The Conserved C-Terminus of the Citrate (CitP) and Malate (MleP) Transporters of Lactic Acid Bacteria Is Involved in Substrate Recognition

The membrane potential-generating transporters CitP of Leuconostoc mesenteroides and MleP of Lactococcus lactis are homologous proteins with 48% identical residues that catalyze citrate-lactate and malate-lactate exchange, respectively. The two transporters are highly specific for substrates containing a 2-hydroxycarboxylate motif (HO-CR2-COO-) in which substitutions of the R groups are tolerated well. Differences in substrate specificity between MleP and CitP are based on subtle changes in the interaction of the protein with the R groups affecting both binding and translocation properties. The conserved, 46-residue long C-terminal region of the transporters containing the C-terminal putative transmembrane segment XI was investigated for its role in substrate recognition by constructing chimeric transporters. Replacement of the C-terminal region of MleP with that of CitP and vice versa did not alter the exchange kinetics with the substrates malate and citrate, indicating that the main interactions between the proteins and di- and tricarboxylate substrates were not altered. In contrast, the interaction of the proteins with the monocarboxylate substrates mandelate and 2-hydroxyisovalerate changed in a complementary manner. The affinity of CitP for the S-enantiomers of these substrates was at least 1 order of magnitude lower than observed for MleP. Introduction of the C-terminal residues of MleP in CitP resulted in a higher affinity and vice versa. Interchanging the C-termini had a more complicated effect on the R-enantiomers, affecting different kinetic parameters with different substrates, indicating multiple interactions of the R groups at this side of the binding pocket. It is suggested that the binding pocket is located between transmembrane segment XI and the other transmembrane segments of the transporters

Photochemical activation of TRPA1 channels in neurons and animals

Optogenetics is a powerful research tool because it enables high-resolution optical control of neuronal activity. However, current optogenetic approaches are limited to transgenic systems expressing microbial opsins and other exogenous photoreceptors. Here, we identify optovin, a small molecule that enables repeated photoactivation of motor behaviors in wild type animals. Surprisingly, optovin's behavioral effects are not visually mediated. Rather, photodetection is performed by sensory neurons expressing the cation channel TRPA1. TRPA1 is both necessary and sufficient for the optovin response. Optovin activates human TRPA1 via structure-dependent photochemical reactions with redox-sensitive cysteine residues. In animals with severed spinal cords, optovin treatment enables control of motor activity in the paralyzed extremities by localized illumination. These studies identify a light-based strategy for controlling endogenous TRPA1 receptors in vivo, with potential clinical and research applications in non-transgenic animals, including humans

Substrate recognition by the 2-hydroxycarboxylate transport proteins

The object of the research described in this thesis was to identify substrate-protein interactions that provide affinity and specificity for CitPLEME from Lc.mesenteroides and MlePLALA form L.lactis. The ability of these transporters to transport substrates that differ in structure and charge implied specific requirements for the substrate binding site. ... Zie: Chapter 1

Arg-425 of the Citrate Transporter CitP Is Responsible for High Affinity Binding of Di- and Tricarboxylates

The citrate transporter of Leuconostoc mesenteroides (CitP) catalyzes exchange of divalent anionic citrate from the medium for monovalent anionic lactate, which is an end product of citrate degradation. The exchange generates a membrane potential and thus metabolic energy for the cell. The mechanism by which CitP transports both a divalent and a monovalent substrate was the subject of this investigation. Previous studies indicated that CitP is specific for substrates containing a 2-hydroxycarboxylate motif, HO-CR2-COO-. CitP has a high affinity for substrates that have a “second” carboxylate at one of the R groups, such as divalent citrate and (S)-malate. Monovalent anionic substrates that lack this second carboxylate were found to bind with a low affinity. In the present study we have constructed site-directed mutants, changing Arg-425 into a lysine or a cysteine residue. By using two substrates, i.e. (S)-malate and 2-hydroxyisobutyrate, the substrate specificity of the mutants was analyzed. In both mutants the affinity for divalent (S)-malate was strongly decreased, whereas the affinity for monovalent 2-hydroxyisobutyrate was not. The largest effect was seen when the arginine was changed into the neutral cysteine, which reduced the affinity for (S)-malate over 50-fold. Chemical modification of the R425C mutant with the sulfhydryl reagent 2-aminoethyl methanethiosulfonate, which restores the positive charge at position 425, dramatically reactivated the mutant transporter. The R425C and R425K mutants revealed a substrate protectable inhibition by other sulfhydryl reagents and the lysine reagent 2,4,6-trinitrobenzene sulfonate, respectively. It is concluded that Arg-425 complexes the charged carboxylate present in divalent substrates but that is absent in monovalent substrates, and thus plays an important role in the generation of the membrane potential.

Piezo1, a mechanically activated ion channel, is required for vascular development in mice

Mechanosensation is perhaps the last sensory modality not understood at the molecular level. Ion channels that sense mechanical force are postulated to play critical roles in a variety of biological processes including sensing touch/pain (somatosensation), sound (hearing), and shear stress (cardiovascular physiology); however, the identity of these ion channels has remained elusive. We previously identified Piezo1 and Piezo2 as mechanically activated cation channels that are expressed in many mechanosensitive cell types. Here, we show that Piezo1 is expressed in endothelial cells of developing blood vessels in mice. Piezo1-deficient embryos die at midgestation with defects in vascular remodeling, a process critically influenced by blood flow. We demonstrate that Piezo1 is activated by shear stress, the major type of mechanical force experienced by endothelial cells in response to blood flow. Furthermore, loss of Piezo1 in endothelial cells leads to deficits in stress fiber and cellular orientation in response to shear stress, linking Piezo1 mechanotransduction to regulation of cell morphology. These findings highlight an essential role of mammalian Piezo1 in vascular development during embryonic development

Zinc activates damage-sensing TRPA1 ion channels.

Zinc is an essential biological trace element. It is required for the structure or function of over 300 proteins, and it is increasingly recognized for its role in cell signaling. However, high concentrations of zinc have cytotoxic effects, and overexposure to zinc can cause pain and inflammation through unknown mechanisms. Here we show that zinc excites nociceptive somatosensory neurons and causes nociception in mice through TRPA1, a cation channel previously shown to mediate the pungency of wasabi and cinnamon through cysteine modification. Zinc activates TRPA1 through a unique mechanism that requires zinc influx through TRPA1 channels and subsequent activation via specific intracellular cysteine and histidine residues. TRPA1 is highly sensitive to intracellular zinc, as low nanomolar concentrations activate TRPA1 and modulate its sensitivity. These findings identify TRPA1 as an important target for the sensory effects of zinc and support an emerging role for zinc as a signaling molecule that can modulate sensory transmission