Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm² in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.     

The pathway and spatial scale for MscS inactivation

The mechanosensitive channel of small conductance (MscS) is a bacterial tension-driven osmolyte release valve with homologues in many walled eukaryotic organisms. When stimulated by steps of tension in excised patches, Escherichia coli MscS exhibits transient opening followed by reversible adaptation and then complete inactivation. Here, we study properties of the inactivation transition, which renders MscS nonconductive and tension insensitive. Using special pressure protocols we demonstrate that adaptation and inactivation are sequential processes with opposite tension dependencies. In contrast to many eukaryotic channels, which inactivate from the open state, MscS inactivates primarily from the closed state because full openings by preconditioning pulses do not influence the degree of inactivation, and saturating tensions keeping channels open prevent inactivation. The easily opened A98S mutant lacks inactivation completely, whereas the L111S mutant with a right-shifted activation curve inactivates silently before reaching the threshold for opening. This suggests that opening and inactivation are two independent tension-driven pathways, both starting from the closed state. Analysis of tension dependencies for inactivation and recovery rates estimated the in-plane expansion (ΔA) associated with inactivation as 8.5 nm(2), which is about half of the area change for opening. Given that the interhelical contact between the outer TM1-TM2 pairs and the core TM3s is the force-transmitting path from the periphery to the gate, the determined ΔA now can be used as a constraining parameter for the models of the inactivated state in which the lipid-facing TM1-TM2 pairs are displaced and uncoupled from the gate.     

Concentration dependent effect of GsMTx4 on mechanosensitive channels of small conductance in <Emphasis Type="Italic">E. coli</Emphasis> spheroplasts

The spider peptide GsMTx4, at saturating concentration of 5 μM, is an effective and specific inhibitor for stretch-activated mechanosensitive (MS) channels found in a variety of eukaryotic cells. Although the structure of the peptide has been solved, the mode of action remains to be determined. Because of its amphipathic structure, the peptide is proposed to interact with lipids at the boundaries of the MS channel proteins. In addition, GsMTx4 has antimicrobial effects, inhibiting growth of several species of bacteria in the range of 5–64 μM. Previous studies on prokaryotic MS channels, which serve as model systems to explore the principle of MS channel gating, have shown that various amphipathic compounds acting at the protein–lipid interface affect MS channel gating. We have therefore analyzed the effect of different concentrations of extracellular GsMTx4 on MS channels of small conductance, MscS and MscK, in the cytoplasmic membrane of wild-type E. coli spheroplasts using the patch-clamp technique. Our study shows that the peptide GsMTx4 exhibits a biphasic response in which peptide concentration determines inhibition or potentiation of activity in prokaryotic MS channels. At low peptide concentrations of 2 and 4 μM the gating of the prokaryotic MS channels was hampered, manifested by a decrease in pressure sensitivity. In contrast, application of peptide at concentrations of 12 and 20 μM facilitated prokaryotic MS channel opening by increasing the pressure sensitivity.     

Functional design of bacterial mechanosensitive channels. Comparisons and contrasts illuminated by random mutagenesis

MscS and MscL are mechanosensitive channels found in bacterial plasma membranes that open large pores in response to membrane tension. These channels function to alleviate excess cell turgor invoked by rapid osmotic downshock. Although much is known of the structure and molecular mechanisms underlying MscL, genes correlating with MscS activity have only recently been identified. Previously, it was shown that eliminating the expression of Escherichia coli yggB removed a major portion of MscS activity. YggB is distinct from MscL by having no obvious structural similarity. Here we have reconstituted purified YggB in proteoliposomes and have successfully detected MscS channel activity, confirming that purified YggB protein encodes MscS activity. Additionally, to define functional regions of the channel protein, we have randomly mutagenized the structural gene and isolated a mutant that evokes a gain-of-function phenotype. Physiological experiments demonstrate that the mutated channel allows leakage of solutes from the cell, suggesting inappropriate channel opening. Interestingly, this mutation is analogous in position and character to mutations yielding a similar phenotype in MscL. Hence, although MscS and MscL mechanosensitive channels are structurally quite distinct, there may be analogies in their gating mechanisms.     

Human PIEZO1: Removing Inactivation

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.     

Human PIEZO1: Removing Inactivation

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.     

Structure and mechanism in prokaryotic mechanosensitive channels

Mechanosensitive channels function as electromechanical switches with the capability to sense the physical state of lipid bilayers. The X-ray crystal structures of MscL and MscS offer a unique opportunity to identify the types of protein motions associated with the opening and closing of these structurally unrelated channels, while providing the framework to address a mechanism of tension sensing that is defined by channel-lipid interactions. Recent functional, structural and dynamic data offer fresh insights into the molecular basis of gating for these membrane proteins.     

The mechanosensitive ion channel Piezo1 is inhibited by the peptide GsMTx4

Cells can respond to mechanical stress by gating mechanosensitive ion channels (MSCs). The cloning of Piezo1, a eukaryotic cation selective MSC, defines a new system for studying mechanical transduction at the cellular level. Because Piezo1 has electrophysiological properties similar to those of endogenous cationic MSCs that are selectively inhibited by the peptide GsMTx4, we tested whether the peptide targets Piezo1 activity. Extracellular GsMTx4 at micromolar concentrations reversibly inhibited ～80% of the mechanically induced current of outside-out patches from transfected HEK293 cells. The inhibition was voltage insensitive, and as seen with endogenous MSCs, the mirror image d enantiomer inhibited like the l. The rate constants for binding and unbinding based on Piezo1 current kinetics provided association and dissociation rates of 7.0 × 10(5) M(-1) s(-1) and 0.11 s(-1), respectively, and a K(D) of ～155 nM, similar to values previously reported for endogenous MSCs. Consistent with predicted gating modifier behavior, GsMTx4 produced an ～30 mmHg rightward shift in the pressure-gating curve and was active on closed channels. In contrast, streptomycin, a nonspecific inhibitor of cationic MSCs, showed the use-dependent inhibition characteristic of open channel block. The peptide did not block currents of the mechanical channel TREK-1 on outside-out patches. Whole-cell Piezo1 currents were also reversibly inhibited by GsMTx4, and although the off rate was nearly identical to that of outside-out patches, differences were observed for the on rate. The ability of GsMTx4 to target the mechanosensitivity of Piezo1 supports the use of this channel in high-throughput screens for pharmacological agents and diagnostic assays.     

Two Families of Mechanosensitive Channel Proteins

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 α-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Two families of mechanosensitive channel proteins.

Mechanosensitive (MS) channels that provide protection against hypoosmotic shock are found in the membranes of organisms from the three domains of life: bacteria, archaea, and eucarya. Two families of ubiquitous MS channels are recognized, and these have been designated the MscL and MscS families. A high-resolution X-ray crystallographic structure is available for a member of the MscL family, and extensive molecular genetic, biophysical, and biochemical studies conducted in many laboratories have allowed postulation of a gating mechanism allowing the interconversion of a tightly closed state and an open state that controls transmembrane ion and metabolite fluxes. In contrast to the MscL channel proteins, which are of uniform topology, the much larger MscS family includes protein members with topologies that are predicted to vary from 3 to 11 alpha-helical transmembrane segments (TMSs) per polypeptide chain. Sequence analyses reveal that the three C-terminal TMSs of MscS channel proteins are conserved among family members and that the third of these three TMSs exhibits a 20-residue motif that is shared by the channel-forming TMS (TMS 1) of the MscL proteins. We propose that this C-terminal TMS in MscS family homologues serves as the channel-forming helix in a homooligomeric structure. The presence of a conserved residue pattern for the putative channel-forming TMSs in the MscL and MscS family proteins suggests a common structural organization, gating mechanism, and evolutionary origin.     

Electrophysiological Characterization of the Mechanosensitive Channel MscCG in Corynebacterium glutamicum

Corynebacterium glutamicum MscCG, also referred to as NCgl1221, exports glutamate when biotin is limited in the culture medium. MscCG is a homolog of Escherichia coli MscS, which serves as an osmotic safety valve in E. coli cells. Patch-clamp experiments using heterogeneously expressed MscCG have shown that MscCG is a mechanosensitive channel gated by membrane stretch. Although the association of glutamate secretion with the mechanosensitive gating has been suggested, the electrophysiological characteristics of MscCG have not been well established. In this study, we analyzed the mechanosensitive gating properties of MscCG by expressing it in E. coli spheroplasts. MscCG is permeable to glutamate, but is also permeable to chloride and potassium. The tension at the midpoint of activation is 6.68 ± 0.63 mN/m, which is close to that of MscS. The opening rates at saturating tensions and closing rates at zero tension were at least one order of magnitude slower than those observed for MscS. This slow kinetics produced strong opening-closing hysteresis in response to triangular pressure ramps. Whereas MscS is inactivated under sustained stimulus, MscCG does not undergo inactivation. These results suggest that the mechanosensitive gating properties of MscCG are not suitable for the response to abrupt and harmful changes, such as osmotic downshock, but are tuned to execute slower processes, such as glutamate export.     

Bacterial mechanosensitive channels as a paradigm for mechanosensory transduction

Research on bacterial mechanosensitive (MS) channels has since their discovery been at the forefront of the MS channel field due to extensive studies of the structure and function of MscL and MscS, two of the several different types of MS channels found in bacteria. Just a few years after these two MS channels were cloned their 3D structure was solved by X-ray crystallography. Today, the repertoire of multidisciplinary approaches used in experimental and theoretical studies following the cloning and crystallographic determination of the MscL and MscS structure has expanded by including electronparamagnetic resonance (EPR) and F?rster resonance energy transfer (FRET) spectroscopy aided by computational modelling employing molecular dynamics as well as Brownian dynamics simulations, which significantly advanced the understanding of structural determinants of the gating and conduction properties of these two MS channels. These extensive multidisciplinary studies of MscL and MscS have greatly contributed to elucidation of the basic physical principles of MS channel gating by mechanical force. This review summarizes briefly the major experimental and conceptual advancements, which helped in establishing MscL and MscS as a major paradigm of mechanosensory transduction in living cells.     

Electrophysiological Characterization of the Mechanosensitive Channel MscCG in Corynebacterium glutamicum

Corynebacterium glutamicum MscCG, also referred to as NCgl1221, exports glutamate when biotin is limited in the culture medium. MscCG is a homolog of Escherichia coli MscS, which serves as an osmotic safety valve in E. coli cells. Patch-clamp experiments using heterogeneously expressed MscCG have shown that MscCG is a mechanosensitive channel gated by membrane stretch. Although the association of glutamate secretion with the mechanosensitive gating has been suggested, the electrophysiological characteristics of MscCG have not been well established. In this study, we analyzed the mechanosensitive gating properties of MscCG by expressing it in E. coli spheroplasts. MscCG is permeable to glutamate, but is also permeable to chloride and potassium. The tension at the midpoint of activation is 6.68 ± 0.63 mN/m, which is close to that of MscS. The opening rates at saturating tensions and closing rates at zero tension were at least one order of magnitude slower than those observed for MscS. This slow kinetics produced strong opening-closing hysteresis in response to triangular pressure ramps. Whereas MscS is inactivated under sustained stimulus, MscCG does not undergo inactivation. These results suggest that the mechanosensitive gating properties of MscCG are not suitable for the response to abrupt and harmful changes, such as osmotic downshock, but are tuned to execute slower processes, such as glutamate export.     

From membrane tension to channel gating: A principal energy transfer mechanism for mechanosensitive channels

Mechanosensitive (MS) channels are evolutionarily conserved membrane proteins that play essential roles in multiple cellular processes, including sensing mechanical forces and regulating osmotic pressure. Bacterial MscL and MscS are two prototypes of MS channels. Numerous structural studies, in combination with biochemical and cellular data, provide valuable insights into the mechanism of energy transfer from membrane tension to gating of the channel. We discuss these data in a unified two‐state model of thermodynamics. In addition, we propose a lipid diffusion‐mediated mechanism to explain the adaptation phenomenon of MscS.     

Energetics of gating MscS by membrane tension in azolectin liposomes and giant spheroplasts

Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations.     

Energetics of gating MscS by membrane tension in azolectin liposomes and giant spheroplasts

Mechanosensitive (MS) ion channels are molecular sensors that detect and transduce signals across prokaryotic and eukaryotic cell membranes arising from external mechanical stimuli or osmotic gradients. They play an integral role in mechanosensory responses including touch, hearing, and proprioception by opening or closing in order to facilitate or prevent the flow of ions and organic osmolytes. In this study we use a linear force model of MS channel gating to determine the gating membrane tension (γ) and the gating area change (ΔA) associated with the energetics of MscS channel gating in giant spheroplasts and azolectin liposomes. Analysis of Boltzmann distribution functions describing the dependence of MscS channel gating on membrane tension indicated that the gating area change (ΔA) was the same for MscS channels recorded in both preparations. The comparison of the membrane tension (γ) gating the channel, however, showed a significant difference between the MscS channel activities in these two preparations.     

Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.     

On the mechanism of CFTR inhibition by a thiazolidinone derivative

The effects of a thiazolidinone derivative, 3-[(3-trifluoromethyl)phenyl]-5-[(4-carboxyphenyl)methylene]-2-thioxo-4-thiazolidinone (or CFTRinh-172), on cystic fibrosis transmembrane conductance regulator (CFTR) gating were studied in excised inside-out membrane patches from Chinese hamster ovary cells transiently expressing wild-type and mutant CFTR. We found that the application of CFTRinh-172 results in an increase of the mean closed time and a decrease of the mean open time of the channel. A hyperbolic relationship between the closing rate and [CFTRinh-172] suggests that CFTRinh-172 does not act as a simple pore blocker. Interestingly, the potency of inhibition increases as the open time of the channel is increased with an IC50 in the low nanomolar range for CFTR channels locked in an open state for tens of seconds. Our studies also provide evidence that CFTRinh-172 can bind to both the open state and the closed state. However, at least one additional step, presumably reflecting inhibitor-induced conformational changes, is required to shut down the conductance after the binding of the inhibitor to the channel. Using the hydrolysis-deficient mutant E1371S as a tool as the closing rate of this mutant is dramatically decreased, we found that CFTRinh-172-dependent inhibition of CFTR channel gating, in two aspects, mimics the inactivation of voltage-dependent cation channels. First, similar to the recovery from inactivation in voltage-gated channels, once CFTR is inhibited by CFTRinh-172, reopening of the channel can be seen upon removal of the inhibitor in the absence of adenosine triphosphate (ATP). Second, ATP induced a biphasic current response on inhibitor-bound closed channels as if the ATP-opened channels "inactivate" despite a continuous presence of ATP. A simplified six-state kinetic scheme can well describe our data, at least qualitatively. Several possible structural mechanisms for the effects of CFTRinh-172 will be discussed.     

Redox reagents and divalent cations alter the kinetics of cystic fibrosis transmembrane conductance regulator channel gating.

Gating of the cystic fibrosis Cl(-) channel requires hydrolysis of ATP by its nucleotide binding folds, but how this process controls the kinetics of channel gating is poorly understood. In the present work we show that the kinetics of channel gating and presumably the rate of ATP hydrolysis depends on the species of divalent cation present and the oxidation state of the protein. With Ca(2+) as the dominant divalent cation instead of Mg(2+), the open burst duration of the channel is increased approximately 20-fold, and this change is reversible upon washout of Ca(2+). In contrast, "soft" divalent cations such as Cd(2+) interact covalently with cystic fibrosis transmembrane conductance regulator (CFTR). These metals decrease both opening and closing rates of the channel, and the effects are not reversed by washout. Oxidation of CFTR channels with a variety of oxidants resulted in a similar slowing of channel gating. In contrast, reducing agents had the opposite effect, increasing both opening and closing rates of the channel. In cell-attached patches, CFTR channels exhibit both oxidized and reduced types of gating, raising the possibility that regulation of the redox state of the channel may be a physiological mode of control of CFTR channel activity.     

The protective effect of osmoprotectant TMAO on bacterial mechanosensitive channels of small conductance MscS/MscK under high hydrostatic pressure

Activity of the bacterial mechanosensitive channels of small conductance MscS/MscK of E. coli was investigated under high hydrostatic pressure (HHP) using the “flying-patch” patch-clamp technique. The channels were gated by negative pipette voltage and their open probability was measured at HHP of 0.1 to 80 MPa. The channel open probability decreased with increasing HHP. When the osmolyte methylamine N-oxide (TMAO) was applied to the cytoplasmic side of the inside-out excised membrane patches of E. coli giant spheroplasts the inhibitory effect of HHP on the channel activity was suppressed at pressures of up to 40 MPa. At 40 MPa and above the channel open probability decreased in a similar fashion with or without TMAO. Our study suggests that TMAO helps to counteract the effect of HHP up to 40 MPa on the MscS/MscK open state by “shielding” the cytoplasmic domain of the channels.