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Red Fluorescent Protein pH Biosensor to Detect Concentrative Nucleoside Transport*

  • Danielle E. Johnson
    Footnotes
    Affiliations
    Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7
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  • Hui-wang Ai
    Footnotes
    Affiliations
    Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
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  • Peter Wong
    Footnotes
    Affiliations
    Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
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  • James D. Young
    Footnotes
    Affiliations
    Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7
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  • Robert E. Campbell
    Affiliations
    Department of Chemistry, University of Alberta, Edmonton T6G 2G2, Canada
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  • Joseph R. Casey
    Correspondence
    To whom correspondence should be addressed: Dept. of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada. Tel.: 780-492-7203; Fax: 780-492-8915;
    Footnotes
    Affiliations
    Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7
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  • Author Footnotes
    * This work was supported in part by a Canadian Institutes of Health Research operating grant (to J. R. C.), by financial support from the Canada Foundation for Innovation, the Natural Sciences and Engineering Research Council, and Alberta Ingenuity (to R. E. C.), and by support from the National Cancer Institute of Canada (to J. D. Y.).
    The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2.
    1 Both authors contributed equally to this work.
    2 Supported by an Alberta Heritage Foundation for Medical Research (AHFMR) studentship.
    3 Present address: The Scripps Research Institute, La Jolla, CA 92037.
    4 Supported by the Natural Sciences and Engineering Research Council, undergraduate student research award.
    5 AHFMR Scientists.
Open AccessPublished:June 03, 2009DOI:https://doi.org/10.1074/jbc.M109.019042
      Human concentrative nucleoside transporter, hCNT3, mediates Na+/nucleoside and H+/nucleoside co-transport. We describe a new approach to monitor H+/uridine co-transport in cultured mammalian cells, using a pH-sensitive monomeric red fluorescent protein variant, mNectarine, whose development and characterization are also reported here. A chimeric protein, mNectarine fused to the N terminus of hCNT3 (mNect.hCNT3), enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in HEK293 cells expressing mNect.hCNT3 or mNect.hCNT3-F563C, an inactive hCNT3 mutant. Free cytosolic mNect, mNect.hCNT3, and the traditional pH-sensitive dye, BCECF, reported cytosolic pH similarly in pH-clamped HEK293 cells. Cells were incubated at the permissive pH for H+-coupled nucleoside transport, pH 5.5, under both Na+-free and Na+-containing conditions. In mNect.hCNT3-expressing cells (but not under negative control conditions) the rate of acidification increased in media containing 0.5 mm uridine, providing the first direct evidence for H+-coupled uridine transport. At pH 5.5, there was no significant difference in uridine transport rates (coupled H+ flux) in the presence or absence of Na+ (1.09 ± 0.11 or 1.18 ± 0.32 mm min−1, respectively). This suggests that in acidic Na+-containing conditions, 1 Na+ and 1 H+ are transported per uridine molecule, while in acidic Na+-free conditions, 1 H+ alone is transported/uridine. In acid environments, including renal proximal tubule, H+/nucleoside co-transport may drive nucleoside accumulation by hCNT3. Fusion of mNect to hCNT3 provided a simple, self-referencing, and effective way to monitor nucleoside transport, suggesting an approach that may have applications in assays of transport activity of other H+-coupled transport proteins.
      Nucleosides are hydrophilic molecules that require transport proteins to mediate their movement across the plasma membrane (
      • Griffith D.A.
      • Jarvis S.M.
      ). Human (h)
      The abbreviations used are: h
      human
      NT
      nucleoside transport protein
      C
      concentrative
      E
      equilibrative
      mNect.hCNT3
      mNectarine fused to the N terminus of hCNT3
      EIPA
      5-(N-ethyl-N-isopropyl)amiloride
      NHE1
      Na+/H+ exchanger isoform 1
      FP
      fluorescent protein
      RFP
      red fluorescent protein
      mRFP
      monomeric RFP
      avGFP
      A. victoria green fluorescent protein
      pKa
      equal to the pH at which the fluorescence is half-maximal in intensity
      Discosoma RFP
      tetrameric Discosoma RFP
      YFP
      yellow FP
      enhanced avGFP
      enhanced GFP
      SGLT1
      Na+/glucose cotransporter isoform 1
      PEPT1/2
      proton-dependent oligopeptide transporters isoforms 1/2
      MCT1
      monocarboxylate transporter isoform 1
      PNGaseF
      peptide: N-glycosidase F
      MES
      4-morpholineethanesulfonic acid
      BCECF-AM
      3′,6′-bis(acetyloxy)-5(or 6)-[[(acetyloxy)methoxy]carbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-2′,7′-dipropanoic acid 2′,7′-bis[(acetyloxy)methyl] ester.
      7The abbreviations used are: h
      human
      NT
      nucleoside transport protein
      C
      concentrative
      E
      equilibrative
      mNect.hCNT3
      mNectarine fused to the N terminus of hCNT3
      EIPA
      5-(N-ethyl-N-isopropyl)amiloride
      NHE1
      Na+/H+ exchanger isoform 1
      FP
      fluorescent protein
      RFP
      red fluorescent protein
      mRFP
      monomeric RFP
      avGFP
      A. victoria green fluorescent protein
      pKa
      equal to the pH at which the fluorescence is half-maximal in intensity
      Discosoma RFP
      tetrameric Discosoma RFP
      YFP
      yellow FP
      enhanced avGFP
      enhanced GFP
      SGLT1
      Na+/glucose cotransporter isoform 1
      PEPT1/2
      proton-dependent oligopeptide transporters isoforms 1/2
      MCT1
      monocarboxylate transporter isoform 1
      PNGaseF
      peptide: N-glycosidase F
      MES
      4-morpholineethanesulfonic acid
      BCECF-AM
      3′,6′-bis(acetyloxy)-5(or 6)-[[(acetyloxy)methoxy]carbonyl]-3-oxo-spiro[isobenzofuran-1(3H),9′-[9H]xanthene]-2′,7′-dipropanoic acid 2′,7′-bis[(acetyloxy)methyl] ester.
      nucleoside transport (NT) proteins catalyze the vectorial transport of nucleosides, using either concentrative (C) or equilibrative (E) mechanisms (
      • Young J.D.
      • Cheeseman C.I.
      • Mackey J.R.
      • Cass C.E.
      • Baldwin S.A.
      ). hCNTs use either a Na+ or H+ gradient to accumulate nucleosides against their concentration gradient, whereas hENTs mediate facilitated diffusion of nucleosides down their concentration gradient (
      • Elwi A.N.
      • Damaraju V.L.
      • Baldwin S.A.
      • Young J.D.
      • Sawyer M.B.
      • Cass C.E.
      ). Nucleoside transporters also transport anti-cancer and anti-viral drugs, and cellular expression of nucleoside transporters is important in cancer therapy as well as in the treatment of cardiovascular, parasitic, and viral diseases (
      • King A.E.
      • Ackley M.A.
      • Cass C.E.
      • Young J.D.
      • Baldwin S.A.
      ,
      • Zhang J.
      • Visser F.
      • King K.M.
      • Baldwin S.A.
      • Young J.D.
      • Cass C.E.
      ).
      Members of the SLC28 family of concentrative nucleoside transporters (CNTs) divide into two phylogenetic subfamilies: hCNT1/2 belonging to one subfamily, and hCNT3 to the other (
      • Ritzel M.W.
      • Yao S.Y.
      • Huang M.Y.
      • Elliott J.F.
      • Cass C.E.
      • Young J.D.
      ,
      • Ritzel M.W.
      • Yao S.Y.
      • Ng A.M.
      • Mackey J.R.
      • Cass C.E.
      • Young J.D.
      ,
      • Ritzel M.W.
      • Ng A.M.
      • Yao S.Y.
      • Graham K.
      • Loewen S.K.
      • Smith K.M.
      • Ritzel R.G.
      • Mowles D.A.
      • Carpenter P.
      • Chen X.Z.
      • Karpinski E.
      • Hyde R.J.
      • Baldwin S.A.
      • Cass C.E.
      • Young J.D.
      ). Cation substitution and charge/flux ratio studies suggest that hCNT1/2 couple the inward movement of nucleoside to the Na+ electrochemical gradient with a 1:1 stoichiometry, whereas hCNT3 can couple nucleoside transport to either the Na+ gradient (2 Na+:1 nucleoside) or a H+ gradient (1 H+:1 nucleoside) in the absence of Na+ (
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). The 2:1 coupling ratio of hCNT3 allows it to develop a trans-membrane nucleoside concentration gradient up to 10-fold higher than that of hCNT1 or hCNT2 (
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Damaraju V.L.
      • Elwi A.N.
      • Hunter C.
      • Carpenter P.
      • Santos C.
      • Barron G.M.
      • Sun X.
      • Baldwin S.A.
      • Young J.D.
      • Mackey J.R.
      • Sawyer M.B.
      • Cass C.E.
      ). At pH 5.5, hCNT3 also transports uridine in the presence of Na+ with a 2 cation:1 nucleoside stoichiometry, which raises the possibility that 1 H+ and 1 Na+ may be transported per nucleoside molecule in these conditions (
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Damaraju V.L.
      • Elwi A.N.
      • Hunter C.
      • Carpenter P.
      • Santos C.
      • Barron G.M.
      • Sun X.
      • Baldwin S.A.
      • Young J.D.
      • Mackey J.R.
      • Sawyer M.B.
      • Cass C.E.
      ,
      • Slugoski M.D.
      • Smith K.M.
      • Mulinta R.
      • Ng A.M.
      • Yao S.Y.
      • Morrison E.L.
      • Lee Q.O.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). Up to this point, however, there has been no direct demonstration that hCNT3 can transport H+.
      Concentrative nucleoside transport has previously been investigated using the Xenopus laevis oocyte expression system and both electrophysiology (two-microelectrode voltage clamp technique) and radioisotope flux measurements (
      • Ritzel M.W.
      • Yao S.Y.
      • Huang M.Y.
      • Elliott J.F.
      • Cass C.E.
      • Young J.D.
      ,
      • Ritzel M.W.
      • Yao S.Y.
      • Ng A.M.
      • Mackey J.R.
      • Cass C.E.
      • Young J.D.
      ,
      • Ritzel M.W.
      • Ng A.M.
      • Yao S.Y.
      • Graham K.
      • Loewen S.K.
      • Smith K.M.
      • Ritzel R.G.
      • Mowles D.A.
      • Carpenter P.
      • Chen X.Z.
      • Karpinski E.
      • Hyde R.J.
      • Baldwin S.A.
      • Cass C.E.
      • Young J.D.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Smith K.M.
      • Mulinta R.
      • Ng A.M.
      • Yao S.Y.
      • Morrison E.L.
      • Lee Q.O.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). Electrophysiological experiments are advantageous in that they measure the current induced by addition of substrate in real-time, but they are time-consuming and require specialized equipment and skills. Radioisotope flux assays measure the accumulation of radiolabeled substrate. The need for radiolabeled substrate restricts the range of permeants able to be studied. In addition, radioisotope flux assays are not done in real-time and are labor-intensive, requiring large numbers of oocytes.
      An attractive alternative approach for the study of hCNT3 would be to measure pH in the immediate vicinity of its intracellular face during H+/nucleoside co-transport. These measurements could take advantage of the remarkable progress achieved in the development of genetically encoded fluorophores (
      • Giepmans B.N.
      • Adams S.R.
      • Ellisman M.H.
      • Tsien R.Y.
      ). Indeed, all members of the extended family of homologues and variants of the Aequorea victoria green fluorescent protein (avGFP) exhibit pH-dependent changes in their fluorescent intensity. The spectral changes that occur upon a change in pH can be intensiometric (
      • Miesenböck G.
      • De Angelis D.A.
      • Rothman J.E.
      ), excitation ratiometric (
      • Miesenböck G.
      • De Angelis D.A.
      • Rothman J.E.
      ), emission ratiometric (
      • Hanson G.T.
      • McAnaney T.B.
      • Park E.S.
      • Rendell M.E.
      • Yarbrough D.K.
      • Chu S.
      • Xi L.
      • Boxer S.G.
      • Montrose M.H.
      • Remington S.J.
      ), or both excitation and emission ratiometric (
      • Bizzarri R.
      • Arcangeli C.
      • Arosio D.
      • Ricci F.
      • Faraci P.
      • Cardarelli F.
      • Beltram F.
      ). The apparent pKa (pKa′, equal to the pH at which the fluorescence is half-maximal in intensity) for a specific fluorescent protein (FP) is acutely dependent on specific amino acid substitutions in close proximity to the chromophore and can range from less than 3 (
      • Ai H.W.
      • Shaner N.C.
      • Cheng Z.
      • Tsien R.Y.
      • Campbell R.E.
      ,
      • Subach O.M.
      • Gundorov I.S.
      • Yoshimura M.
      • Subach F.V.
      • Zhang J.
      • Grüenwald D.
      • Souslova E.A.
      • Chudakov D.M.
      • Verkhusha V.V.
      ) to greater than 8 (
      • Abad M.F.
      • Di Benedetto G.
      • Magalhães P.J.
      • Filippin L.
      • Pozzan T.
      ). Variants with pKa′ values that are relatively close to intracellular pH values (i.e. ∼7.3 for the mammalian cytosol (
      • Llopis J.
      • McCaffery J.M.
      • Miyawaki A.
      • Farquhar M.G.
      • Tsien R.Y.
      )) are particularly useful as genetically encoded biosensors for dynamic measurement of proton concentrations in living cells.
      A major development in the area of FP technology has been the identification (
      • Matz M.V.
      • Fradkov A.F.
      • Labas Y.A.
      • Savitsky A.P.
      • Zaraisky A.G.
      • Markelov M.L.
      • Lukyanov S.A.
      ) and subsequent optimization (
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      ,
      • Campbell R.E.
      • Tour O.
      • Palmer A.E.
      • Steinbach P.A.
      • Baird G.S.
      • Zacharias D.A.
      • Tsien R.Y.
      ) of red fluorescent protein (RFP) homologues of avGFP. The first (monomeric RFP 1 (mRFP1)) (
      • Campbell R.E.
      • Tour O.
      • Palmer A.E.
      • Steinbach P.A.
      • Baird G.S.
      • Zacharias D.A.
      • Tsien R.Y.
      ) and second (the mFruit series) (
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      ) generation-optimized RFPs, derived from tetrameric Discosoma RFP (
      • Matz M.V.
      • Fradkov A.F.
      • Labas Y.A.
      • Savitsky A.P.
      • Zaraisky A.G.
      • Markelov M.L.
      • Lukyanov S.A.
      ), suffer from relatively low brightness relative to other common hues of FP. For example, of the three most red-shifted second generation mFruit variants (mTangerine, mStrawberry, and mCherry) (
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      ), the brightest (mStrawberry) has only 44% of the intrinsic brightness (proportional to the product of extinction coefficient (ϵ) and quantum yield (Φ)) of the popular yellow FP (YFP) Citrine (
      • Griesbeck O.
      • Baird G.S.
      • Campbell R.E.
      • Zacharias D.A.
      • Tsien R.Y.
      ) and 76% of the brightness of enhanced avGFP. This limitation has been partially addressed by third generation mRFPs, specifically mApple and TagRFP-T, with fluorescent brightness values on par with, or better than, that of enhanced avGFP (
      • Shaner N.C.
      • Lin M.Z.
      • McKeown M.R.
      • Steinbach P.A.
      • Hazelwood K.L.
      • Davidson M.W.
      • Tsien R.Y.
      ).
      Generally speaking, the most red-shifted RFPs derived from Discosoma RFP are relatively pH-insensitive, with the majority of variants having pKa′ values < 5 (
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      ,
      • Shaner N.C.
      • Lin M.Z.
      • McKeown M.R.
      • Steinbach P.A.
      • Hazelwood K.L.
      • Davidson M.W.
      • Tsien R.Y.
      ). A notable exception is the recently reported mApple variant with a pKa′ of 6.5 (
      • Shaner N.C.
      • Lin M.Z.
      • McKeown M.R.
      • Steinbach P.A.
      • Hazelwood K.L.
      • Davidson M.W.
      • Tsien R.Y.
      ). The more blue-shifted of the mFruit variants (i.e. mOrange) also have pKa′ values of 6.5 (
      • Shaner N.C.
      • Campbell R.E.
      • Steinbach P.A.
      • Giepmans B.N.
      • Palmer A.E.
      • Tsien R.Y.
      ). Several variants of mRFP1 with pKa′ values >7.5 have been previously reported (
      • Jach G.
      • Pesch M.
      • Richter K.
      • Frings S.
      • Uhrig J.F.
      ).
      Here we report the engineering of a pH-sensitive mFruit variant through multiple rounds of directed evolution by random mutagenesis. This RFP, called mNectarine, is appropriate to measure physiological pH changes in mammalian cells, because it has a pKa′ of 6.9. We have developed a new method to measure H+/nucleoside co-transport in mammalian cells, which utilizes hCNT3's H+ coupling characteristics and the pH sensitivity of mNectarine. We fused mNectarine to the cytosolic N terminus of hCNT3 to generate mNect.hCNT3. Fusion of the fluorescent H+ sensor to hCNT3 enabled measurement of pH at the intracellular surface of hCNT3, and provided insight into the mechanism of hCNT3 H+/uridine co-transport.

      DISCUSSION

      We utilized hCNT3's H+-coupling characteristics to develop a new method to assay nucleoside transport in cultured mammalian cells. This assay was made possible by engineering a new pH-sensitive red fluorescent protein, mNectarine. mNectarine, fused to the N terminus of hCNT3, was able to reliably report on changes in pH at the intracellular surface of hCNT3 in real-time. Addition of uridine to mNect.hCNT3-expressing cells, in both Na+-free and Na+-containing conditions elicited an increase in the rate of acidification that was not seen in the negative control (mNect.hCNT3-F563C-expressing cells). Our data are consistent with hCNT3-mediated Na+/H+/nucleoside co-transport under acid conditions.
      Using mNect.hCNT3 to measure uridine flux by monitoring H+ co-transport is advantageous, because it enables direct measurement of changes in intracellular H+ concentration, which, up until now, had only been inferred from H+ activation of [14C]uridine influx and pH-dependent uridine-evoked currents in oocytes (
      • Ritzel M.W.
      • Ng A.M.
      • Yao S.Y.
      • Graham K.
      • Loewen S.K.
      • Smith K.M.
      • Ritzel R.G.
      • Mowles D.A.
      • Carpenter P.
      • Chen X.Z.
      • Karpinski E.
      • Hyde R.J.
      • Baldwin S.A.
      • Cass C.E.
      • Young J.D.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Ng A.M.
      • Yao S.Y.
      • Smith K.M.
      • Lin C.C.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). The present experiments therefore provide the first direct evidence that hCNT3 transports H+. Because we did not directly measure uridine flux in these experiments, it is formally possible that the mNect.hCNT3 assay measures an hCNT3-mediated H+ flux unrelated to uridine transport. We consider this unlikely, because under similar extracellular acid medium conditions uptake of radioactive nucleoside was observed in oocytes (
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ).
      We assessed the reliability of mNect.hCNT3 as a reporter for hCNT3 H+/nucleoside co-transport, by measuring the Km of mNect.hCNT3 for uridine. The Km determined here (72 ± 24 μm) was between two previously published values for hCNT3 expressed in oocytes (110 ± 10 and 62 ± 5 μm) (
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Smith K.M.
      • Mulinta R.
      • Ng A.M.
      • Yao S.Y.
      • Morrison E.L.
      • Lee Q.O.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ), indicating that mNect.hCNT3 provides an accurate method to measure hCNT3 kinetics.
      This study illuminates the mechanism of nucleoside transport under conditions of extracellular acid and Na+. Previous investigations in X. laevis oocytes demonstrated that uridine was transported with high efficiency in both Na+-containing medium at pH 5.5, and Na+-free medium at pH 5.5, although to a somewhat lesser extent under Na+-free conditions (
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Smith K.M.
      • Mulinta R.
      • Ng A.M.
      • Yao S.Y.
      • Morrison E.L.
      • Lee Q.O.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). hCNT3 co-transport of nucleoside with Na+ has been verified outside oocytes, using yeast and mammalian cells (
      • Zhang J.
      • Tackaberry T.
      • Ritzel M.W.
      • Raborn T.
      • Barron G.
      • Baldwin S.A.
      • Young J.D.
      • Cass C.E.
      ,
      • Toan S.V.
      • To K.K.
      • Leung G.P.
      • de Souza M.O.
      • Ward J.L.
      • Tse C.M.
      ,
      • Errasti-Murugarren E.
      • Pastor-Anglada M.
      • Casado F.J.
      ). In the current study, there was H+ movement in both acidic Na+-containing and acidic Na+-free medium, with no significant difference in transport rate between the two conditions. This result indicates that H+ is co-transported with uridine in acidic Na+-containing buffer. Charge/flux ratio experiments in oocytes revealed that hCNT3 functions with 2:1 cation:uridine stoichiometry in both acidic and alkaline Na+-containing medium, compared with a 1:1 cation:uridine stoichiometry in acidic Na+-free medium (
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Smith K.M.
      • Mulinta R.
      • Ng A.M.
      • Yao S.Y.
      • Morrison E.L.
      • Lee Q.O.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). Because we find similar rates of H+ movement in the presence and absence of Na+, the present data support a model in which 1 H+ and 1 Na+ are transported per uridine molecule in acidic Na+-containing medium, compared with 1 H+ per uridine molecule in the absence of Na+, and 2 Na+ per uridine molecule in alkaline conditions.
      mNect.hCNT3 can be used to measure the transport rate for any nucleoside or nucleoside drug that is co-transported with H+, so any range of substrates can be assayed for transport, even if a radioactive analogue is unavailable. This characteristic, along with utilizing a pH-sensitive fluorescent reporter, opens up the possibility of high throughput assays in which a wide range of possible substrates could be added to mNect.hCNT3-transfected cells grown in multiwell plates while monitoring fluorescence changes over time with a multiwell plate fluorometer. mNect.hCNT3 also provides the possibility of high throughput screening for inhibitors, as no high affinity CNT inhibitors have yet been identified. In contrast to hCNT3 assays performed using X. laevis oocytes (
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Ng A.M.
      • Yao S.Y.
      • Smith K.M.
      • Lin C.C.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ), the mNect.hCNT3 assay enables studies of hCNT3 regulation in a physiologically relevant mammalian cell context.
      Interestingly, hCNT3 exhibits markedly different selectivity characteristics for symport of physiological nucleosides and therapeutic nucleoside drugs in acidic Na+-free or alkaline Na+-containing conditions (
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). For example, in acidic Na+-free conditions, transport of guanosine, 3′-azido-3′-deoxythymidine and 2′,3′-dideoxycytidine are almost completely abolished (
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Ng A.M.
      • Yao S.Y.
      • Smith K.M.
      • Lin C.C.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). This suggests that binding of Na+ or H+ induces cation-specific conformational changes in hCNT3 (
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ). Differential H+ or Na+ binding to the SGLT1 Na+/glucose transporter also leads to cation-specific conformational changes (
      • Hirayama B.A.
      • Loo D.D.
      • Wright E.M.
      ). Using mNect.hCNT3, it will be possible to measure transport of nucleosides and nucleoside drugs in acidic Na+-containing conditions. In conjunction with corresponding electrophysiological recordings in hCNT3-expressing oocytes (
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Loewen S.K.
      • Ng A.M.
      • Yao S.Y.
      • Chen X.Z.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Smith K.M.
      • Mulinta R.
      • Ng A.M.
      • Yao S.Y.
      • Morrison E.L.
      • Lee Q.O.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ,
      • Slugoski M.D.
      • Ng A.M.
      • Yao S.Y.
      • Smith K.M.
      • Lin C.C.
      • Zhang J.
      • Karpinski E.
      • Cass C.E.
      • Baldwin S.A.
      • Young J.D.
      ), it may be possible to discern an intermediate conformation where both Na+ and H+ are bound.
      The concentrative nucleoside transporters are primarily expressed in epithelial cells where they are involved in absorption, secretion, distribution, and elimination of nucleosides and nucleoside analogs (
      • Young J.D.
      • Cheeseman C.I.
      • Mackey J.R.
      • Cass C.E.
      • Baldwin S.A.
      ,
      • Elwi A.N.
      • Damaraju V.L.
      • Baldwin S.A.
      • Young J.D.
      • Sawyer M.B.
      • Cass C.E.
      ,
      • Damaraju V.L.
      • Elwi A.N.
      • Hunter C.
      • Carpenter P.
      • Santos C.
      • Barron G.M.
      • Sun X.
      • Baldwin S.A.
      • Young J.D.
      • Mackey J.R.
      • Sawyer M.B.
      • Cass C.E.
      ). hCNT3 is especially abundant in the kidney, and may be the key CNT at the apical surface of the proximal tubule (
      • Elwi A.N.
      • Damaraju V.L.
      • Baldwin S.A.
      • Young J.D.
      • Sawyer M.B.
      • Cass C.E.
      ,
      • Damaraju V.L.
      • Elwi A.N.
      • Hunter C.
      • Carpenter P.
      • Santos C.
      • Barron G.M.
      • Sun X.
      • Baldwin S.A.
      • Young J.D.
      • Mackey J.R.
      • Sawyer M.B.
      • Cass C.E.
      ). The ability of hCNT3 to couple nucleoside movement to either a Na+ and/or H+ gradient may be advantageous in the kidney, because the lumen of the proximal tubule is acidic, and hCNT3 could take advantage of the H+ gradient to maximize its transport rate in these conditions. Both hCNT3 and SGLT1, which have similar transport kinetics, are present in the intestine, which also has an acidic luminal environment, particularly in the more proximal regions (
      • Hirayama B.A.
      • Loo D.D.
      • Wright E.M.
      ). Thus, the H+-coupling characteristic of hCNT3 may be physiologically and pharmacologically important (
      • Elwi A.N.
      • Damaraju V.L.
      • Baldwin S.A.
      • Young J.D.
      • Sawyer M.B.
      • Cass C.E.
      ,
      • Smith K.M.
      • Slugoski M.D.
      • Cass C.E.
      • Baldwin S.A.
      • Karpinski E.
      • Young J.D.
      ).
      The use of pH-sensitive fluorescent protein fusions as an assay of transport activity extends beyond hCNT3. mNectarine could be fused to any transporter that induces a change in intracellular pH. Such transporters could include the H+-coupled PepT1/2 peptide transporters (
      • Mackenzie B.
      • Loo D.D.
      • Fei Y.
      • Liu W.J.
      • Ganapathy V.
      • Leibach F.H.
      • Wright E.M.
      ,
      • Chen X.Z.
      • Zhu T.
      • Smith D.E.
      • Hediger M.A.
      ), SGLT1 (
      • Hirayama B.A.
      • Loo D.D.
      • Wright E.M.
      ), and the MCT1 monocarboxylate transporter (
      • Becker H.M.
      • Deitmer J.W.
      ), or any of the transport proteins involved in the regulation of pHi, including Cl/HCO3 exchangers (
      • McMurtrie H.L.
      • Cleary H.J.
      • Alvarez B.V.
      • Loiselle F.B.
      • Sterling D.
      • Morgan P.E.
      • Johnson D.E.
      • Casey J.R.
      ), NHEs (
      • Slepkov E.R.
      • Rainey J.K.
      • Sykes B.D.
      • Fliegel L.
      ), or sodium bicarbonate co-transporters (
      • Romero M.F.
      ). PepT1 (
      • Daniel H.
      • Kottra G.
      ), MCT1 (
      • Poole R.C.
      • Sansom C.E.
      • Halestrap A.P.
      ), NHE1 (
      • Slepkov E.R.
      • Rainey J.K.
      • Sykes B.D.
      • Fliegel L.
      ), and the SLC4 family members (including Cl/HCO3 exchangers and sodium bicarbonate co-transporters) (
      • Romero M.F.
      ), all have at least one intracellular terminus, so an mNectarine fusion could be constructed. SGLT1 does not have intracellular N or C termini, but FP fusions could possibly be made in the large cytosolic loop (
      • Wright E.M.
      • Turk E.
      ). Fusion of mNectarine to these proteins would report on changes in pH local to the transporter and would be more specific than simply measuring pH changes in the bulk cytosol with a pH-sensitive intracellular dye. Moreover, the self-referencing nature of an mNect.transporter fusion ensures that transport activity is measured only in the cells expressing the transport protein. This would be particularly beneficial in studying pH microdomains or metabolons (
      • Becker H.M.
      • Deitmer J.W.
      ,
      • Sterling D.
      • Reithmeier R.A.
      • Casey J.R.
      ).
      Furthermore, the spectral characteristics of mNectarine make it ideal for use as a partner with other fluorescent reporters. Even more tantalizing, is the prospect of pairing one mNectarine fusion protein with another pH-sensitive fluorescent protein fusion. This would allow simultaneous measurement of pH in two different regions of a cell. For example, mNectarine can be paired with the green fluorescent protein deGFP4 (
      • Hanson G.T.
      • McAnaney T.B.
      • Park E.S.
      • Rendell M.E.
      • Yarbrough D.K.
      • Chu S.
      • Xi L.
      • Boxer S.G.
      • Montrose M.H.
      • Remington S.J.
      ,
      • Johnson D.E.
      • Casey J.R.
      ), because they exhibit spectrally distinct wavelengths, which do not exhibit cross-talk (not shown).
      D. E. Johnson, H.-w. Ai, P. Wong, J. D. Young, R. E. Campbell, and J. R. Casey, our observations.
      Indeed, ongoing studies are investigating the phenomenon of differential pH surrounding pH regulatory transport proteins, resulting from unstirred layer effects.
      In conclusion, we have developed a method to measure H+/uridine co-transport in mammalian cells by fusing the pH-sensitive fluorescent protein, mNectarine, to the N terminus of hCNT3. mNectarine is a bright, pH-sensitive mRFP with a pKa well suited to measurement of physiological pH changes. Fusion to hCNT3 created a self-reporting probe of nucleoside transport that can be expressed in mammalian cells. Furthermore, mNect.hCNT3 reports on nucleoside transport in real-time and enables measurement of the transportability of any substrate that is coupled to H+ flux. Fusion to the pH-reporting mNectarine is an approach that could be extended to high throughput assays and could be used to report on the transport activity of any pH transporter. Taken together, our findings demonstrate that mNect.hCNT3 is a valid reporter of H+/nucleoside co-transport, and support a transport mechanism where 1 H+ and 1 Na+ are co-transported with uridine in acidic Na+-containing conditions.

      Acknowledgments

      The cDNA for mCherry2 was kindly provided by Nathan C. Shaner and Roger Y. Tsien (University of California, San Diego).

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