Human monocarboxylate transporters accept and relay protons via the bound substrate for selectivity and activity at physiological pH.

Human monocarboxylate/H+ transporters, MCT, facilitate the transmembrane translocation of vital weak acid metabolites, mainly l-lactate. Tumors exhibiting a Warburg effect rely on MCT activity for l-lactate release. Recently, high-resolution MCT structures revealed binding sites for anticancer drug candidates and the substrate. Three charged residues, Lys 38, Asp 309, and Arg 313 (MCT1 numbering) are essential for substrate binding and initiation of the alternating access conformational change. However, the mechanism by which the proton cosubstrate binds and traverses MCTs remained elusive. Here, we report that substitution of Lys 38 by neutral residues maintained MCT functionality in principle, yet required strongly acidic pH conditions for wildtype-like transport velocity. We determined pH-dependent biophysical transport properties, Michaelis-Menten kinetics, and heavy water effects for MCT1 wildtype and Lys 38 mutants. Our experimental data provide evidence for the bound substrate itself to accept and shuttle a proton from Lys 38 to Asp 309 initiating transport. We have shown before that substrate protonation is a pivotal step in the mechanisms of other MCT-unrelated weak acid translocating proteins. In connection with this study, we conclude that utilization of the proton binding and transfer capabilities of the transporter-bound substrate is probably a universal theme for weak acid anion/H+ cotransport.

Lactic Acid Permeability of Aquaporin-9 Enables Cytoplasmic Lactate Accumulation via an Ion Trap.

(1) Background: Human aquaporin-9 (AQP9) conducts several small uncharged metabolites, such as glycerol, urea, and lactic acid. Certain brain tumors were shown to upregulate AQP9 expression, and the putative increase in lactic acid permeability was assigned to severity. (2) Methods: We expressed AQP9 and human monocarboxylate transporter 1 (MCT1) in yeast to determine the uptake rates and accumulation of radiolabeled l-lactate/l-lactic acid in different external pH conditions. (3) Results: The AQP9-mediated uptake of l-lactic acid was slow compared to MCT1 at neutral and slightly acidic pH, due to low concentrations of the neutral substrate species. At a pH corresponding to the pKa of l-lactic acid, uptake via AQP9 was faster than via MCT1. Substrate accumulation was fundamentally different between AQP9 and MCT1. With MCT1, an equilibrium was reached, at which the intracellular and extracellular l-lactate/H+ concentrations were balanced. Uptake via AQP9 was linear, theoretically yielding orders of magnitude of higher substrate accumulation than MCT1. (4) Conclusions: The selectivity of AQP9 for neutral l-lactic acid establishes an ion trap for l-lactate after dissociation. This may be physiologically relevant if the transmembrane proton gradient is steep, and AQP9 acts as the sole uptake path on at least one side of a polarized cell.

Cysteine 159 delineates a hinge region of the alternating access monocarboxylate transporter 1 and is targeted by cysteine-modifying inhibitors.

Monocarboxylate transporter isoforms 1-4, MCT, of the solute carrier SLC16A family facilitate proton-coupled transport of l-lactate. Growth of tumors that exhibit the Warburg effect, that is, high rates of anaerobic glycolysis despite availability of oxygen, relies on swift l-lactate export, whereas oxygenic cancer cells import circulating l-lactate as a fuel. Currently, MCTs are viewed as promising anticancer targets. Small-molecule inhibitors have been found, and, recently, high-resolution protein structures have been obtained. Key questions, however, regarding the exact binding sites of cysteine-modifying inhibitors and the substrate translocation cycle lack a conclusive experimental basis. Here, we report Cys159 of the ubiquitous human MCT1 to reside in a critical hinge region of the alternating access-type transporter. We identified Cys159 as the binding site of the organomercurial pCMBS. The inhibitory effect of pCMBS was proposed to be indirect via modification of the chaperone basigin. We provide evidence that pCMBS locks MCT1 in its outward open conformation in a wedge-like fashion. We corroborated this finding using smaller cysteine-modifying reagents that size-dependently inhibited l-lactate transport. The smallest modifiers targeted additional cysteines as shown by a C159S mutant. We found a Cys399/Cys400 pair to constitute the second hinge of the transporter that tolerated only individual replacement by serine. The hinge cysteines, in particular the selectively addressable Cys159, provide natural anchors for placing probes into MCTs to report, for instance, on the electrostatics or hydration upon binding of the transported l-lactate substrate and the proton cosubstrate.

Analysis of the Ordering Effects in Anthraquinone Thin Films and Its Potential Application for Sodium Ion Batteries.

The ordering effects in anthraquinone (AQ) stacking forced by thin-film application and its influence on dimer solubility and current collector adhesion are investigated. The structural characteristics of AQ and its chemical environment are found to have a substantial influence on its electrochemical performance. Computational investigation for different charged states of AQ on a carbon substrate obtained via basin hopping global minimization provides important insights into the physicochemical thin-film properties. The results reveal the ideal stacking configurations of the individual AQ-carrier systems and show ordering effects in a periodic supercell environment. The latter reveals the transition from intermolecular hydrogen bonding toward the formation of salt bridges between the reduced AQ units and a stabilizing effect upon the dimerlike rearrangement, while the strong surface-molecular interactions in the thin-film geometries are found to be crucial for the formed dimers to remain electronically active. Both characteristics, the improved current collector adhesion and the stabilization due to dimerization, are mutual benefits of thin-film electrodes over powder-based systems. This hypothesis has been further investigated for its potential application in sodium ion batteries. Our results show that AQ thin-film electrodes exhibit significantly better specific capacities (233 vs 87 mAh g-1 in the first cycle), Coulombic efficiencies, and long-term cycling performance (80 vs 4 mAh g-1 after 100 cycles) over the AQ powder electrodes. By augmenting the experimental findings via computational investigations, we are able to suggest design strategies that may foster the performance of industrially desirable powder-based electrode materials.

A sub-4 Kelvin radio frequency linear multipole wire trap.

A linear cryogenic 16-pole wire ion trap has been developed and constructed for cryogenic ion spectroscopy at temperatures below 4 K. The trap is temperature-variable, can be operated with different buffer gases, and offers large optical access perpendicular to the ion beam direction. The housing geometry enables temperature measurement during radio frequency operation. The effective trapping potential of the wire-based radio frequency trap is described and compared to conventional multipole ion trap designs. Furthermore, time-of-flight mass spectra of multiple helium tagged protonated glycine ions that are extracted from the trap are presented, which prove very low ion temperatures and suitable conditions for sensitive spectroscopy.

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.

Perylenetetracarboxylic Diimide as Diffusion-Less Electrode Material for High-Rate Organic Na-Ion Batteries.

In this work 3,4,9,10-perylenetetracarboxylic diimide (PTCDI) is investigated as electrode material for organic Na-ion batteries. Since PTCDI is a widely used industrial pigment, it may turn out to be a cost-effective, abundant, and environmentally benign cathode material for secondary Na-ion batteries. Among other carbonyl pigments, PTCDI is especially interesting due to its high Na-storage capacity in combination with remarkable high rate capabilities. The detailed analysis of cyclic voltammetry measurements reveals a diffusion-less mechanism, suggesting that Na-ion storage in the PTCDI film allows for exceptionally fast charging/discharging rates. This finding is further corroborated by galvanostatic sodiation measurements at high rates of 17 C (2.3 A g-1 ), showing that 57 % of the theoretically possible capacity of PTCDI, or 78 mAh g-1 , are attained in only 3.5 min charging time.

Vibrational Predissociation Spectroscopy of Cold Protonated Tryptophan with Different Messenger Tags.

Vibrational spectra of protonated tryptophan were recorded by predissociation of H2 messenger tags using cryogenic ion traps. We explore the issue of messenger induced spectral changes by solvating TrpH+(H2) n with n = 1-5 to obtain single photon vibrational spectra of TrpH+ and of its partly deuterated isotopomer in the spectral region of 800-4400 cm-1. Depending on the number of messenger molecules, the spectra of several conformational isomers associated with multiple H2 binding locations along with two natural conformations of TrpH+ were found using the two photon MS3IR2 conformational hole burning method. Most probable messenger positions were established by comparison with predictions from DFT calculations on various candidate structures. Mechanical anharmonicity effects associated with the charged amino group were modeled by Born-Oppenheimer ab initio molecular dynamics. The spectra of TrpH+(H2O) m=1,2, recorded by infrared multiphoton dissociation (IRMPD), reveal broad features in the NH stretching region of the NH3+ group, indicating strong hydrogen bonding in acceptor-donor configuration with the benzene ring for the first water molecule, while the second water appears to attach to a less strongly perturbing site, yielding unique transitions associated with the free OH stretching fundamentals. We discuss the structural deformations induced by the water molecules and compare our results to recent experiments on similar hydrated cationic systems.