Red fluorescent protein pH biosensor to detect concentrative nucleoside transport

Abstract

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.

FIGURE 1.

pH dependence of mNectarine fluorescence. A, fluorescence excitation (left, emission at 600 nm) and emission (right, excitation at 550 nm) intensity for mNectarine, recorded at various pH (actual pH values presented in B). Arrows represent the direction of change with increasing pH. B, plot of relative fluorescence intensity versus pH for mNectarine, with excitation at 550 nm and emission at 600 nm. The curve represents the best of fit of the data obtained using a form of the Henderson-Hasselbalch equation. C, absorbance spectra of mNectarine at various pH. Arrows represent the direction of change with increasing pH. These spectra were subjected to a global four-component linear regression analysis (supplemental Fig. S2). D, relative absorbance intensities of each component of the spectra in C plotted as a function of pH. The four components are the red fluorescence excitation profile (, orange-absorbing) and three Gaussian curves centered at 489 nm (⬡, cyan-absorbing), 453 nm (♢, blue-absorbing), and 387 nm (□, violet-absorbing).

FIGURE 2.

Expression of mNect.hCNT3 in HEK293 cells and analysis of glycosylation status. HEK293 cells were transiently transfected with vector (pcDNA3.1), mNectarine.hCNT3, or mNectarine.hCNT3-F563C cDNA. Lysates from the cells were treated with (+) or without (−) the deglycosylation enzyme, PNGaseF. Samples were immunoblotted and probed with a polyclonal anti-RFP antibody.

FIGURE 3.

Spectral characterization of mNectarine expressed in HEK293 cells. HEK293 cells were transfected with mNectarine cDNA, and cell lysates were prepared. A, excitation (ex.) and emission (em.) scans of the lysates were collected at respective fixed emission and excitation wavelengths 590 and 550 nm. B, fluorescence excitation spectra (λ_em = 573 nm) of intact mNect-transfected HEK293 cells clamped at pH 7.5, 7.0, and 6.5 with nigericin/high potassium (black lines). Excitation spectra of vector-alone-transfected cells are shown as gray lines, with clamped pH values of experiments indicated. C, relationship between fluorescence (λ_ex = 558 nm and λ_em = 573 nm) and clamped cytosolic pH, in mNect-transfected HEK293 cells (data from B).

FIGURE 4.

Correction for mNectarine photobleaching. HEK293 cells were transiently transfected with free cytosolic mNectarine cDNA, and fluorescence was monitored (λ_ex = 550 nm and λ_em = 573 nm). A and B, cells were perfused with nigericin/high potassium solution, pH 7.0, to clamp intracellular pH. A, raw mNectarine fluorescence at pH 7.0, declining as a result of photobleaching. B, mNectarine fluorescence at pH 7.0. Data from panel A has been corrected for photobleaching, using the approach described in methods. C and D, HEK293 cells transfected with mNect cDNA were perfused consecutively with nigericin/high potassium solutions at pHs indicated in the black bars above the curves. To minimize the period of sample illumination, samples were perfused without illumination for 480 s in each solution prior to collecting fluorescence data. Above or below each trace are the average pH values for each perfusion interval, calculated from the fluorescence data. C, data not corrected for photobleaching. D, data from panel C corrected for photobleaching, using the approach described under “Experimental Procedures.”

FIGURE 5.

Measurement of intracellular pH, using cytosolic mNectarine, mNect.hCNT3, and BCECF. HEK293 cells were transiently co-transfected with cytosolic mNectarine and hCNT3 cDNA (A) or mNect.hCNT3 cDNA (B) or hCNT3 cDNA (C). hCNT3-transfected cells were incubated with the pH-sensitive dye BCECF-AM (C). Cells were perfused with nigericin/high potassium calibration solutions at pH values indicated in the black bars above the curves to calibrate fluorescence to pH. To minimize the period of sample illumination, samples were perfused without illumination for 480 s in each solution prior to collecting fluorescence data. Average pH values for each perfusion interval (calculated from the fluorescence data) are indicated above or below the trace. A and B, mNect fluorescence (λ_ex = 550 nm and λ_em = 573 nm) was corrected for photobleaching. C, BCECF fluorescence was monitored at λ_ex = 440 nm and 502.5 nm and λ_em = 528.7 nm.

FIGURE 6.

hCNT3 mediated H⁺/uridine co-transport measured by mNectarine-hCNT3. HEK293 cells were transiently transfected with mNect.hCNT3 cDNA (A) or with mNect.hCNT3-F563C cDNA (B). mNect.hCNT3 fluorescence was monitored at λ_ex = 550 nm and λ_em = 573 nm. Cells were perfused consecutively with Na⁺-free MBSS buffer, pH 7.5 (open bar), in which NaCl is replaced by choline chloride, Na⁺-free MBSS buffer, pH 5.5 (gray bar), and Na⁺-free MBSS buffer with 0.5 mm uridine, pH 5.5 (black bar). At the end of each experiment photobleaching-corrected fluorescence values were calibrated to pH via the nigericin/high potassium technique (not shown in figure) (35). A, pH_i transients observed for mNectarine.hCNT3-transfected cells. B, pH_i transients observed for mNect.hCNT3-F563C-transfected cells. C, quantification of the change in rate of acidification (proton flux in units of mm·min⁻¹) upon switching to uridine-containing medium. Error bars represent standard error (n = 5). Asterisk indicates significant difference (p < 0.0001).

FIGURE 7.

Kinetics of H⁺/uridine co-transport in HEK293 cells. HEK293 cells were transiently transfected with mNect.hCNT3 cDNA. mNect.hCNT3 fluorescence was monitored at λ_ex = 550 nm and λ_em = 573 nm. Cells were perfused consecutively with Na⁺-free MBSS buffer, pH 7.5, in which NaCl is replaced by choline chloride, Na⁺-free MBSS buffer, pH 5.5, and Na⁺-free MBSS buffer, pH 5.5, containing varying concentrations of uridine from 0 to 960 μm. At the end of each experiment pH was calibrated via the nigericin/high potassium technique, and fluorescence values were calibrated to pH. The change in the rate of acidification (proton flux in units of mm·min⁻¹) was quantified upon switching to uridine-containing medium at each concentration of uridine. This graph is a representative example of three separate experiments used to calculate the K_m of mNect.hCNT3 for uridine.

FIGURE 8.

Effect of Na⁺ on H⁺/uridine co-transport. HEK293 cells were transiently transfected with mNectarine.hCNT3 cDNA (A and B) or with mNect.hCNT3-F563C cDNA (C). mNect.hCNT3 fluorescence was monitored at λ_ex = 550 nm and λ_em = 573 nm. Cells were perfused with MBSS buffer, pH 5.5 (gray bars), or MBSS buffer, pH 5.5, containing 0.5 mm uridine (black bars). Perfusion with Na⁺-containing MBSS buffer is indicated by hatched bars, and perfusion with Na⁺-free MBSS buffer, in which NaCl is replaced by choline chloride (ChCl), is indicated by un-hatched bars. Open bar indicates perfusion with Na⁺-free MBSS, pH 7.5. All buffers contained 5 μm EIPA. D, quantification of the change in rate of acidification (proton flux in units of mm·min⁻¹) upon switching to uridine-containing medium. Error bars represent standard error (n = 3). N.S. indicates non-significant difference (p = 0.81).