Sasaki S et al. (2015), Involvement of Histidine Residue His382 in pH R...

XB-ART-50564

PLoS One 2015 Apr 22;104:e0122738. doi: 10.1371/journal.pone.0122738.

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Involvement of Histidine Residue His382 in pH Regulation of MCT4 Activity.

Sasaki S , Kobayashi M , Futagi Y , Ogura J , Yamaguchi H , Iseki K .

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Monocarboxylate transporter 4 (MCT4) is a pH-dependent bi-directional lactate transporter. Transport of lactate via MCT4 is increased by extracellular acidification. We investigated the critical histidine residue involved in pH regulation of MCT4 function. Transport of lactate via MCT4 was measured by using a Xenopus laevis oocyte expression system. MCT4-mediated lactate transport was inhibited by Zn2+ in a pH physiological condition but not in an acidic condition. The histidine modifier DEPC (diethyl pyrocarbonate) reduced MCT4 activity but did not completely inactivate MCT4. After treatment with DEPC, pH regulation of MCT4 function was completely knocked out. Inhibitory effects of DEPC were reversed by hydroxylamine and suppressed in the presence of excess lactate and Zn2+. Therefore, we performed an experiment in which the extracellular histidine residue was replaced with alanine. Consequently, the pH regulation of MCT4-H382A function was also knocked out. Our findings demonstrate that the histidine residue His382 in the extracellular loop of the transporter is essential for pH regulation of MCT4-mediated substrate transport activity.

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Species referenced: Xenopus laevis
Genes referenced: mcts1 slc16a3

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	Fig 2. Inhibitory effects of DEPC on MCT4-mediated uptake of lactate.(A) Oocytes expressing MCT4 and MCT1 were washed and incubated at 25°C with DEPC at different doses. After 10 min, the oocytes were washed twice and incubated with a transport buffer (pH 5.5) containing 0.1 mM lactate (0.1 μCi/ml) for 10 min. (B) Oocytes were subjected to a 10-min pre-incubation in a transport buffer (pH 7.5) containing 2.5 mM DEPC at 25°C. The oocytes were washed and uptake of 0.1 mM lactate (0.1 μCi/ml) was measured for 10 min at the pH values indicated. Values for water-injected oocytes were subtracted from the values for MCT4-expressing oocytes. Each value is the mean±S.E. of three experiments.
	Fig 3. Determination of the kind of DEPC-sensitive residue.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.
	Fig 4. Effects of various compounds on DEPC modification.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.
	Fig 5. Nicotinic acid transport via MCT4.(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, Km was estimated as the x-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.
	Fig 6. Putative topology of human MCT4.The histidine residues of MCT4 are shown in red.
	Fig 7. Transport of lactate via MCT4-mutant.(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 via 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 via 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.
	Fig 8. His382 is extracellular pH sensor.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.
	Fig 1. Inhibitory effects of metals on MCT4-mediated lactate uptake.(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.

References [+] :

Baird, Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue. 2006, Pubmed, Xenbase

Baird, Evidence for allosteric regulation of pH-sensitive System A (SNAT2) and System N (SNAT5) amino acid transporter activity involving a conserved histidine residue. 2006, Pubmed , Xenbase
Boll, A cluster of proton/amino acid transporter genes in the human and mouse genomes. 2003, Pubmed , Xenbase
Clemmitt, Facilitated downstream processing of a histidine-tagged protein from unclarified E. coli homogenates using immobilized metal affinity expanded-bed adsorption. 2000, Pubmed
Emoto, H(+)-linked transport of salicylic acid, an NSAID, in the human trophoblast cell line BeWo. 2002, Pubmed
Fei, Expression cloning of a mammalian proton-coupled oligopeptide transporter. 1994, Pubmed , Xenbase
Fei, Identification of the histidyl residue obligatory for the catalytic activity of the human H+/peptide cotransporters PEPT1 and PEPT2. 1997, Pubmed , Xenbase
Fujisawa, The extracellular pH dependency of transport activity by human oligopeptide transporter 1 (hPEPT1) expressed stably in Chinese hamster ovary (CHO) cells: a reason for the bell-shaped activity versus pH. 2006, Pubmed
Gunshin, Cloning and characterization of a mammalian proton-coupled metal-ion transporter. 1997, Pubmed , Xenbase
Hemdan, Surface topography of histidine residues: a facile probe by immobilized metal ion affinity chromatography. 1989, Pubmed
Hosoya, MCT1-mediated transport of L-lactic acid at the inner blood-retinal barrier: a possible route for delivery of monocarboxylic acid drugs to the retina. 2001, Pubmed
Ju, Zn2+ inhibits glycine transport by glycine transporter subtype 1b. 2004, Pubmed , Xenbase
Lam-Yuk-Tseung, Iron transport by Nramp2/DMT1: pH regulation of transport by 2 histidines in transmembrane domain 6. 2003, Pubmed
Lin, Human monocarboxylate transporter 2 (MCT2) is a high affinity pyruvate transporter. 1998, Pubmed
Metzner, Influence of a proton gradient on the transport kinetics of the H+/amino acid cotransporter PAT1 in Caco-2 cells. 2006, Pubmed
Metzner, Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1. 2008, Pubmed
Miles, Modification of histidyl residues in proteins by diethylpyrocarbonate. 1977, Pubmed
Miyauchi, Functional identification of SLC5A8, a tumor suppressor down-regulated in colon cancer, as a Na(+)-coupled transporter for short-chain fatty acids. 2004, Pubmed , Xenbase
Plata, Zebrafish Slc5a12 encodes an electroneutral sodium monocarboxylate transporter (SMCTn). A comparison with the electrogenic SMCT (SMCTe/Slc5a8). 2007, Pubmed , Xenbase
Qiu, Identification of an intestinal folate transporter and the molecular basis for hereditary folate malabsorption. 2006, Pubmed , Xenbase
Sasaki, Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4). 2013, Pubmed , Xenbase
Sasaki, Functional characterization of 5-oxoproline transport via SLC16A1/MCT1. 2015, Pubmed , Xenbase
Shimada, Functional characteristics of H+ -dependent nicotinate transport in primary cultures of astrocytes from rat cerebral cortex. 2006, Pubmed
Sloane, Expression and purification of a recombinant metal-binding T4 lysozyme fusion protein. 1996, Pubmed
Tachikawa, Retinal transfer of nicotinate by H+ -monocarboxylate transporter at the inner blood-retinal barrier. 2011, Pubmed
Thwaites, H+-coupled nutrient, micronutrient and drug transporters in the mammalian small intestine. 2007, Pubmed
Unal, The functional roles of the His247 and His281 residues in folate and proton translocation mediated by the human proton-coupled folate transporter SLC46A1. 2009, Pubmed , Xenbase
Zong, A single histidine residue determines the pH sensitivity of the pacemaker channel HCN2. 2001, Pubmed