methodology; K. and 0.7 mm in MDA-MB-231 cells, macrophages, and HEK293 cells expressing Brofaromine recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a for pyruvate of 4.2 mm, as opposed to >150 mm reported previously. Parallel assays with the pH-sensitive dye 2,7-bis-(carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) indicated that previous estimates based on substrate-induced acidification were severely biased by confounding pH-regulatory mechanisms. Numerical simulation using revised kinetic parameters revealed that MCT4, but not the related Brofaromine transporters MCT1 and MCT2, endows cells with the ability to export lactate in high-lactate microenvironments. In conclusion, MCT4 is a high-affinity lactate transporter with physiologically relevant affinity for pyruvate. family of H+-coupled monocarboxylate transporters MCTs of which MCT1 (of MCT4 for lactate is more than 1 order of magnitude higher than the levels of lactate prevailing in tissues and even within hypoxic tumors (6, 15). Taken at face value, this means that MCT4 runs at a small fraction of its capacity. In contrast, MCT1 has a of 3C5 mm. Kinetic transport parameters are determined by measuring initial rates of uptake at increasing concentrations of radiolabeled substrate. Unfortunately, this is not practical for MCTs in mammalian cells, because uptake is too fast, demanding high levels of radioactivity and sophisticated stop-flow devices. The introduction of pH-sensitive dyes in the 1980s revolutionized the field by permitting MCT activity determinations with high spatiotemporal resolution (16, 17). With the additional advantage that any substrate could be studied with the same probe, most of what we know about the function of the monocarboxylate transporters was learned from substrate-induced acidification. However, there was a caveat. To obtain detectable acidifications, experiments had to be done in the absence of bicarbonate. As demonstrated below, pH buffering is a major confounding factor when MCTs are characterized using pH dyes. Genetically-encoded FRET nanosensors have been recently used by several laboratories to directly monitor lactate and pyruvate dynamics in various cell types, and (18,C27). During the characterization of a MCT4-rich cell line with a FRET sensor we detected robust transport at low lactate concentrations. The present manuscript describes a set of experiments prompted by that observation. Results MCT4 mediates monocarboxylate transport in MDA-MB-231 cells To study the functional properties of MCT4, we expressed the genetically-encoded FRET lactate sensor Laconic (18) in MDA-MB-231 cells, a human breast cancer cell line conspicuous for its high levels of MCT4 and absence of MCT1 (28, 29). Fig. 1shows MDA-LAC, a cell line generated with MDA-MB-231 cells stably expressing Laconic. Fig. 1shows that the abundance of MCT4 in these cells is almost as high as that achieved by overexpressing MCT4 in HEK293 cells Brofaromine under the Brofaromine strong cytomegalovirus promoter, and that MCT4 levels are not diminished by expression of the FRET sensor. Exposure of MDA-MB-231 cells to a lactate load caused a rapid increase in intracellular lactate, demonstrative of high permeability (Fig. 1and and represents 50 m. shows mean S.E. (3 separate preparations). and MDA-MB-231 cells expressing Laconic were exposed to 10 mm lactate and then to 1 1 m AR-C155858 and/or 250 m pCMBS as indicated, mean S.E. (10 cells from single experiments, representative of three experiments for each protocol). Open in a separate window Figure 2. MCT4 mediates the influx of lactate in MDA-MB-231 cells. CD63 The uptake of 10 mm lactate by MDA-LAC cells was monitored before and during exposure to MCT inhibitors, mean S.E. (10 cells from single experiments). show the initial rates of uptake and the rate of accumulation elicited by the inhibitor itself, mean S.E. (30 cells in three experiments). *, < Brofaromine 0.05 in the Tukey's test. show rates of lactate uptake (mean S.E. of 30C50 cells in three experiments; *, < 0.05 in the Mann-Whitney test). lactate efflux from WT and MCT4-KO MDA-MB-231 cells was monitored immediately after extracellular lactate (1 mm) was replaced by 6 mm sodium oxamate (mean S.E. of 30C50 cells in three experiments; *, < 0.05 in the Mann-Whitney test). the uptake of 10 mm lactate in the presence of 1 m AR-C155858 was measured in HEK293, HEK-MCT4, and HEK-MCT4-MCT4KO cells. Data from 30 cells in three experiments (mean S.E.; *, < 0.05 in the Mann-Whitney test). summarize the results of three experiments (mean S.E. of 30C50 cells in three experiments; *, < 0.05 in the Mann-Whitney test). MCT4 of MDA-MB-231 cells.