Red fluorescent protein pH biosensor to detect concentrative nucleoside transport.
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.
Johnson
Danielle E
DE
Membrane Protein Research Group, Department of Physiology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada.
Ai
Hui-Wang
HW
Wong
Peter
P
Young
James D
JD
Campbell
Robert E
RE
Casey
Joseph R
JR
D001692:Biological Transport; D015374:Biosensing Techniques; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D005818:Genetic Engineering; D006031:Glycosylation; D006801:Humans; D006863:Hydrogen-Ion Concentration; D042541:Intracellular Space / Q000378:metabolism; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism*; D050505:Mutant Proteins / Q000378:metabolism; D009705:Nucleosides / Q000378:metabolism*; D038761:Photobleaching; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014529:Uridine / Q000378:metabolism; D000097573:Red Fluorescent Protein
D005452:Fluoresceins; D008164:Luminescent Proteins; D050505:Mutant Proteins; D009705:Nucleosides; D011522:Protons; D011993:Recombinant Fusion Proteins; D014529:Uridine; D000097573:Red Fluorescent Protein; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein