Characterization and protein expression of a novel monocarboxylate transporter ((KF)MCT) from killifish (Fundulus heteroclitus).

摘要

Monocarboxylate transporters (MCTs) are important proteins in facilitating the transmembrane movement of carbohydrates and have been studied extensively in a plethora of mammalian tissues. However, studies characterizing MCTs from lower vertebrate models are considerably lacking, especially when considering the vital role monocarboxylates play in maintaining a metabolic balance under various environmental and physiological conditions. This is the first ever study to identify, confirm and characterize a MCT in teleosts. Using RT-PCR and degenerate primers designed against conserved regions of known MCT sequences, we were able to isolate a full length putative killifish (Fundulus heteroclitus) MCT (KFMCT) sequence, similar to MCT2 in higher vertebrates. The function of the injected KFMCT mRNA was confirmed by the protein expression in Xenopus laevis oocytes. A yellow fluorescence fusion protein (KFMCT-YFP) was used to confirm expression of the sequence and its localization. Transport kinetics of the full length KFMCT was studied using 3H-lactate and 14C-beta-hydroxybutyrate in the oocytes system. The tissue specific MCT protein levels were examined in killifish in vivo normoxia and acute exposure to environmental hypoxic (0.5mg L-1) conditions for 2, 4, 8, 16 h using a KFMCT gene specific antibody.; Functional translated KFMCT proteins were located at the oocyte plasma membrane. KFMCT cRNA injected oocytes revealed that KFMCT preferentially transports pyruvate (Ki = 0.50+/-0.19 mM, and 0.56+/-0.020 mM, in the presence of lactate and beta-hydroxybutyrate, respectively), followed by lactate (Km of 1.99+/-0.34 mM), and beta-hydroxybutyrate (Km of 3.82+/-2.22 mM), suggesting KFMCT is a high affinity monocarboxylate transporter. Monocarboxylate uptake was accelerated with a decrease in extracellular pH and inhibited by known MCT inhibitors, such as CINN (alpha-cyano-4-hydroxycinnamic acid) and DIDS (4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid). The partial depolarization of oocytes membranes induced by elevated extracellular potassium resulted in a 77% decrease in lactate uptake suggesting KFMCT transport activity may be voltage sensitive.; KFMCT protein expression increased in liver and brain after 4 h and in muscle after 16 h of acute hypoxia. The time dependent variation of KFMCT protein expression in different tissues may reflect the tissue specific metabolic status and needs under hypoxic conditions.; We conclude that KFMCT possesses substrate kinetics similar to its mammalian counterparts. The transient increase in protein expression of KFMCT after acute environmental hypoxia is feasibly a trait adapted by hypoxia tolerant species such as the killifish to cope with metabolic needs when operating under anaerobic conditions in its estuarine habitat where hypoxic episodes are a regular short term occurrence.