAcetylcholine

500 ms

200 pA

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r=0.0366 min-1

+nic acute

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+nicotine

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Lamp1

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alpha4

Mock

HA merge

SKD1(EQ)-HA

A

B

-nic+nic (500nM)

******

***

0

150

50

100

alpha4beta2

alpha4beta2SKD1

alpha4beta2

SKD1(EQ)

surf

ace

nA

Ch

R(p

erce

nt

con

tro

l)

200

250

300

350C

A

0

25

50

75

100

125

150

175

200

untreated bfa(100µM)

-nic+nic (500nM)

B

beta2 calnexin merge

surf

ace

nA

Ch

R(p

erce

nt

con

tro

l)

0

25

50

75

100

125

150

175

tota

l[3 H

]ep

ibat

idin

eb

ind

ing

(per

cen

tco

ntr

ol)

untreated bfa(100µM)

200

C

-nic+nic (500nM)

******

NS

**