Untersuchung des Beitrags der Substratsammelantenne Sammelantenne zum Protonen/Laktat-Cotransport (in PfFNT und MCT1)

Some cells rely intensively on the coupled activity of glycolysis and lactate dehydrogenase to constantly generate ATP. This process releases lactate and protons, the accumulation of which would become detrimental to the cell´s survival. Therefore, the removal of these metabolites is critical to maintain the cells energy generation. This is ensured by monocarboxylate transporters (MCT), such as the human MCT and the Plasmodium falciparum formate nitrite transporter (PfFNT). In the case of human MCT, it has been established that their transport activity was modulated by partner proteins: its chaperone Basigin and carbonic anhydrases enzymes. The surface of such proteins act as proton and substrate collecting antennas, generating microenvironments of greater substrate concentration close to the transport sites. This work set out to investigate how such antennas were involved in the modulation of the monocarboxylate transport functionality of MCT1 and PfFNT. Initial attempts of expressing fusion constructs of carbonic anhydrase, Basigin chaperone and MCT1 transporter proved unsuccessful. Then, alternative methods of protein production were explored to observe interaction between the transporter and the proton antenna. Moreover, this work identified that C-terminal poly-Histidine tag initially intended for protein purification and identification would affect the transport capacity of such MCT1 transporter. This work also hypothesized that the PfFNT C-terminal helix, highly conserved among all human-infecting Plasmodia, plays the role of an endogenous proton collecting antenna facilitating the proton/lactate cotransport. Experimental results suggested that this terminus does modulate the substrate transport (radiolabeled lactate influx capacity was increased in acidic extracellular pH upon its deletion), but it remains to be determined whenever this collecting antenna increases the local concentration of protons or lactate

Local Attraction of Substrates and Co-Substrates Enhances Weak Acid and Base Transmembrane Transport

The transmembrane transport of weak acid and base metabolites depends on the local pH conditions that affect the protonation status of the substrates and the availability of co-substrates, typically protons. Different protein designs ensure the attraction of substrates and co-substrates to the transporter entry sites. These include electrostatic surface charges on the transport proteins and complexation with seemingly transport-unrelated proteins that provide substrate and/or proton antenna, or enzymatically generate substrates in place. Such protein assemblies affect transport rates and directionality. The lipid membrane surface also collects and transfers protons. The complexity in the various systems enables adjustability and regulation in a given physiological or pathophysiological situation. This review describes experimentally shown principles in the attraction and facilitation of weak acid and base transport substrates, including monocarboxylates, ammonium, bicarbonate, and arsenite, plus protons as a co-substrate

Fluorescence Cross-Correlation Spectroscopy Yields True Affinity and Binding Kinetics of Plasmodium Lactate Transport Inhibitors

Blocking lactate export in the parasitic protozoan Plasmodium falciparum is a novel strategy to combat malaria. We discovered small drug-like molecules that inhibit the sole plasmodial lactate transporter, PfFNT, and kill parasites in culture. The pentafluoro-3-hydroxy-pent-2-en-1-one BH296 blocks PfFNT with nanomolar efficiency but an in vitro selected PfFNT G107S mutation confers resistance against the drug. We circumvented the mutation by introducing a nitrogen atom as a hydrogen bond acceptor site into the aromatic ring of the inhibitor yielding BH267.meta. The current PfFNT inhibitor efficiency values were derived from yeast-based lactate transport assays, yet direct affinity and binding kinetics data are missing. Here, we expressed PfFNT fused with a green fluorescent protein in human embryonic kidney cells and generated fluorescent derivatives of the inhibitors, BH296 and BH267.meta. Using confocal imaging, we confirmed the location of the proposed binding site at the cytosolic transporter entry site. We then carried out fluorescence cross-correlation spectroscopy measurements to assign true Ki-values, as well as kon and koff rate constants for inhibitor binding to PfFNT wildtype and the G107S mutant. BH296 and BH267.meta gave similar rate constants for binding to PfFNT wildtype. BH296 was inactive on PfFNT G107S, whereas BH267.meta bound the mutant protein albeit with weaker affinity than to PfFNT wildtype. Eventually, using a set of PfFNT inhibitor compounds, we found a robust correlation of the results from the biophysical FCCS binding assay to inhibition data of the functional transport assay

Basigin drives intracellular accumulation of l-lactate by harvesting protons and substrate anions.

Transmembrane transport of l-lactate by members of the monocarboxylate transporter family, MCT, is vital in human physiology and a malignancy factor in cancer. Interaction with an accessory protein, typically basigin, is required to deliver the MCT to the plasma membrane. It is unknown whether basigin additionally exerts direct effects on the transmembrane l-lactate transport of MCT1. Here, we show that the presence of basigin leads to an intracellular accumulation of l-lactate 4.5-fold above the substrate/proton concentrations provided by the external buffer. Using basigin truncations we localized the effect to arise from the extracellular Ig-I domain. Identification of surface patches of condensed opposite electrostatic potential, and experimental analysis of charge-affecting Ig-I mutants indicated a bivalent harvesting antenna functionality for both, protons and substrate anions. From these data, and determinations of the cytosolic pH with a fluorescent probe, we conclude that the basigin Ig-I domain drives lactate uptake by locally increasing the proton and substrate concentration at the extracellular MCT entry site. The biophysical properties are physiologically relevant as cell growth on lactate media was strongly promoted in the presence of the Ig-I domain. Lack of the domain due to shedding, or misfolding due to breakage of a stabilizing disulfide bridge reversed the effect. Tumor progression according to classical or reverse Warburg effects depends on the transmembrane l-lactate distribution, and this study shows that the basigin Ig-I domain is a pivotal determinant