Real‐time measurement of transport activity of the human concentrative nucleoside transporter, hCNT3, using a fluorescent reporter

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−/HCO3− 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.