Supplementary files_YOno_BMJ open_2018

Supplementary files_YOno_BMJ open_201

Transport of lactate <i>via</i> MCT4-mutant.

<p>(A) Oocytes injected with MCT4-WT cRNA, MCT4-H382A cRNA, or water were incubated with 0.1 mM lactate (0.1 μCi/ml) for 10 min. (B) Sensitivities against 5 mM zinc of lactate transport activities <i>via</i> MCT4 with a histidine residue mutant. Uptake of 0.1 mM lactate (0.1 μCi/ml) was measured at pH 7.5 for 10 min. (C) Wild-type and histidine mutant MCT4 cRNA-injected oocytes were incubated for 10 min with/without 2.5 mM DEPC in a buffer of pH 7.5 at 25°C. Uptake of 0.1 mM lactate (0.1 μCi/ml) was measured for 10 min at pH 5.5. (D) pH regulation of lactate uptake <i>via</i> wild-type and mutant MCT4. Uptake of 0.1 mM lactate was measured for 10 min at the indicated pH by oocytes expressing proteins of interest. Values are presented as percentages of uptake measured at pH 5.5. All data are presented as means±S.E. of three experiments.</p

Effects of various compounds on DEPC modification.

<p>Oocytes were pre-incubated for 10 min in a transport buffer (pH 7.5) containing 2.5 mM DEPC at 25°C in the absence or presence of 100 mM lactate, 100 mM propionic acid, 50 mM nicotinic acid, 20 mM salicylic acid, 10 mM ibuprofen and 5 mM zinc. After treatment with DEPC, the oocytes were rinsed twice with a transport buffer not including a radiolabeled compound. Oocytes were additionally incubated twice for 5 min each time with the transport buffer. The oocytes were incubated for 10 min in a transport buffer of pH 5.5 containing 0.1 mM lactate (0.1 μCi/ml). Data are presented as means±S.E. of three experiments. MCT4-specific uptake was calculated by subtracting the uptake in water-injected oocytes from the uptake in MCT4 cRNA-injected oocytes.</p

Inhibitory effects of metals on MCT4-mediated lactate uptake.

<p>(A) MCT4-mediated uptake of 0.1 mM lactate (0.1 μCi/ml) was measured in a transport buffer of pH 7.5 containing 5 mM of each metal for 10 min. The inset shows the effect of 5 mM zinc under an acidic condition (pH 5.5). (B) MCT1-mediated uptake of 0.1 mM lactate (0.1 μCi/ml) was measured in a transport buffer of pH 7.5 containing 5 mM of each metal for 10 min. The background uptake values of water-injected oocytes were subtracted. Data are presented as means±S.E. of three experiments.</p

His382 is extracellular pH sensor.

<p>Taken together, our findings demonstrate that the non-conserved residue His382 in the extracellular loop of MCT4 is essential for pH regulation of MCT4-mediated lactate transport activity. The results of this study should be useful for development of a drug delivery system using MCT4.</p

Putative topology of human MCT4.

<p>The histidine residues of MCT4 are shown in red.</p

Determination of the kind of DEPC-sensitive residue.

<p>Oocytes were pre-incubated for 10 min in a transport buffer in the presence or absence of DEPC (2.5 mM). After DEPC treatment, the oocytes were additionally incubated for 1 h in a transport buffer in the presence or absence of hydroxylamine (50 mM). The oocytes were incubated for 10 min in a transport buffer of pH 5.5 containing 0.1 mM lactate (0.1 μCi/ml). Data are presented as means±S.E. of three experiments. MCT4-specific uptake was calculated by subtracting the uptake in water-injected oocytes from the uptake in MCT4 cRNA-injected oocytes.</p

Nicotinic acid transport <i>via</i> MCT4.

<p>(A) MCT4 cRNA-injected or water-injected oocytes were incubated for 5 min at 25°C with 10 nM nicotinic acid (0.5 μCi/ml) at pH 5.5 or 7.5. Data are presented as means±S.E. of three experiments. Uptake of nicotinic acid was measured with 5-min incubation at 25°C in a transport buffer of pH 5.5 (B) or pH 7.5 (C) in the presence of an increasing dose of nicotinic acid. The background uptake values of water-injected oocytes were subtracted. Only the MCT4-specific uptake was used for kinetic analysis. Results are Hanes plots for which each value is the mean value for three experiments, and lines were fitted using the least-squares method: from each line, <i>K</i>_m was estimated as the <i>x</i>-axis intercept. (D) Dose dependency of MCT4-mediated lactate inhibition of nicotinic acid uptake. The nicotinic acid uptake was measured at pH 5.5 for 5 min. Data are presented as means±S.E. of three experiments.</p

Uptake of nicotinic and salicylic acids in MCT4 and MCT1 cRNA-injected oocytes and water-injected oocytes

<p>Uptake of salicylic acid and nicotinic acid (100 μM: labeled + unlabeled) in MCT4 and MCT1 cRNA-injected oocytes and water-injected oocytes was measured with 5-min incubation at 25°C in a transport buffer of pH 5.5. Data are presented as means±S.E. of three experiments.</p><p>Uptake of nicotinic and salicylic acids in MCT4 and MCT1 cRNA-injected oocytes and water-injected oocytes</p

Kinetic parameters of lactate transport via MCT4.

<p>*Taken from reference 1.</p><p>Kinetic parameters of lactate transport via MCT4.</p