The cytoplasmic cage domain of the mechanosensitive channel MscS is a sensor of macromolecular crowding

Cells actively regulate the macromolecular excluded volume of the cytoplasm to maintain the reciprocal fraction of free aqueous solution that is optimal for intracellular processes. However, the mechanisms whereby cells sense this critical parameter remain unclear. The mechanosensitive channel of small conductance (MscS channel), which is the major regulator of turgor in bacteria, mediates efflux of small osmolytes in response to increased membrane tension. At moderate sustained tensions produced by a decrease in external osmolarity, MscS undergoes slow adaptive inactivation; however, it inactivates abruptly in the presence of cytoplasmic crowding agents. To understand the mechanism underlying this rapid inactivation, we combined extrapolated and equilibrium molecular dynamics simulations with electrophysiological analyses of MscS mutants to explore possible transitions of MscS and generated models of the resting and inactivated states. Our models suggest that the coupling of the gate formed by TM3 helices to the peripheral TM1–TM2 pairs depends on the axial position of the core TM3 barrel relative to the TM1–TM2 shaft and the state of the associated hollow cytoplasmic domain (“cage”). They also indicate that the tension-driven inactivation transition separates the gate from the peripheral helices and promotes kinks in TM3s at G113 and that this conformation is stabilized by association of the TM3b segment with the β domain of the cage. We found that mutations destabilizing the TM3b–β interactions preclude inactivation and make the channel insensitive to crowding agents and voltage; mutations that strengthen this association result in a stable closed state and silent inactivation. Steered simulations showed that pressure exerted on the cage bottom in the inactivated state reduces the volume of the cage in the cytoplasm and at the same time increases the footprint of the transmembrane domain in the membrane, implying coupled sensitivity to both membrane tension and crowding pressure. The cage, therefore, provides feedback on the increasing crowding that disengages the gate and prevents excessive draining and condensation of the cytoplasm. We discuss the structural mechanics of cells surrounded by an elastic cell wall where this MscS-specific feedback mechanism may be necessary.     

Gain-of-function mutations reveal expanded intermediate states and a sequential action of two gates in MscL

The tension-driven gating transition in the large mechanosensitive channel MscL proceeds through detectable states of intermediate conductance. Gain-of-function (GOF) mutants with polar or charged substitutions in the main hydrophobic gate display altered patterns of subconducting states, providing valuable information about gating intermediates. Here we present thermodynamic analysis of several GOF mutants to clarify the nature and position of low-conducting conformations in the transition pathway. Unlike wild-type (WT) MscL, which predominantly occupies the closed and fully open states with very brief substates, the mild V23T GOF mutant frequently visits a multitude of short-lived subconducting states. Severe mutants V23D and G22N open in sequence: closed (C) --> low-conducting substate (S) --> open (O), with the first subtransition occurring at lower tensions. Analyses of equilibrium state occupancies as functions of membrane tension show that the C-->S subtransition in WT MscL is associated with only a minor conductance increment, but the largest in-plane expansion and free energy change. The GOF substitutions strongly affect the first subtransition by reducing area ((Delta)A) and energy ((Delta)E) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger (Delta)E associated with the second (S-->O) subtransition, but a (Delta)A similar to WT. The area changes indicate that closed conformations of GOF mutants are physically preexpanded. The tension dependencies of rate constants for channel closure (k(off)) predict different positions of rate-limiting barriers on the energy-area profiles for WT and GOF MscL. The data support the two-gate mechanism in which the first subtransition (C-->S) can be viewed as opening of the central (M1) gate, resulting in an expanded water-filled "leaky" conformation. Strong facilitation of this step by polar GOF substitutions suggests that separation of M1 helices associated with hydration of the pore in WT MscL is the major energetic barrier for opening. Mutants with a stabilized S1 gate demonstrate impeded transitions from low-conducting substates to the fully open state, whereas extensions of S1-M1 linkers result in a much higher probability of reverse O-->S transitions. These data strongly suggest that the bulk of conductance gain in the second subtransition (S-->O) occurs through the opening of the NH2-terminal (S1) gate and the linkers are coupling elements between the M1 and S1 gates.     

Effects of GsMTx4 on bacterial mechanosensitive channels in inside-out patches from giant spheroplasts

GsMTx4 is a 34-residue peptide isolated from the tarantula Grammostola spatulata folded into an inhibitory cysteine knot and it selectively affects gating of some mechanosensitive channels. Here we report the effects of cytoplasmic GsMTx4 on the two bacterial channels, MscS and MscL, in giant Escherichia coli spheroplasts. In excised inside-out patches, GsMTx4 sensitized both channels to tension by increasing the opening rate and decreasing the closing rate. With ascending and descending pressure ramps, GsMTx4 increased the gating hysteresis for MscS, a consequence of slower gating kinetics. Quantitative kinetic analysis of the primary C↔O transition showed that the hysteresis is a result of the decreased closing rate. The gating barrier location relative to the open state energy well was unaffected by GsMTx4. A reconstructed energy profile suggests that the peptide prestresses the resting state of MscS, lowering the net barrier to opening and stabilizes the open conformation by ∼8 kT. In excised patches, both MscL and MscS exhibit reversible adaptation, a process separable from inactivation for MscS. GsMTx4 decreased the rate of reversible adaptation for both channels and the MscS recovery rate from the inactivation. These measurements support a mechanism where GsMTx4 binds to the lipid interface of the channel, increasing the local stress that is sensed by the channels and stabilizing the expanded conformations.     

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.     

Negative and positive temperature dependence of potassium leak in MscS mutants: Implications for understanding thermosensitive channels

Bacterial mechanosensitive channel of small conductance (MscS) is a protein, whose activity is modulated by membrane tension, voltage and cytoplasmic crowding. MscS is a homoheptamer and each monomer consists of three transmembrane helices (TM1-3). Hydrophobic pore of the channel is made of TM3s surrounded by peripheral TM1/2s. MscS gating is a complex process, which involves opening and inactivation in response to the increase of membrane tension. A number of MscS mutants were isolated. Among them mutants affecting gating have been found including gain-of-function (GOF) and loss-of-function (LOF) that open at lower or at higher thresholds, respectively. Previously, using an in vivo screen we isolated multiple MscS mutants that leak potassium and some of them were GOF or LOF. Here we show that for a subset of these mutants K⁺ leak is negatively (NTD) or positively (PTD) temperature dependent. We show that temperature reliance of these mutants does not depend on how MS gating is affected by a particular mutation. Instead, we argue that NTD or PTD leak is due to the opposite allosteric coupling of the structures that determine the temperature dependence to the channel gate. In PTD mutants an increased hydration of the pore vestibule is directly coupled to the increase in the channel conductance. In NTD mutants, at higher temperatures an increased hydration of peripheral structures leads to complete separation of TM3 and a pore collapse.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations: From tension sensing to channel opening

One of the ultimate goals of the study on mechanosensitive (MS) channels is to understand the biophysical mechanisms of how the MS channel protein senses forces and how the sensed force induces channel gating. The bacterial MS channel MscL is an ideal subject to reach this goal owing to its resolved 3D protein structure in the closed state on the atomic scale and large amounts of electrophysiological data on its gating kinetics. However, the structural basis of the dynamic process from the closed to open states in MscL is not fully understood. In this study, we performed molecular dynamics (MD) simulations on the initial process of MscL opening in response to a tension increase in the lipid bilayer. To identify the tension-sensing site(s) in the channel protein, we calculated interaction energy between membrane lipids and candidate amino acids (AAs) facing the lipids. We found that Phe78 has a conspicuous interaction with the lipids, suggesting that Phe78 is the primary tension sensor of MscL. Increased membrane tension by membrane stretch dragged radially the inner (TM1) and outer (TM2) helices of MscL at Phe78, and the force was transmitted to the pentagon-shaped gate that is formed by the crossing of the neighboring TM1 helices in the inner leaflet of the bilayer. The radial dragging force induced radial sliding of the crossing portions, leading to a gate expansion. Calculated energy for this expansion is comparable to an experimentally estimated energy difference between the closed and the first subconductance state, suggesting that our model simulates the initial step toward the full opening of MscL. The model also successfully mimicked the behaviors of a gain of function mutant (G22N) and a loss of function mutant (F78N), strongly supporting that our MD model did simulate some essential biophysical aspects of the mechano-gating in MscL.     

Analysis of Hyperekplexia Mutations Identifies Transmembrane Domain Rearrangements That Mediate Glycine Receptor Activation

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes and are therapeutic targets for a wide range of clinical indications. Elucidating the structural differences between their closed and open states may help in designing improved drugs that bias receptors toward the desired conformational state. We recently showed that two new hyperekplexia mutations, Q226E and V280M, induced spontaneous activity in α1 glycine receptors. Gln-226, located near the top of transmembrane (TM) 1, is closely apposed to Arg-271 at the top of TM2 in the neighboring subunit. Using mutant cycle analysis, we inferred that Q226E induces activation via an enhanced electrostatic attraction to Arg-271. This would tilt the top of TM2 toward TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against Ile-225 at the top of TM1 in the neighboring subunit. We infer that this steric repulsion would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the x-ray atomic structures of closed and open state bacterial pLGICs, we propyose that the model of pLGIC activation as outlined here may be broadly applicable across the eukaryotic pLGIC receptor family.     

Gated Access to the Pore of a P2X Receptor: STRUCTURAL IMPLICATIONS FOR CLOSED-OPEN TRANSITIONS*

P2X receptors are ligand-gated cation channels that transition from closed to open states upon binding ATP. The crystal structure of the closed zebrafish P2X4.1 receptor directly reveals that the ion-conducting pathway is formed by three transmembrane domain 2 (TM2) α-helices, each being provided by the three subunits of the trimer. However, the transitions in TM2 that accompany channel opening are incompletely understood and remain unresolved. In this study, we quantified gated access to Cd²⁺ at substituted cysteines in TM2 of P2X2 receptors in the open and closed states. Our data for the closed state are consistent with the zebrafish P2X4.1 structure, with isoleucines and threonines (Ile-332 and Thr-336) positioned one helical turn apart lining the channel wall on approach to the gate. Our data for the open state reveal gated access to deeper parts of the pore (Thr-339, Val-343, Asp-349, and Leu-353), suggesting the closed channel gate is between Thr-336 and Thr-339. We also found unexpected interactions between native Cys-348 and D349C that result in tight Cd²⁺ binding deep within the intracellular vestibule in the open state. Interpreted with a P2X2 receptor structural model of the closed state, our data suggest that the channel gate opens near Thr-336/Thr-339 and is accompanied by movement of the pore-lining regions, which narrow toward the cytosolic end of TM2 in the open state. Such transitions would relieve the barrier to ion flow and render the intracellular vestibule less splayed during channel opening in the presence of ATP.     

Detection of the opening of the bundle crossing in KcsA with fluorescence lifetime spectroscopy reveals the existence of two gates for ion conduction

The closed KcsA channel structure revealed a crossing of the cytosolic ends of the transmembrane helices blocking the permeation pathway. It is generally agreed that during channel opening this helical bundle crossing has to widen in order to enable access to the inner cavity. Here, we address the question of whether the opening of the inner gate is sufficient for ion conduction, or if a second gate, located elsewhere, may interrupt the ion flow. We used fluorescence lifetime measurements on KcsA channels labeled with tetramethylrhodamine at residues in the C-terminal end of TM2 to report on the opening of the lower pore region. We found two populations of channels with different fluorescence lifetimes, whose relative distribution agrees with the open probability of the channel. The absolute fraction of channels found with an open bundle crossing is too high to explain the low open probability of the KcsA-WT channel. We found the same distribution as in the WT channel between open and closed bundle crossing for two KcsA mutants, A73E and E71A, which significantly increase open probability at low pH. These two results strongly suggest that a second gate in the ion permeation pathway exists. The location of the mutations A73E and E71A suggests that the second gate may be the selectivity filter, which resides in an inactivated state under steady-state conditions. Since the long closed times observed in KcsA-WT are not present in KcsA-A73E or -E71A, we propose that KcsA-WT remains predominantly in a state with an open bundle crossing but closed (inactivated) second gate, while the mutations A73E and E71A sharply decrease the tendency to enter in the inactivated state, and as a consequence, the second gate is predominantly open at steady state. The ability to monitor the opening of the bundle crossing optically enables the direct recording of the movement of the pore helices while the channel is functioning.     

Genetic screen for potassium leaky small mechanosensitive channels (MscS) in Escherichia coli: recognition of cytoplasmic β domain as a new gating element

Mechanosensitive membrane channels in bacteria respond to the mechanical stretching of the membrane. They will open when bacteria are subjected to rapid osmotic down shock. MscS is a bacterial mechanosensitive channel of small conductance. It is a heptameric membrane protein whose transmembrane part, including the gate and its kinetics, has been well characterized. MscS has a large cytoplasmic domain of a cage-like shape that changes its conformation upon gating, but its involvement in gating is not understood. We screened MscS for mutations that cause potassium leak in Escherichia coli strains deficient in potassium transport systems. We did a phenotypic analysis of single and multiple mutants and recorded the single channel activities of some of them. After these analyses, we attributed the effects of a number of mutations to particular functional states of the channel. Our screen revealed that MscS leaks potassium in a desensitized and in an inactivated state. It also appeared that the lower part of TM3 (transmembrane, pore-forming helix) and the cytoplasmic β domain are tightly packed in the inactivated state but are dissociated in the open state. We attribute the TM3-β interaction to stabilization of the inactivated state in MscS and to the control of tight closure of its membrane pore.     

Three-dimensional architecture of membrane-embedded MscS in the closed conformation

The mechanosensitive channel of small conductance (MscS) is part of a coordinated response to osmotic challenges in Escherichia coli. MscS opens as a result of membrane tension changes, thereby releasing small solutes and effectively acting as an osmotic safety valve. Both the functional state depicted by its crystal structure and its gating mechanism remain unclear. Here, we combine site-directed spin labeling, electron paramagnetic resonance spectroscopy, and molecular dynamics simulations with novel energy restraints based on experimental electron paramagnetic resonance data to investigate the native transmembrane (TM) and periplasmic molecular architecture of closed MscS in a lipid bilayer. In the closed conformation, MscS shows a more compact TM domain than in the crystal structure, characterized by a realignment of the TM segments towards the normal of the membrane. The previously unresolved NH₂-terminus forms a short helical hairpin capping the extracellular ends of TM1 and TM2 and is in close interaction with the bilayer interface. The present three-dimensional model of membrane-embedded MscS in the closed state represents a key step in determining the molecular mechanism of MscS gating.     

Status of the intracellular gate in the activated-not-open state of shaker K+ channels

Voltage-dependent K⁺ channels like Shaker use an intracellular gate to control ion flow through the pore. When the membrane voltage becomes more positive, these channels traverse a series of closed conformations before the final opening transition. Does the intracellular gate undergo conformational changes before channel opening? To answer this question we introduced cysteines into the intracellular end of the pore and studied their chemical modification in conditions favoring each of three distinct states, the open state, the resting closed state, and the activated-not-open state (the closed state adjacent to the open state). We used two independent ways to isolate the channels in the activated-not-open state. First, we used mutations in S4 (ILT; Smith-Maxwell, C.J., J.L. Ledwell, and R.W. Aldrich. 1998. J. Gen. Physiol. 111:421–439; Ledwell, J.L., and R.W. Aldrich. 1999. J. Gen. Physiol. 113:389–414) that separate the final opening step from earlier charge-movement steps. Second, we used the open channel blocker 4-aminopyridine (4-AP), which has been proposed to promote closure of the intracellular gate and thus specifically to stabilize the activated-not-open state of the channels. Supporting this proposed mechanism, we found that 4-AP enters channels only after opening, remaining trapped in closed channels, and that in the open state it competes with tetraethylammonium for binding. Using these tools, we found that in the activated-not-open state, a cysteine located at a position considered to form part of the gate (Shaker 478) showed higher reactivity than in either the open or the resting closed states. Additionally, we have found that in this activated state the intracellular gate continued to prevent access to the pore by molecules as small as Cd²⁺ ions. Our results suggest that the intracellular opening to the pore undergoes some rearrangements in the transition from the resting closed state to the activated-not-open state, but throughout this process the intracellular gate remains an effective barrier to the movement of potassium ions through the pore.     

Retinal release from opsin in molecular dynamics simulations

The crystal structures of opsin in the ligand-free and the G-protein-interacting states showed two inter-helical openings between transmembrane (TM) helices TM1 and TM7 and between TM5 and TM6 near the extracellular side that were thought to serve as the retinal uptake and release gates. However, it is unclear which opening is for 11-cis-retinal uptake or all-trans-retinal release although speculations have been proposed based on the structural features of opsin and retinal. In this work, we simulated the exit process of all-trans-retinal from the ligand-free opsin structure by the classical molecular dynamics (MD) and random acceleration molecular dynamics (RAMD). In the 64 ns classical MD simulation, retinal remained in the receptor but moved significantly toward the TM5-TM6 opening and almost inserted into the opening after 50 ns. Complete exit was observed in 114 out of 160 RAMD trajectories with the TM5-TM6 opening being the predominant egress gate while egress from the TM1-TM7 opening was observed in only a few trajectories when relatively large acceleration was applied and large structural alteration of the protein resulted. These results suggest that photolyzed all-trans-retinal is likely released through the TM5-TM6 opening. Based on the unidirectional mechanism of retinal exchange suggested by experiment, we speculate that the TM1-TM7 opening serves as the 11-cis-retinal uptake gate. The spatial occupancy maps of retinal computed from the 160 RAMD trajectories further indicated that retinal experienced significant interactions with the receptor during the exit process. The implications of these findings for disease mechanisms of rhodopsin mutants are discussed.     

Thermodynamic coupling between activation and inactivation gating in potassium channels revealed by free energy molecular dynamics simulations

The amount of ionic current flowing through K(+) channels is determined by the interplay between two separate time-dependent processes: activation and inactivation gating. Activation is concerned with the stimulus-dependent opening of the main intracellular gate, whereas inactivation is a spontaneous conformational transition of the selectivity filter toward a nonconductive state occurring on a variety of timescales. A recent analysis of multiple x-ray structures of open and partially open KcsA channels revealed the mechanism by which movements of the inner activation gate, formed by the inner helices from the four subunits of the pore domain, bias the conformational changes at the selectivity filter toward a nonconductive inactivated state. This analysis highlighted the important role of Phe103, a residue located along the inner helix, near the hinge position associated with the opening of the intracellular gate. In the present study, we use free energy perturbation molecular dynamics simulations (FEP/MD) to quantitatively elucidate the thermodynamic basis for the coupling between the intracellular gate and the selectivity filter. The results of the FEP/MD calculations are in good agreement with experiments, and further analysis of the repulsive, van der Waals dispersive, and electrostatic free energy contributions reveals that the energetic basis underlying the absence of inactivation in the F103A mutation in KcsA is the absence of the unfavorable steric interaction occurring with the large Ile100 side chain in a neighboring subunit when the intracellular gate is open and the selectivity filter is in a conductive conformation. Macroscopic current analysis shows that the I100A mutant indeed relieves inactivation in KcsA, but to a lesser extent than the F103A mutant.     

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.     

Adaptive MscS gating in the osmotic permeability response in E. coli: the question of time

Microorganisms adapt to osmotic downshifts by releasing small osmolytes through mechanosensitive (MS) channels. We want to understand how the small mechanosensitive channel's (MscS) activation and inactivation, both driven by membrane tension, optimize survival in varying hypoosmotic shock situations. By measuring light scattering with a stopped-flow device, we estimate bacterial swelling time as 30-50 ms. A partial solute equilibration follows within 150-200 ms, during which optical responses from cells with WT MscS deviate from those lacking MS channels. MscS opening rates estimated in patch clamp show the channels readily respond to tensions below the lytic limit with a time course faster than 20 ms and close promptly upon tension release. To address the role of the tension-insensitive inactivated state in vivo, we applied short, long, and two-step osmotic shock protocols to WT, noninactivating G113A, and fast-inactivating D62N mutants. WT and G113A showed a comparable survival in short 1 min 800 mOsm downshock experiments, but G113A was at a disadvantage under a long 60 min shock. Preshocking cells carrying WT MscS for 15 s to 15 min with a 200 mOsm downshift did not sensitize them to the final 500 mOsm drop in osmolarity of the second step. However, these two-step shocks induced death in D62N more than just a one-step 700 mOsm downshift. We conclude MscS is able to activate and exude osmolytes faster than lytic pressure builds inside the cell under abrupt shock. During prolonged shocks, gradual inactivation prevents continuous channel activity and assists recovery. Slow kinetics of inactivation in WT MscS ensures that mild shocks do not inactivate the entire population, leaving some protection should conditions worsen.     

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.     

Cross talk between activation and slow inactivation gates of Shaker potassium channels

This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs(+) ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.