Characterisation of human monocarboxylate transporter 4 substantiates its role in lactic acid efflux from skeletal muscle

1. Monocarboxylate transporter (MCT) 4 is the major monocarboxylate transpo rter isoform present in white skeletal muscle and is responsible for the ef flux of the lactic acid produced by glycolysis. Here we report the characte risation of MCT4 expressed in Xenopus oocytes. 2. The protein was correctly targeted to the plasma membrane and rates of s ubstrate transport were determined from the rate of intracellular acidifica tion monitored with the pH-sensitive dye 2',7'-bis-(carboxyethyl)-5(6)-carb oxyfluorescein (BCECF). 3. In order to validate the technique, the kinetics of monocarboxylate tran sport were measured in oocytes expressing MCT1. K-m values determined for L -lactate, D-lactate and pyruvate of 4.4, > 60 and 2.1 mM, respectively, wer e similar to those determined previously in tumour cells. 4. Comparison of the time course of [C-14]lactate accumulation with the rat e of intracellular acidification monitored with BCECF suggests that the lat ter reflects pH changes close to the plasma membrane associated with transp ort, whilst the former may include diffusion-limited movement of lactate in to the bulk cytosol. 5. Km values of MCT4 for these substrates were found to be 28, 519 and 153 mM, respectively, and for a range of other monocarboxylates values were at least an order of magnitude higher than for MCT1. V-max values appeared to be similar for all substrates. 6. K-0.5 values of MCT4 (determined at 30 mM L-lactate) for inhibition by a lpha -cyano-4-hydroxycinnamate (991 muM), phloretin (41 muM), 5-nitro-2-(3- phenylpropylamino)benzoate (240 muM), p-chloromercuribenzene sulphonate (21 muM) and 3-isobutyl-1-methylxanthine (970 muM, partial inhibition) were al so substantially higher than for MCT1. No inhibition of MCT4 by 2 mM 4,4'-d iisothiocyanostilbene-2,2'-disulphonate was observed. 7. The properties of MCT4 are consistent with published data on giant sarco lemmal vesicles in which MCT4 is the dominant MCT isoform, and are appropri ate for the proposed role of MCT4 in mediating the efflux from the cell of glycolytically derived lactic acid but not pyruvate.