Biology subjects

Moller, E.

Publications and source records attributed to Moller, E..

5 recordsLinked to original sources

bioRxiv2024

On the lipid dependence of bacterial mechanosensitive channel gating in situ

For bacterial mechanosensitive channels acting as turgor-adjusting osmolyte release valves, membrane tension is the primary stimulus driving opening transitions. Because tension is transmitted through the surrounding lipid bilayer, it is possible that the presence or absence of different lipid species may influence the function of these channels. In this work, we characterize the lipid dependence of chromosome-encoded MscS and MscL in E. coli strains with genetically altered lipid composition. We use two previously generated strains that lack one or two major lipid species (PE, PG, or CL) and engineer a third strain that is highly enriched in CL due to the presence of hyperactive cardiolipin synthase ClsA. We characterize the functional behavior of these channels using patch-clamp and quantify the relative tension midpoints, closing rates, inactivation depth, and the rate of recovery back to the closed state. We also measure the osmotic survival of lipid-deficient strains, which characterizes the functional consequences of lipid-mediated channel function at the cell level. We find that the opening and closing behavior of MscS and MscL tolerate the absence of specific lipid species remarkably well. The lack of cardiolipin (CL), however, reduces the active MscS population relative to MscL and decreases the closing rate, slightly increasing the propensity of MscS toward inactivation and slowing the recovery process. The data points to the robustness of the osmolyte release system and the importance of cardiolipin for the adaptive behavior of MscS.

biophysics↗

bioRxiv2024

Polymer-solubilized structure of the mechanosensitive channel MscS suggests the role of protein-lipid interactions in the functional gating cycle

Interpretations of experimental conformations of mechanosensitive channels gated by force from lipids become more reliable when native lipids are preserved in the structures. Escherichia coli MscS is an adaptive osmolyte release valve that regulates turgor in osmotically stressed cells. MscS promptly opens under abrupt super-threshold tensions in the membrane, but at lower and more gradually applied tensions, it silently inactivates from the closed state. A central question has been whether to assign the commonly observed non-conductive conformation with splayed peripheral helices to a closed or inactivated state. We present a 3-[A] MscS cryo-EM structure obtained in Glyco-DIBMA polymers, which avoid complete lipid removal. Within the complex, we observe densities for endogenous phospholipids intercalating between the peripheral and pore-lining helices. The lipidomic analysis shows a 2-3 fold enrichment of phosphatidylglycerol in Glyco-DIBMA-extracted MscS samples. The computed pressure of these lipids on the protein surface enforces the characteristic kinks in the pore-lining helices, sterically stabilizing the separation of the peripheral helices. Mutations of residues coordinating lipids in the crevices eliminate inactivation, allowing us to classify this group of structures as the inactivated state. Our study reveals a novel inactivation mechanism in which intercalated lipids physically decouple the tension-sensing helices from the gate.

biophysics↗

bioRxiv2023

MscS is a critical component for osmotic survival of Vibrio cholerae

Like other intestinal bacteria, the facultative pathogen Vibrio cholerae adapts to a wide range of osmotic environments. Under drastic osmotic down-shifts, Vibrio avoids mechanical rupture by rapidly releasing excessive metabolites through mechanosensitive (MS) channels that belong to two major types, low-threshold MscS and high-threshold MscL. To investigate each channels individual contribution to V. choleraes osmotic permeability response, we generated individual{Delta} mscS, {Delta}mscL, and double{Delta} mscL {Delta}mscS mutants in V. cholerae O395 and characterized their tension-dependent activation in patch-clamp experiments, as well as their millisecond-scale osmolyte release kinetics using a stopped-flow light scattering technique. We additionally generated numerical models reflecting the kinetic competition of osmolyte release with water influx. Both mutants lacking MscS exhibited delayed osmolyte release kinetics and decreased osmotic survival rates compared to WT. The{Delta} mscL mutant showed comparable release kinetics to WT, but a higher osmotic survival, while {Delta}mscS had low survival, comparable to the double{Delta} mscL {Delta}mscS mutant. By analyzing release kinetics following rapid medium dilution, we illustrate the sequence of events and define the set of parameters that characterize discrete phases of the osmotic response. Osmotic survival rates are directly correlated to the extent and duration of cell swelling, the rate of osmolyte release and the onset time, and the completeness of the post-shock membrane resealing. Not only do the two channels interact functionally during the resealing phase, but there is also a compensatory up-regulation of MscS in the{Delta} mscL strain suggesting some transcriptional crosstalk. The data reveal the advantage of the low-threshold MscS channel in curbing tension surges, without which MscL becomes toxic, and the role of MscS in the proper termination of the osmotic permeability response in Vibrio.

biophysics↗

bioRxiv2023

MscS inactivation and recovery are slow voltage-dependent processes sensitive to interactions with lipids

Mechanosensitive channel MscS, the major bacterial osmolyte release valve, shows a characteristic adaptive behavior. With a sharp onset of activating tension, the channel population readily opens, but under prolonged action of moderate near-threshold tension, it inactivates. The inactivated state is non-conductive and tension-insensitive, which suggests that the gate gets uncoupled from the lipid-facing domains. The kinetic rates for tension-driven opening-closing transitions are 4-6 orders of magnitude higher than the rates for inactivation and recovery. Here we show that inactivation is augmented and recovery is slowed down by depolarization. Hyperpolarization, conversely, impedes inactivation and speeds up recovery. We then address the question of whether protein-lipid interactions may set the rates and influence voltage dependence of inactivation and recovery. Mutations of conserved arginines 46 and 74 anchoring the lipid-facing helices to the inner membrane leaflet to tryptophans do not change the closing transitions, but instead change the kinetics of both inactivation and recovery and essentially eliminate their voltage-dependence. Uncharged polar substitutions (S or Q) for these anchors produce functional channels but increase the inactivation and reduce the recovery rates. The data suggest that it is not the activation and closing transitions, but rather the inactivation and recovery pathways that involve substantial rearrangements of the protein-lipid boundary associated with the separation of the lipid-facing helices from the gate. The discovery that hyperpolarization robustly assists MscS recovery indicates that membrane potential can regulate osmolyte release valves by putting them either on the ready or standby mode depending on the cells metabolic state.

biophysics↗

bioRxiv2023

Mechanosensitive channel MscS is critical for termination of the bacterial hypoosmotic permeability response

Free-living microorganisms are subjected to drastic changes in osmolarity. To avoid lysis under sudden osmotic down-shock, bacteria quickly expel small metabolites through the tension-activated channels MscL, MscS, and MscK. We examined five chromosomal knockout strains, {Delta}mscL, {Delta}mscS, a double knockout {Delta}mscS {Delta}mscK, and a triple knockout {Delta}mscL {Delta}mscS {Delta}mscK in comparison to the wild-type parental strain. Stopped-flow experiments confirmed that both MscS and MscL mediate fast osmolyte release and curb cell swelling, but osmotic viability assays indicated that they are not equivalent. MscS alone was capable of rescuing the cell population, but in some strains MscL did not rescue and additionally became toxic in the absence of both MscS and MscK. Furthermore, MscS was upregulated in the {Delta}mscL strain, suggesting either a cross-talk between the two genes/proteins or the influence of cell mechanics on mscS expression. The data shows that for the proper termination of the permeability response, the high-threshold (MscL) and the low-threshold (MscS/MscK) channels must act sequentially. In the absence of low-threshold channels, at the end of the release phase, MscL should stabilize membrane tension at around 10 mN/m. Patch-clamp protocols emulating the tension changes during the release phase indicated that the non-inactivating MscL, residing at its own tension threshold, flickers and produces a protracted leakage. The MscS/MscK population, when present, stays open at this stage to reduce tension below the MscL threshold and silence the large channel. When MscS reaches its own threshold, it inactivates and thus ensures proper termination of the hypoosmotic permeability response. This functional interplay between the high- and low-threshold channels is further supported by the compromised osmotic survival of bacteria expressing non-inactivating MscS mutants. Summary (for the table of contents)The kinetics of hypotonic osmolyte release from E. coli is analyzed in conjunction with bacterial survival. It is shown that MscL, the high-threshold emergency release valve, rescues bacteria from down-shocks only in the presence of MscS, MscK or other low-threshold channels that are necessary to pacify MscL at the end of the release phase.

biophysics↗