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• W2037610035 abstract "The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768–9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 μs. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates. The human norepinephrine (NE) transporter (hNET) attenuates neuronal signaling by rapid NE clearance from the synaptic cleft, and NET is a target for cocaine and amphetamines as well as therapeutics for depression, obsessive-compulsive disorder, and post-traumatic stress disorder. In spite of its central importance in the nervous system, little is known about how NET substrates, such as NE, 1-methyl-4-tetrahydropyridinium (MPP+), or amphetamine, interact with NET at the molecular level. Nor do we understand the mechanisms behind the transport rate. Previously we introduced a fluorescent substrate similar to MPP+, which allowed separate and simultaneous binding and transport measurement (Schwartz, J. W., Blakely, R. D., and DeFelice, L. J. (2003) J. Biol. Chem. 278, 9768–9777). Here we use this substrate, 4-(4-(dimethylamino)styrl)-N-methyl-pyridinium (ASP+), in combination with green fluorescent protein-tagged hNETs to measure substrate-transporter stoichiometry and substrate binding kinetics. Calibrated confocal microscopy and fluorescence correlation spectroscopy reveal that hNETs, which are homomultimers, bind one substrate molecule per transporter subunit. Substrate residence at the transporter, obtained from rapid on-off kinetics revealed in fluorescence correlation spectroscopy, is 526 μs. Substrate residence obtained by infinite dilution is 1000 times slower. This novel examination of substrate-transporter kinetics indicates that a single ASP+ molecule binds and unbinds thousands of times before being transported or ultimately dissociated from hNET. Calibrated fluorescent images combined with mass spectroscopy give a transport rate of 0.06 ASP+/hNET-protein/s, thus 36,000 on-off binding events (and 36 actual departures) occur for one transport event. Therefore binding has a low probability of resulting in transport. We interpret these data to mean that inefficient binding could contribute to slow transport rates. Adrenergic signaling in the central nervous system modulates learning and memory, the fight-or-flight response, and the reception of pain (2Valentino R.J. Foote S.L. Aston-Jones G. Brain Res. 1983; 270: 363-367Crossref PubMed Scopus (503) Google Scholar). Noradrenaline (norepinephrine, NE) 1The abbreviations used are: NE, norepinephrine; hNET, human norepinephrine transporter; MPP+, 1-methyl-4-tetrahydropyridinium; FCS, fluorescence correlation spectroscopy; FLIM, fluorescence lifetime imaging microscopy; TIRF, total internal reflection fluorescence; DS, desipramine; DiO, 3,3′-dioctadecyloxacarbocyanine perchlorate; GFP, green fluorescent protein; FRAP, fluorescence recovery after photobleach; KRH, Krebs-Ringer-Hepes. is the principal transmitter in postganglionic sympathetic neurons (3Trendelenburg A.U. Gaiser E.G. Cox S.L. Meyer A. Starke K. J. Neurochem. 1999; 73: 1431-1438Crossref PubMed Scopus (11) Google Scholar), and in the brainstem NE regulates autonomic function (4Foote S. Aston-Jones G. Pharmacology and Physiology of Central Noradrenergic Systems. Psychopharmacology: The Fourth Generation of Progress. Raven Press Ltd., New York1995Google Scholar). Noradrenergic dysfunction is associated with mood disorders and depression (5Schildkraut J.J. Gordon E.K. Durell J. J. Psychiatr. Res. 1965; 3: 213-228Crossref PubMed Scopus (91) Google Scholar, 6Clark M.S. Russo A.F. Brain Res. Brain Res. Protoc. 1998; 2: 273-285Crossref PubMed Scopus (8) Google Scholar), post-traumatic stress disorder (7Maes M. Lin A.H. Verkerk R. Delmeire L. Van Gastel A. Van der Planken M. Scharpe S. Neuropsychopharmacology. 1999; 20: 188-197Crossref PubMed Scopus (82) Google Scholar), hypertension, diabetes, cardiomyopathy, and heart failure (8Backs J. Haunstetter A. Gerber S.H. Metz J. Borst M.M. Strasser R.H. Kubler W. Haass M. J. Mol. Cell Cardiol. 2001; 33: 461-472Abstract Full Text PDF PubMed Scopus (92) Google Scholar). After stimulated or spontaneous NE release, NETs rapidly clear NE from the synaptic cleft via efficient transport system attenuating signaling (9Axelrod J. Kopin I.J. Prog. Brain Res. 1969; 31: 21-32Crossref PubMed Scopus (79) Google Scholar), and recycling 90% of synaptic NE (10Blakely R.D. J. Neurosci. 2001; 21: 8319-8323Crossref PubMed Google Scholar). A polymorphism in hNET causing decreased uptake correlates with orthostatic intolerance (11Garland E.M. Hahn M.K. Ketch T.P. Keller N.R. Kim C.H. Kim K.S. Biaggioni I. Shannon J.R. Blakely R.D. Robertson D. Ann. N. Y. Acad. Sci. 2002; 971: 506-514Crossref PubMed Scopus (18) Google Scholar, 12Robertson D. Flattem N. Tellioglu T. Carson R. Garland E. Shannon J.R. Jordan J. Jacob G. Blakely R.D. Biaggioni I. Ann. N. Y. Acad. Sci. 2001; 940: 527-543Crossref PubMed Scopus (54) Google Scholar, 13Shannon J.R. Flattem N.L. Jordan J. Jacob G. Black B.K. Biaggioni I. Blakely R.D. Robertson D. N. Engl. J. Med. 2000; 342: 541-549Crossref PubMed Scopus (477) Google Scholar). hNETs are targets for cocaine, antidepressants, amphetamines, and neurotoxins (14Pacholczyk T. Blakely R.D. Amara S.G. Nature. 1991; 350: 350-354Crossref PubMed Scopus (760) Google Scholar, 15Sacchetti G. Bernini M. Bianchetti A. Parini S. Invernizzi R.W. Samanin R. Br. J. Pharmacol. 1999; 128: 1332-1338Crossref PubMed Scopus (95) Google Scholar). Drugs that block NETs or replace NE anomalously increase NE levels and prolong signaling. In particular, the neurotoxin 1-methyl-4-tetrahydropyridinium (MPP+), an monoamine oxidase (MAO-B) metabolite of 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine, inhibits mitochondrial respiration and induces Parkinson disease symptoms (16Javitch J.A. D'Amato R.J. Strittmatter S.M. Snyder S.H. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 2173-2177Crossref PubMed Scopus (1148) Google Scholar). Understanding hNET and how substrates and drugs interact with this transporter are fundamental questions in neuroscience. The mechanism of NE transport and drug interfere are only partially understood, one obstacle being few data on substrate-transporter interactions. NET belongs to a gene family that uses the Na+ gradient to transport transmitters (14Pacholczyk T. Blakely R.D. Amara S.G. Nature. 1991; 350: 350-354Crossref PubMed Scopus (760) Google Scholar). Hydropathy plots and antibody accessibility data imply that NETs have 12 transmembrane domains with intracellular N and C termini and a large extracellular loop between transmembrane domains II and III (14Pacholczyk T. Blakely R.D. Amara S.G. Nature. 1991; 350: 350-354Crossref PubMed Scopus (760) Google Scholar, 17Savchenko V. Sung U. Blakely R.D. Mol. Cell Neurosci. 2003; 24: 1131-1150Crossref PubMed Scopus (38) Google Scholar). A single gene encodes hNET (18Hahn M.K. Robertson D. Blakely R.D. J. Neurosci. 2003; 23: 4470-4478Crossref PubMed Google Scholar); however, biochemical evidence predicts that NETs are multimers (19Kocabas A.M. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (50) Google Scholar). Furthermore, related transporters for serotonin (SERT) and dopamine (DAT) function as multimers (20Kilic F. Rudnick G. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 3106-3111Crossref PubMed Scopus (189) Google Scholar, 21Ramsey I.S. DeFelice L.J. J. Biol. Chem. 2002; 277: 14475-14482Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar), also suggested by fluorescence resonance energy transfer (22Schmid J.A. Scholze P. Kudlacek O. Freissmuth M. Singer E.A. Sitte H.H. J. Biol. Chem. 2001; 276: 3805-3810Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar) and cross-linking experiments (23Hastrup H. Sen N. Javitch J.A. J. Biol. Chem. 2003; 278: 45045-45048Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). Directional cross-linking and Zn2+ binding suggest that DAT consists of homodimers (24Hastrup H. Karlin A. Javitch J.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 10055-10060Crossref PubMed Scopus (173) Google Scholar, 25Norgaard-Nielsen K. Norregaard L. Hastrup H. Javitch J.A. Gether U. FEBS Lett. 2002; 524: 87-91Crossref PubMed Scopus (34) Google Scholar, 26Torres G.E. Carneiro A. Seamans K. Fiorentini C. Sweeney A. Yao W.D. Caron M.G. J. Biol. Chem. 2003; 278: 2731-2739Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar), and Kocabas et al. (19Kocabas A.M. Rudnick G. Kilic F. J. Neurochem. 2003; 85: 1513-1520Crossref PubMed Scopus (50) Google Scholar) measured oligomerization by co-immunoprecipitation of tagged NETs. Thus, monoamine transporters function as homomultimers (27Sitte H.H. Farhan H. Javitch J.A. Mol. Intervent. 2004; 4: 38-47Crossref PubMed Scopus (110) Google Scholar), but the number of bound substrates per functional unit is unknown. In vitro studies in tissue culture, resealed membrane vesicles, cultured cells, and synaptosomes demonstrate that NE accumulation saturates at micromolar concentrations, but Na+ and Cl- dependence saturate in the millimolar range (28Bonisch H. Fuchs G. Graefe K.H. Naunyn-Schmiedebergs Arch. Pharmacol. 1986; 332: 131-134Crossref PubMed Scopus (16) Google Scholar). The accepted mechanism for co-transporters is the fixed stoichiometry, alternating access model (29DeFelice L.J. Trends Neurosci. 2004; 27: 352-359Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar), which predicts NET transports 1NE:1Na:1Cl roughly once per second (30Friedrich U. Bonisch H. Naunyn-Schmiedebergs Arch. Pharmacol. 1986; 333: 246-252Crossref PubMed Scopus (55) Google Scholar, 31Gu H.H. Wall S. Rudnick G. J. Biol. Chem. 1996; 271: 6911-6916Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar); however, NET-mediated currents are 100 times larger than predicted by this model (32Galli A. DeFelice L.J. Duke B.J. Moore K.R. Blakely R.D. J. Exp. Biol. 1995; 198: 2197-2212Crossref PubMed Google Scholar). Similar currents exist in native cells (33Larsson H.P. Picaud S.A. Werblin F.S. Lecar H. Biophys. J. 1996; 70: 733-742Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 34Bruns D. Engert F. Lux H.D. Neuron. 1993; 10: 559-572Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 35Bruns D. Methods Enzymol. 1998; 296: 593-607Crossref PubMed Scopus (6) Google Scholar, 36Jayanthi L.D. Vargas G. DeFelice L.J. Br. J. Pharmacol. 2002; 135: 1927-1934Crossref PubMed Scopus (19) Google Scholar, 37Ingram S.L. Prasad B.M. Amara S.G. Nat. Neurosci. 2002; 5: 971-978Crossref PubMed Scopus (186) Google Scholar, 38Carvelli L. McDonald P.W. Blakely R.D. Defelice L.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 16046-16051Crossref PubMed Scopus (115) Google Scholar), and the charge-to-substrate ratio is an important key to mechanism (39Rudnick G. Methods Enzymol. 1998; 296: 233-247Crossref PubMed Scopus (37) Google Scholar). Here we use fluorescence methodology and patch clamp to compare ASP+ binding, ASP+ transport, and ASP+-induced current. After adding ASP+, hNET-expressing cells intensify fluorescence initially to binding and secondarily to transport (1Schwartz J.W. Blakely R.D. DeFelice L.J. J. Biol. Chem. 2003; 278: 9768-9777Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Here we use morphological markers to distinguish plasma membrane-localized ASP+ (bound to the cell surface) from mitochondrial ASP+ (transported into the cell). Using fluorescence lifetime imaging microscopy (FLIM), total internal reflection fluorescence (TIRF) microscopy, and fluorescence correlation microscopy (FCS) with ASP+ and GFP-tagged hNETs, we directly measure substrate-transporter kinetics, substrate-transporter stoichiometry, transporter, and substrate surface density, transport rates, and charge/substrate ratios. These data provide a novel model of neurotransmitter co-transport, in which the rate-limiting step is not exclusively large conformation changes but also inefficient binding, expressed as the low probability of transport following binding. Reagents—Experiments were at room temperature unless noted. ASP+ and DiO were from Molecular Probes (Eugene, OR), soluble recombinant EGFP from Clontech (Palo Alto, CA), and other chemicals from Sigma. Images were processed with MetaMorph software (Universal Imaging Corp., Downington, PA). P. Bissel at Virginia Polytechnic synthesized deuterated ASP+. Methyliodide-d3 (84 ml, 1.4 mmol) was added to trans-4-[4-(dimethylamino)-styryl]pyridine (100 mg, 0.45 mmol) in N,N-dimethylformamide (2 ml), and added at 4 h to 20 ml of ether and filtered. The crude product crystallized from methanol gave pyridinium salt as purple needles (111 mg, 67%, m.p. 256 °C), validated by 1H NMR and 13C NMR. Cell Culture—HEK293 cells were kept in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (v/v), 2 mm glutamine, 100 IU/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Stable lines expressing hNET (hNET-293) are previously described (32Galli A. DeFelice L.J. Duke B.J. Moore K.R. Blakely R.D. J. Exp. Biol. 1995; 198: 2197-2212Crossref PubMed Google Scholar, 40Ramamoorthy S. Giovanetti E. Qian Y. Blakely R.D. J. Biol. Chem. 1998; 273: 2458-2466Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). N-terminal GFP-tagged hNET cDNA was a gift from S. Amara. Stable GFP-tagged hNET (GFP-hNET) cells (GFP-hNET-293) were generated by G418 antibiotic selection. HEK293 were FuGENE 6 transfected with GFP-hNET-293 cDNA. At 24 h, transfected cells were selected (G418 250 μg/ml) and colonies were examined for ASP+ or NE uptake. Stable lines were maintained under G418 selection. Radiometric and Mass Spectrometric Transport Assay—hNET-293 cells were plated on poly-l-lysine-coated 24-well plates, 105 cells per well, 3 days before transport assays (90% confluence on 3rd day, media removed by aspiration). Cells were preincubated 10 min in Krebs-Ringer-Hepes (KRH, mm: 130 NaCl, 1.3 KCl, 2.2 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 10 Hepes, pH 7.4) with/without 10 μm desipramine (DS) before [3H]MPP+ or ASP+ exposure to define nonspecific uptake, well above the DS IC50. Between 0.5 and 10 μm there is no significance difference in DS displacement of substrate (data not shown, see Ref. 1Schwartz J.W. Blakely R.D. DeFelice L.J. J. Biol. Chem. 2003; 278: 9768-9777Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). After 5 min, cells were washed 3 times at 4 °C with KRH, and accumulated [3H]MPP+ was determined by liquid scintillation of 1% (w/v) SDS-solubilized cells; [ASP+] was determined by liquid chromatography and tandem mass spectrometry (LC/MS/MS). An electrospray (+ESI) LC-MS assay with selected reaction monitoring determined [ASP+] in hNET-293 cells (incubated in 2 μm ASP+ for 5 min with/without DS). Cells with ASP+ were solubilized in 1% SDS and treated with control [D]3-ASP+ and acetonitrile. Precipitated proteins were pelleted and discarded. Acetonitrile from the supernatant was evaporated and SDS was removed by polymeric anion exchange trap. High performance liquid chromatography was done on a Zorbax SB-C18, 5 μm, 2.1 × 50-mm column in water:acetonitrile gradient buffered with 10 mm ammonium acetate. Mass spectrometric analysis was performed on a Finnigan TSQ-7000 triple quadrapole mass spectrometer with a standard API-1 electrospray ionization source with a 100-μl inner diameter deactivated fused silica capillary. Nitrogen was used for both sheath and auxiliary gas in the mass spectrometer in the selected reaction monitoring mode, to quantify ASP+ (mass to charge ratio, m/z 239 > 223) with [D]3-ASP+ (m/z 242 > 226) as an internal standard. No other significant chromatographic peaks were present in cell extracts at the retention time of ASP+ or internal standard. The lower limit measurement was 150 fm on a column (30 nm in cell extract). Absolute recovery of ASP+ from cell extracts was greater than 70%. The polymeric strong anion exchange trap reduced SDS content from 1 to ∼0.03%. Peak area ratios were evaluated using linear regression weighted with the inverse square of [ASP+]. Calibration range was 150–5000 fmol on the column (30–1000 nm ASP+ in original samples). Evaluation of multipoint calibrations in triplicate yielded concentrations of standards within 10% of the theoretical value. Microscopy—Cells were plated on 35-mm glass-bottom Petri dishes (MatTek, Ashland, MA) coated with poly-l-lysine 3 days before experimentation. Confocal and Spectra Imaging—Confocal images were taken as described previously (1Schwartz J.W. Blakely R.D. DeFelice L.J. J. Biol. Chem. 2003; 278: 9768-9777Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar): culture medium was aspirated and cells immediately mounted on a Zeiss 510 confocal microscope centrally focused on a monolayer using differential interference contrast. Autofluorescence was established from images taken 10 s before adding ASP+. The argon laser was tuned to 488 nm; emitted light was filtered with a 580-nm lp filter (λmax = 610 nm). ASP+ accumulation was measured from average pixel intensity of time-resolved fluorescent images in a differential interference contrast-identified region. Average pixel intensity was used to normalize the data. Membrane-localized ASP+ is defined by a line-scan (3 pixel width) corresponding to a differential interference contrast image. HEK293 cells endogenously accumulate ASP+ (41Stachon A. Schlatter E. Hohage H. Cell. Physiol. Biochem. 1996; 6: 72-91Crossref Scopus (26) Google Scholar); NET-mediated ASP+ accumulation is defined as hNET-293 minus HEK293 fluorescence. Spectral images were collected using the Meta detector on the Zeiss 510. Emitted light was divided into 10-nm segments ranging from 550 to 700 nm. The average pixel intensity in a region of interest was used to generate an ASP+ spectrum. FLIM and Lifetime Spectroscopy—FLIM experiments were performed at the University of Illinois, Champaign-Urbana, in collaboration with N. Barry and E. Gratton (42Hanson K.M. Behne M.J. Barry N.P. Mauro T.M. Gratton E. Clegg R.M. Biophys. J. 2002; 83: 1682-1690Abstract Full Text Full Text PDF PubMed Scopus (233) Google Scholar): hNET-293 cells were exposed to 2 μm ASP+ for 5 min before imaging, and in 2 μm ASP+ several intensity and lifetime images were collected. Lifetimes were established by measuring phase and modulation shifts for ASP+ fluorescence excited by a Ti-sapphire laser at 80 MHz tuned to 920 nm. Calculations for heterdyne frequency measurements are described in Refs. 43Jameson D.M. Gratton E. Hall R.D. Appl. Spectrosc. Rev. 1984; 20: 55-106Crossref Scopus (346) Google Scholar, 44Gratton E. Jameson D.M. Hall R.D. Annu. Rev. Biophys. Bioeng. 1984; 13: 105-124Crossref PubMed Scopus (246) Google Scholar, 45Alcala J.R. Gratton E. Jameson D.M. Anal. Instr. 1985; 14: 225-250Crossref Scopus (10) Google Scholar. The electronic delay was calibrated using 50 nm rhodamine. Lifetime spectroscopy was performed using a Life Spec spectrometer (Edinburgh, Scotland) coupled to a Ti-sapphire laser (Coherent, Santa Clara, CA) producing femtosecond pulses at 1 MHz. TIRF Microscopy—TIRF images were collected on an Olympus IX-70 with TIRF illumination (Olympus, Melville, NY) and dual-view multi-image acquisition (Optical Insights, Santa Fe, NM) with an Orca ER camera (Hamamstu, Bridgewater, NJ). Channel cross-talk (GFP signal in the ASP+ window, or vice versa) was measured by 10-s images taken before ASP+ exposure, and no cross-talk occurred. FCS—Simultaneous GFP-hNET and ASP+ measurements were taken on a Zeiss 510 with confocal II FCS module. GFP-hNET and ASP+ were excited with a 488-nm laser, with emitted light separated with 635 nm long pass dichrohic. GFP-hNET and ASP+ emission were filtered using a 505–530 and 585 band pass. No GFP signal was measured in the ASP+ channel, or vice versa. For all FCS data, the z-position was fixed at the cell membrane coverslip face to minimize mitochondrial contributions. If the ASP+ signal increased over the initial 10-s interval, data were discarded. ASP+ binding was equilibrated in <1 s (1Schwartz J.W. Blakely R.D. DeFelice L.J. J. Biol. Chem. 2003; 278: 9768-9777Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). We collected fluorescence for 10 s in 15 replicates, with auto- and cross-correlation functions determined for each replicate. The back aperture was overfilled to minimize one-photon FCS artifacts (46Hess S.T. Webb W.W. Biophys. J. 2002; 83: 2300-2317Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar), and the focal volume was calibrated using 30 nm rhodamine 6G. FCS measures fluctuations in fluorescence that arise from particle diffusion in the optical volume, chemical reactions, or environmental changes, and the autocorrelation G(τ) reveals time constants of these processes. Following the formulation in Ref. 46Hess S.T. Webb W.W. Biophys. J. 2002; 83: 2300-2317Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, we used the following equation.G(τ)=1N(1−FB+FBe−τ/τB1−FB)(∑i=1nfi(1+τ/τDi)1+τ/ω2τDi)eq.1 The fluctuations depend on the number of particles, N, the characteristic diffusion time, τDi, of those particles in the observation volume, a structure parameter, ω, the weighting factor, fi, the dark fraction, FB, and time constant, τB, for dark to bright conversion. A detailed discussion of the FCS theory is described in Ref. 47Bacia K. Schwille P. Methods. 2003; 29: 74-85Crossref PubMed Scopus (206) Google Scholar. Fluorescence Recovery After Photobleach—FRAP measurements were made using a procedure described in Ref. 48Cole N.B. Smith C.L. Sciaky N. Terasaki M. Edidin M. Lippincott-Schwartz J. Science. 1996; 273: 797-801Crossref PubMed Scopus (406) Google Scholar: GFP-hNET images were acquired with a ×40 (NA = 1.3) objective (×4 digital zoom) on a Zeiss 510. Three pre-bleach images were acquired before bleaching a 4 × 17.5-μm strip of membrane, and data were fit to an inhomogeneous diffusion model (49Siggia E.D. Lippincott-Schwartz J. Bekiranov S. Biophys. J. 2000; 79: 1761-1770Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar). Whole Cell Voltage Clamp—hNET-293 cells on glass coverslips were washed 2 times with bath solution and mounted in a perfusion chamber (SD instruments, San Diego, CA) on an Olympus IX-70 microscope. Whole cell voltage clamp was achieved with an Axopatch 200 B amplifier (Axon instruments, San Diego CA), as described in Ref. 32Galli A. DeFelice L.J. Duke B.J. Moore K.R. Blakely R.D. J. Exp. Biol. 1995; 198: 2197-2212Crossref PubMed Google Scholar. Bath solution was (in mm): 130 NaCl, 1.3 KH2PO4, 0.5 MgSO4, 1.5 CaCl2, 10 Hepes, 34 glucose, adjusted to pH 7.35 with NaOH and 300 mosmol liter-1 with glucose. Pipette solution was (in mm): 120 KCl, 2 MgCl2, 0.1 CaCl2, 1 EGTA, 10 Hepes, 30 glucose, adjusted to pH 7.35 with KOH and 270 mosmol liter-1 with glucose. ASP+ or NE were added freshly to the bath solution at 2 and 30 μm, respectively, and perfused with a gravity driven system. All data related to ASP+ solution exchange (except infinite dilution experiments) were taken at 3 s or later, with ASP+ at its full concentration. ASP+ is quenched until bound, further insuring that we monitor only the kinetics of bound and subsequently transported ASP+. Optical Isolation of Plasma Membrane and Mitochondrial Localized ASP+—To investigate ASP+ bound to plasma membrane-localized NET, we used subcellular organelle stains. hNET-293 cells were stained with 500 nm 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO) for 20 min prior to ASP+ exposure. ASP+ is optically isolated from DiO, a membrane-specific marker (Fig. 1, A and B). Imaging DiO-stained cells for 10 s before adding ASP+ showed negligible cross-talk between ASP+ and DiO channels. Initially, ASP+ staining co-localizes with DiO fluorescence (first 3 s, data not shown). After 3 s, we observe intracellular punctuated ASP+ distributions (Fig. 1A) not co-localized with DiO (Fig. 1C). Instead, punctuated intracellular ASP+ co-localizes with a mitochondria stain (Mito-Tracker Green Molecular Probes, Eugene OR, data not shown), whereas plasma membrane ASP+ is still co-localized with DiO (Fig. 1C, yellow). Panel D shows the average pixel intensity along a 3-pixel width line from the bath, through the plasma membrane, to the mitochondria. In the first seconds (black line), fluorescence localizes to the plasma membrane. After 120 s (blue), mitochondrial ASP+ is significant. Subsequent exposure to NE (30 μm) displaces plasma membrane ASP+ without altering mitochondrial fluorescence (Fig. 1D, red). HEK293 cells measured in parallel demonstrate <0.5% membrane binding and <10% mitochondrial staining. Although line scans separate plasma membrane from deep mitochondria, circumference fluorescence may not report pure plasma membrane. Imperfect separation of near-membrane mitochondria from membrane movement results in ∼25% residual fluorescence in the membrane component, because of relatively low spatial resolution of confocal microscopy and cellular movement during the 2-min incubation. With these restrictions, data in Fig. 1A are quantified in panel E. Plasma membrane-localized fluorescence saturates after ∼6 s (blue), whereas mitochondrial ASP+ continues to increase (Fig. 1, red). At 120 s, 30 μm NE displaces plasma membrane ASP+ and arrests accumulation of mitochondrial ASP+. To improve resolution we use TIRF microscopy (below). Transporter Plasma Membrane Distribution—TIRF microscopy examines a narrow evanescent field (<1000 Å) where cells contact the coverslip (50Axelrod D. Traffic. 2001; 2: 764-774Crossref PubMed Scopus (734) Google Scholar) (Fig. 2). Panel A shows ASP-bound to transporters clustered on the surface and localized to thin filaments at pseudopodia. ASP+ exhibits an identical pattern to GFP-hNET (Fig. 2B) and is displaced by 1 μm DS. To evaluate membrane folding as a source of this pattern, we compared DiO HEK293 cells and GFP-hNET-293: only DiO images were uniform, whereas GFP images show patterns. These data demonstrate co-localization of ASP+ and GFP-hNET in the evanescent field (Fig. 2C), although the distribution of the paired molecules varies widely. Time-resolved patterns indicate substrate-transporter complexes constantly migrating on the surface, but total surface expression remains constant. ASP+and GFP-calibrated Images—To determine the stoichiometry of ASP+ to transporters we used FLIM (see “Materials and Methods”). The fluorescence generated from a population of molecules is dependent on: 1) absorbability (molar extinction); 2) path length; 3) excitation light intensity; 4) quantum yield; and 5) concentration. Knowing this enables conversion of ASP+ fluorescence (AFU) to molecular number (51Lakowicz J. Principles of Fluoresence Spectroscopy. 2nd Ed. Kluwer Academic/Plenum Publishers, New York1999Crossref Google Scholar). Cellular ASP+ fluorescence lifetime was determined by fluorescent phase shift and modulation using heterodyne frequency domain measurements (43Jameson D.M. Gratton E. Hall R.D. Appl. Spectrosc. Rev. 1984; 20: 55-106Crossref Scopus (346) Google Scholar, 45Alcala J.R. Gratton E. Jameson D.M. Anal. Instr. 1985; 14: 225-250Crossref Scopus (10) Google Scholar). To determine the cellular ASP+ fluorescence lifetime, hNET-293 cells were exposed to 2 μm ASP+ for 5 min. The intensity image (Fig. 2A) shows that ASP+ binding is strongest along the plasma membrane, but the signal also arises from mitochondrial regions inside the cell. Using the intensity image as a template, the lifetime of ASP+ can be determined from FLIM (Fig. 2B) on the plasma membrane and in the mitochondria. The heterdyne frequency method provides two measurements of the average lifetime in a diverse population, τphase and τmod. The former measures lifetime via changes in phase of the excitation fluorescence, the latter measures changes in frequency modulation of the excitation fluorescence. Average ASP+ lifetimes were equal for plasma membrane and mitochondrial fluorescence (τphase and τmod were 2.5 ± 0.5 and 1.8 ± 0.36 ns, respectively), indicating that an increase in ASP+ qu" @default.
• W2037610035 created "2016-06-24" @default.
• W2037610035 creator A5013928213 @default.
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• W2037610035 creator A5049876115 @default.
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• W2037610035 date "2005-05-01" @default.
• W2037610035 modified "2023-10-09" @default.
• W2037610035 title "Substrate Binding Stoichiometry and Kinetics of the Norepinephrine Transporter" @default.
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