Curcumin activation of a bacterial mechanosensitive channel underlies its membrane permeability and adjuvant properties.
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ABSTRACT:
SUBMITTER: Wray R
PROVIDER: S-EPMC8769312 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Resistance to antibiotics is a serious and worsening threat to human health worldwide, and there is an urgent need to develop new antibiotics that can avert it. One possible solution is the development of compounds that possess multiple modes of action, requiring at least two mutations to acquire resistance. Compound SCH-79797 both avoids resistance and has two mechanisms of action: one inhibiting the folate pathway, and a second described as "membrane permeabilization"; however, the mechanism by which membranes from bacterial cells, but not the host, are disrupted has remained mysterious. The opening of the bacterial mechanosensitive channel of large conductance, MscL, which ordinarily serves the physiological role of osmotic emergency release valves gated by hypoosmotic shock, has been previously demonstrated to affect bacterial membrane permeabilization. MscL allows the rapid permeabilization of both ions and solutes through the opening of the largest known gated pore, which has a diameter of 30 Å. We found that SCH-79797 and IRS-16, a more potent derivative, directly bind to the MscL channel and produce membrane permeabilization as a result of its activation. These findings suggest that possessing or adding an MscL-activating component to an antibiotic compound could help to lower toxicity and evade antibiotic resistance.
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Project description:Capsaicin is an activator of the heat-sensitive TRPV1 (transient receptor potential vanilloid 1) ion channels and has been used as a local analgesic. We found that activation of TRPV1 channels with capsaicin either in dorsal root ganglion neurons or in a heterologous expression system inhibited the mechanosensitive Piezo1 and Piezo2 channels by depleting phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and its precursor phosphatidylinositol 4-phosphate [PI(4)P] from the plasma membrane through Ca(2+)-induced phospholipase Cδ (PLCδ) activation. Experiments with chemically inducible phosphoinositide phosphatases and receptor-induced activation of PLCβ indicated that inhibition of Piezo channels required depletion of both PI(4)P and PI(4,5)P2. The mechanically activated current amplitudes decreased substantially in the excised inside-out configuration, where the membrane patch containing Piezo1 channels is removed from the cell. PI(4,5)P2 and PI(4)P applied to these excised patches inhibited this decrease. Thus, we concluded that Piezo channel activity requires the presence of phosphoinositides, and the combined depletion of PI(4,5)P2 and PI(4)P reduces channel activity. In addition to revealing a role for distinct membrane lipids in mechanosensitive ion channel regulation, these data suggest that inhibition of Piezo2 channels may contribute to the analgesic effect of capsaicin.
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Project description:YggB and MscL are the major mechanosensitive channels in Escherichia coli, and each can rescue the double knockout mutant from osmotic downshock. However, the role of MscL in wild-type bacteria is in question, not only because cells without MscL survive severe osmotic downshocks, but because 1.8 times more suction is required to gate MscL than YggB under patch clamp. Here, we extend previous evidence [Ajouz, B., Berrier, C., Garrigues, A., Besnard, M. & Ghazi, A. (1998) J. Biol. Chem. 273, 26670-26674] to show that downshock gates MscL in vivo even in the presence of YggB. We have made this determination by engineering a channel we can structurally modify in vivo (Leu-19-->Cys MscL). MscLs with charges in their constrictions are known to open easily and transiently to substates and stop cell growth. In this study, we use downshock to stretch this region open to allow attachment of a charged thiosulfonate reagent MTSET(+), thereby creating a toxic channel. Therefore, channel opening can be monitored by loss of colony forming units. By this measure, we find that an approximately 800 mmol/kg downshock from 1,200 mmol/kg medium opens Leu-19-->Cys MscL in the presence of YggB, but a downshock of only approximately 400 mmol/kg is required in the absence of YggB. In parallel, Leu-19-->Cys MscL, stretched open by large sustained suction in the presence of MTSET(+) in voltage-clamped patches, subsequently flickers open with little suction. These observations show that MscL opening is triggered by a specific downshock, even in the presence of YggB, that YggB buffers MscL gating in vivo, and that residue 19 becomes exposed upon channel opening.
| S-EPMC122824 | biostudies-literature
Project description:We outline several principles that we believe define the gating of two bacterial mechanosensitive channels, MscL and MscS. Serving as turgor regulators in bacteria and other walled cells, these molecules are tangible models for studying conformational transitions in membrane proteins driven directly by membrane tension. MscL, a compact pentamer, reversibly opens a gigantic 30-A pore at near-lytic tensions. MscS, a heptameric complex, exhibits transient activation of a smaller pore at moderate tensions, thereby entering a tension-insensitive inactivated state. By comparing the structures and predicted transitions in these channels, we concluded that opening is commonly achieved through tilting and outward motion of the pore-lining helices, which is kinetically limited by hydration of the pore. The intricate adaptive behavior in MscS appears to depend on specific interhelical associations and the flexibility of the pore-lining helices. We discuss physical factors that may direct the transitions and stabilize main functional states in these channels.
| S-EPMC2740535 | biostudies-literature
Project description:Transmembrane channel-like protein isoform 1 (TMC1) is essential for the generation of mechano-electrical transducer currents in hair cells of the inner ear. TMC1 disruption causes hair cell degeneration and deafness in mice and humans. Although thought to be expressed at the cell surface in vivo, TMC1 remains in the endoplasmic reticulum when heterologously expressed in standard cell lines, precluding determination of its roles in mechanosensing and pore formation. Here, we report that the KCNQ1 Kv channel forms complexes with TMC1 and rescues its surface expression when coexpressed in Chinese Hamster Ovary cells. TMC1 rescue is specific for KCNQ1 within the KCNQ family, is prevented by a KCNQ1 trafficking-deficient mutation, and is influenced by KCNE β subunits and inhibition of KCNQ1 endocytosis. TMC1 lowers KCNQ1 and KCNQ1-KCNE1 K+ currents, and despite the surface expression, it does not detectably respond to mechanical stimulation or high salt. We conclude that TMC1 is not intrinsically mechano- or osmosensitive but has the capacity for cell surface expression, and requires partner protein(s) for surface expression and mechanosensitivity. We suggest that KCNQ1, expression of which is not thought to overlap with TMC1 in hair cells, is a proxy partner bearing structural elements or a sequence motif reminiscent of a true in vivo TMC1 hair cell partner. Discovery of the first reported strategy to rescue TMC1 surface expression should aid future studies of the TMC1 function and native partners.
| S-EPMC6478532 | biostudies-literature
Project description:Mechanical and thermal activation of ion channels is central to touch, thermosensation, and pain. The TRAAK/TREK K(2P) potassium channel subfamily produces background currents that alter neuronal excitability in response to pressure, temperature, signaling lipids, and anesthetics. How such diverse stimuli control channel function is unclear. Here we report structures of K(2P)4.1 (TRAAK) bearing C-type gate-activating mutations that reveal a tilting and straightening of the M4 inner transmembrane helix and a buckling of the M2 transmembrane helix. These conformational changes move M4 in a direction opposite to that in classical potassium channel activation mechanisms and open a passage lateral to the pore that faces the lipid bilayer inner leaflet. Together, our findings uncover a unique aspect of K(2P) modulation, indicate a means for how the K(2P) C-terminal cytoplasmic domain affects the C-type gate which lies ∼40Å away, and suggest how lipids and bilayer inner leaflet deformations may gate the channel.
| S-EPMC4270892 | biostudies-literature
Project description:Mechanosensitive channels are detected in all cells and are speculated to play a key role in many functions including osmoregulation, growth, hearing, balance, and touch. In prokaryotic cells, a direct gating of mechanosensitive channels by membrane tension was clearly demonstrated because the purified channels could be functionally reconstituted in a lipid bilayer. No such evidence has been presented yet in the case of mechanosensitive channels from animal cells. TREK-1, a two-pore domain K(+) channel, was the first animal mechanosensitive channel identified at the molecular level. It is the target of a large variety of agents such as volatile anesthetics, neuroprotective agents, and antidepressants. We have produced the mouse TREK-1 in yeast, purified it, and reconstituted the protein in giant liposomes amenable to patch clamp recording. The protein exhibited the expected electrophysiological properties in terms of kinetics, selectivity, and pharmacology. Negative pressure (suction) applied through the pipette had no effect on the channel, but positive pressure could completely and reversibly close the channel. Our interpretation of these data is that the intrinsic tension in the lipid bilayer is sufficient to maximally activate the channel, which can be closed upon modification of the tension. These results indicate that TREK-1 is directly sensitive to membrane tension.
| S-EPMC3779726 | biostudies-literature
Project description:Bacterial mechanosensitive channels gate when the transmembrane turgor rises to levels that compromise the structural integrity of the cell wall. Gating creates a transient large diameter pore that allows hydrated solutes to pass from the cytoplasm at rates close to those of diffusion. In the closed conformation, the channel limits transmembrane solute movement, even that of protons. In the MscS crystal structure (Protein Data Bank entry 2oau ), a narrow, hydrophobic opening is visible in the crystal structure, and it has been proposed that a vapor lock created by the hydrophobic seals, L105 and L109, is the barrier to water and ions. Tryptophan scanning mutagenesis has proven to be a highly valuable tool for the analysis of channel structure. Here Trp residues were introduced along the pore-forming TM3a helix and in selected other parts of the protein. Mutants were investigated for their expression, stability, and activity and as fluorescent probes of the physical properties along the length of the pore. Most Trp mutants were expressed at levels similar to that of the parent (MscS YFF) and were stable as heptamers in detergent in the presence and absence of urea. Fluorescence data suggest a long hydrophobic region with low accessibility to aqueous solvents, extending from L105/L109 to G90. Steady-state fluorescence anisotropy data are consistent with significant homo-Förster resonance energy transfer between tryptophan residues from different subunits within the narrow pore. The data provide new insights into MscS structure and gating.
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Project description:BackgroundVolatile pyrethroid insecticides, such as transfluthrin, have received increasing attention for their potent repellent activities in recent years for controlling human disease vectors. It has been long understood that pyrethroids kill insects by promoting activation and inhibiting inactivation of voltage-gated sodium channels. However, the mechanism of pyrethroid repellency remains poorly understood and controversial.Methodology/principal findingsHere, we show that transfluthrin repels Aedes aegypti in a hand-in-cage assay at nonlethal concentrations as low as 1 ppm. Contrary to a previous report, transfluthrin does not elicit any electroantennogram (EAG) responses, indicating that it does not activate olfactory receptor neurons (ORNs). The 1S-cis isomer of transfluthrin, which does not activate sodium channels, does not elicit repellency. Mutations in the sodium channel gene that reduce the potency of transfluthrin on sodium channels decrease transfluthrin repellency but do not affect repellency by DEET. Furthermore, transfluthrin enhances DEET repellency.Conclusions/significanceThese results provide a surprising example that sodium channel activation alone is sufficient to potently repel mosquitoes. Our findings of sodium channel activation as the principal mechanism of transfluthrin repellency and potentiation of DEET repellency have broad implications in future development of a new generation of dual-target repellent formulations to more effectively repel a variety of human disease vectors.
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Project description:Gram-negative bacteria characteristically are surrounded by an additional membrane layer, the outer membrane. Although outer membrane components often play important roles in the interaction of symbiotic or pathogenic bacteria with their host organisms, the major role of this membrane must usually be to serve as a permeability barrier to prevent the entry of noxious compounds and at the same time to allow the influx of nutrient molecules. This review summarizes the development in the field since our previous review (H. Nikaido and M. Vaara, Microbiol. Rev. 49:1-32, 1985) was published. With the discovery of protein channels, structural knowledge enables us to understand in molecular detail how porins, specific channels, TonB-linked receptors, and other proteins function. We are now beginning to see how the export of large proteins occurs across the outer membrane. With our knowledge of the lipopolysaccharide-phospholipid asymmetric bilayer of the outer membrane, we are finally beginning to understand how this bilayer can retard the entry of lipophilic compounds, owing to our increasing knowledge about the chemistry of lipopolysaccharide from diverse organisms and the way in which lipopolysaccharide structure is modified by environmental conditions.
| S-EPMC309051 | biostudies-literature