Monocarboxylate transporter 4 (MCT4) is a high affinity transporter capable of exporting lactate in high-lactate microenvironments

Yasna Contreras-Baeza,Pamela Y Sandoval,Romina Alarcón,Alex Galaz,Francisca Cortés-Molina,Karin Alegría,Felipe Baeza-Lehnert,Robinson Arce-Molina,Anita Guequén,Carlos A Flores,Alejandro San Martín,L Felipe Barros

doi:10.1074/jbc.ra119.009093

Yasna Contreras-Baeza, Pamela Y Sandoval + Show 10 more

Open Access

https://doi.org/10.1074/jbc.ra119.009093

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Abstract

Monocarboxylate transporter 4 (MCT4) is an H+-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect. At these sites, extracellular lactate contributes to malignancy and immune response evasion. Intriguingly, at 30-40 mm, the reported Km of MCT4 for lactate is more than 1 order of magnitude higher than physiological or even pathological lactate levels. MCT4 is not thought to transport pyruvate. Here we have characterized cell lactate and pyruvate dynamics using the FRET sensors Laconic and Pyronic. Dominant MCT4 permeability was demonstrated in various cell types by pharmacological means and by CRISPR/Cas9-mediated deletion. Respective Km values for lactate uptake were 1.7, 1.2, and 0.7 mm in MDA-MB-231 cells, macrophages, and HEK293 cells expressing recombinant MCT4. In MDA-MB-231 cells MCT4 exhibited a Km 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 Km 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 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.

Highlights

Monocarboxylate transporter 4 (MCT4) is an H؉-coupled symporter highly expressed in metastatic tumors and at inflammatory sites undergoing hypoxia or the Warburg effect
The transport of lactate in most mammalian cells is mediated by members of the slc16a family of Hϩ-coupled monocarboxylate transporters MCTs of which MCT1, MCT2, and MCT4 are widely expressed in healthy tissues [10]
In agreement with the pharmacological evidence, genetic deletion of MCT4 in MDA-MB-231 cells using CRISPR/CAS9 caused higher resting intracellular lactate, reduced lactate entry and exit, and reduced lactate/oxamate exchange (Fig. 3). These results provide pharmacological and genetic evidence that MCT4 is responsible for the bidirectional transport of lactate across the plasma membrane of MDA-MB-231 cells

Summary

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Stitial acidification, which along with lactate itself favors tumor invasiveness and facilitates immune response evasion [5]. Lactate levels were double in cervical tumors with metastatic spread compared with malignancies in patients without metastases [6]. The transport of lactate in most mammalian cells is mediated by members of the slc16a family of Hϩ-coupled monocarboxylate transporters MCTs of which MCT1 (slc16a1), MCT2 (slc16a7), and MCT4 (slc16a3) are widely expressed in healthy tissues [10]. MCT4-endowed cells, both healthy and cancerous, are the strongest lactate producers. Kinetic transport parameters are determined by measuring initial rates of uptake at increasing concentrations of radiolabeled substrate. This is not practical for MCTs in mammalian cells, because uptake is too fast, demanding high levels of radioactivity and sophisticated stop-flow devices. 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

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