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Real‐time measurement of transport activity of the human concentrative nucleoside transporter, hCNT3, using a fluorescent reporter
Author(s) -
Johnson Danielle E,
Young James D,
Campbell Robert E,
Casey Joseph R
Publication year - 2009
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.23.1_supplement.796.22
Subject(s) - nucleoside , uridine , fluorescence , chemistry , biophysics , biochemistry , biology , rna , physics , quantum mechanics , gene
Human concentrative nucleoside transporter, hCNT3, mediates Na + /nucleoside co‐transport, with a low turnover rate (10 nucleoside molecules s −1 ). hCNT3 also supports H + ‐coupled uridine transport, as measured in X. laevis oocytes. We used a new approach to monitor H + /uridine co‐transport in mammalian cells. We fused mNectarine, a new H + ‐sensitive red fluorescent protein, to the N‐terminus of hCNT3 to generate mNect‐hCNT3. Fusion of the fluorescent H + sensor enabled measurement of pH at the intracellular surface of hCNT3. mNectarine fluorescence was monitored in human embryonic kidney 293 cells expressing mNect‐hCNT3 or, as a negative control, cytosolic mNectarine and catalytically dead mutant of the AE1 Cl − /HCO 3 − exchanger, AE1‐P652C. mNectarine fluorescence values were calibrated for pH using the nigericin/high K + clamping method. Cells were incubated at the permissive pH for H + ‐coupled nucleoside transport, pH 5.5, under Na + ‐free conditions. At this pH there was a continuous cytosolic acidification. In mNect‐hCNT3 expressing cells (but not under negative control conditions) the rate of acidification increased upon addition of 0.5 mM uridine, consistent with H + ‐coupled uridine transport. We conclude that mNect‐hCNT3 acts as a sensitive self‐referencing sensor of nucleoside transport. Research is supported by the Canadian Institutes of Health Research and the National Cancer Institute of Canada.