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 pore helix is involved in stabilizing the open state of inwardly rectifying K+ channels

Ion channels can be gated by various extrinsic cues, such as voltage, pH, and second messengers. However, most ion channels display extrinsic cue-independent transitions as well. These events represent spontaneous conformational changes of the channel protein. The molecular basis for spontaneous gating and its relation to the mechanism by which channels undergo activation gating by extrinsic cue stimulation is not well understood. Here we show that the proximal pore helix of inwardly rectifying (Kir) channels is partially responsible for determining spontaneous gating characteristics, affecting the open state of the channel by stabilizing intraburst openings as well as the bursting state itself without affecting K(+) ion-channel interactions. The effect of the pore helix on the open state of the channel is qualitatively similar to that of two well-characterized mutations at the second transmembrane domain (TM2), which stabilize the channel in its activated state. However, the effects of the pore helix and the TM2 mutations on gating were additive and independent of each other. Moreover, in sharp contrast to the two TM2 mutations, the pore helix mutation did not affect the functionality of the agonist-responsive gate. Our results suggest that in Kir channels, the bottom of the pore helix and agonist-induced conformational transitions at the TM2 ultimately stabilize via different pathways the open conformation of the same gate.     

Surface changes of the mechanosensitive channel MscS upon its activation, inactivation, and closing

MscS is a bacterial mechanosensitive channel that shows voltage dependence. The crystal structure of MscS revealed that the channel is a homoheptamer with a large chamber on the intracellular site. Our previous experiments indicated that the cytoplasmic chamber of the channel is not a rigid structure and changes its conformation upon the channel activation. In this study, we have applied various sized cosolvents that are excluded from protein surfaces. It is well known that such cosolvents induce compaction of proteins and prevent thermal fluctuations. It is also known that they shift channel equilibrium to the state of lower volume. We have found that large cosolvents that cannot enter the channel interior accelerate channel inactivation when applied from the cytoplasmic side, but they slow down inactivation when applied from the extracellular side. We have also found that small cosolvents that can enter the channel cytoplasmic chamber prevent the channel from opening, unlike the large ones. These data support our idea that the channel cytoplasmic chamber shrinks upon inactivation but also give new clues about conformational changes of the channel upon transitions between its functional states.     

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.     

Probing the Transition State of the Allosteric Pathway of the Shaker Kv Channel Pore by Linear Free-Energy Relations

Long-range coupling between distant functional elements of proteins may rely on allosteric communication trajectories lying along the protein structure, as described in the case of the Shaker voltage-activated potassium (Kv) channel model allosteric system. Communication between the distant Kv channel activation and slow inactivation pore gates was suggested to be mediated by a network of local pairwise and higher-order interactions among the functionally unique residues that constitute the allosteric trajectory. The mechanism by which conformational changes propagate along the Kv channel allosteric trajectory to achieve pore opening, however, remains unclear. Such conformational changes may propagate in either a concerted or a sequential manner during the reaction coordinate of channel opening. Residue-level structural information on the transition state of channel gating is required to discriminate between these possibilities. Here, we combine patch-clamp electrophysiology recordings of Kv channel gating and analysis using linear free-energy relations, focusing on a select set of residues spanning the allosteric trajectory of the Kv channel pore. We show that all allosteric trajectory residues tested exhibit an open-like conformation in the transition state of channel opening, implying that coupling interactions occur along the trajectory break in a concerted manner upon moving from the closed to the open state. Energetic coupling between the Kv channel gates thus occurs in a concerted fashion in both the spatial and the temporal dimensions, strengthening the notion that such trajectories correspond to pathways of mechanical deformation along which conformational changes propagate.     

Water dynamics and dewetting transitions in the small mechanosensitive channel MscS

The dynamics of confined water in capillaries and nanotubes suggests that gating of ion channels may involve not only changes of the pore geometry, but also transitions between water-filled and empty states in certain locations. The recently solved heptameric structure of the small mechanosensitive channel of Escherichia coli, MscS, has revealed a relatively wide (7-15 A) yet highly hydrophobic transmembrane pore. Continuum estimations based on the properties of pore surface suggest low conductance and a thermodynamic possibility of dewetting. To test the predictions we performed molecular dynamics simulations of MscS filled with flexible TIP3P water. Irrespective to the initial conditions, several independent 6-ns simulations converged to the same stable state with the pore water-filled in the wider part, but predominantly empty in the narrow hydrophobic part, displaying intermittent vapor-liquid transitions. The polar gain-of-function substitution L109S in the constriction resulted in a stable hydration of the entire pore. Steered passages of Cl(-) ions through the narrow part of the pore consistently produced partial ion dehydration and required a force of 200-400 pN to overcome an estimated barrier of 10-20 kcal/mole, implying negligibly low conductance. We conclude that the crystal structure of MscS does not represent an open state. We infer that MscS gate, which is similar to that of the nicotinic ACh receptor, involves a vapor-lock mechanism where limited changes of geometry or surface polarity can locally switch the regime between water-filled (conducting) and empty (nonconducting) states.     

Stabilization of the Conductive Conformation of a Voltage-gated K+ (Kv) Channel: THE LID MECHANISM*

Voltage-gated K⁺ (Kv) channels are molecular switches that sense membrane potential and in response open to allow K⁺ ions to diffuse out of the cell. In these proteins, sensor and pore belong to two distinct structural modules. We previously showed that the pore module alone is a robust yet dynamic structural unit in lipid membranes and that it senses potential and gates open to conduct K⁺ with unchanged fidelity. The implication is that the voltage sensitivity of K⁺ channels is not solely encoded in the sensor. Given that the coupling between sensor and pore remains elusive, we asked whether it is then possible to convert a pore module characterized by brief openings into a conductor with a prolonged lifetime in the open state. The strategy involves selected probes targeted to the filter gate of the channel aiming to modulate the probability of the channel being open assayed by single channel recordings from the sensorless pore module reconstituted in lipid bilayers. Here we show that the premature closing of the pore is bypassed by association of the filter gate with two novel open conformation stabilizers: an antidepressant and a peptide toxin known to act selectively on Kv channels. Such stabilization of the conductive conformation of the channel is faithfully mimicked by the covalent attachment of fluorescein at a cysteine residue selectively introduced near the filter gate. This modulation prolongs the occupancy of permeant ions at the gate. It is this longer embrace between ion and gate that we conjecture underlies the observed stabilization of the conductive conformation. This study provides a new way of thinking about gating.     

Structural changes in the cytoplasmic pore of the Kir1.1 channel during pHi-gating probed by FRET

Kir1.1 channels are important in maintaining K⁺ homeostasis in the kidney. Intracellular acidification reversibly closes the Kir1.1 channel and thus decreases K⁺ secretion. In this study, we used Foster resonance energy transfer (FRET) to determine whether the conformation of the cytoplasmic pore changes in response to intracellular pH (pH_i)-gating in Kir1.1 channels fused with enhanced cyan fluorescent protein (ECFP) and enhanced yellow fluorescent protein (EYFP) (ECFP-Kir1.1-EYFP). Because the fluorescence intensities of ECFP and EYFP were affected at pH_i < 7.4 where pH_i-gating occurs in the ECFP-Kir1.1-EYFP construct, we examined the FRET efficiencies of an ECFP-S219R-EYFP mutant, which is completed closed at pH_i 7.4 and open at pH_i 10.0. FRET efficiency was increased from 25% to 40% when the pH_i was decreased from 10.0 to 7.4. These results suggest that the conformation of the cytoplasmic pore in the Kir1.1 channel changes in response to pH_i gating such that the N- and C-termini move apart from each other at pH_i 7.4, when the channel is open.     

Diverse gating in K channels: Differential role of the pore-helix glutamate in stabilizing the channel pore

The selectivity filter and adjacent regions in the bacterial KcsA and inwardly rectifying K⁺ (Kir) channels reveal significant conformational changes that cause the channel pore to transition from an activated to inactive state (C-type inactivation) once the channel is open. The meshwork of residues stabilizing the pore of KcsA involves Glu71–Asp80 carboxyl–carboxylate interaction ‘behind’ the selectivity filter. Interestingly, the Kir channels do not have this exact interaction, but instead have a Glu–Arg salt bridge where the Glu is in the same position but the Arg is one position N-terminal compared to the Asp in KcsA. Also, the Kir channels lack the Trp that hydrogen bonds to Asp80 in KcsA. Here, the sequence and structural information are combined to understand the dissimilarity in the role of the pore-helix Glu in stabilizing the pore structure in KcsA and Kir channels. This review illustrates that although Glu is quite conserved among both types of channels, the network of interactions is not translatable from one channel to the other; thereby suggesting a unique phenomenon of diverse gating patterns in K⁺ channels.     

Open and shut: Crystal structures of the dodecylmaltoside solubilized mechanosensitive channel of small conductance from Escherichia coli and Helicobacter pylori at 4.4 Å and 4.1 Å resolutions

The mechanosensitive channel of small conductance (MscS) contributes to the survival of bacteria during osmotic downshock by transiently opening large diameter pores for the efflux of cellular contents before the membrane ruptures. Two crystal structures of the Escherichia coli MscS are currently available, the wild type protein in a nonconducting state at 3.7 Å resolution (Bass et al., Science 2002; 298:1582–1587) and the Ala106Val variant in an open state at 3.45 Å resolution (Wang et al., Science 2008; 321:1179–1183). Both structures used protein solubilized in the detergent fos‐choline‐14. We report here crystal structures of MscS from E. coli and Helicobacter pylori solubilized in the detergent β‐dodecylmaltoside at resolutions of 4.4 and 4.2 Å, respectively. While the cytoplasmic domains are unchanged in these structures, distinct conformations of the transmembrane domains are observed. Intriguingly, β‐dodecylmaltoside solubilized wild type E. coli MscS adopts the open state structure of A106V E. coli MscS, while H. pylori MscS resembles the nonconducting state structure observed for fos‐choline‐14 solubilized E. coli MscS. These results highlight the sensitivity of membrane protein conformational equilibria to variations in detergent, crystallization conditions, and protein sequence.     

Ion conduction through MscS as determined by electrophysiology and simulation

The mechanosensitive channel of small conductance (MscS) is a membrane protein thought to act as a safety valve in bacteria, regulating the release of ions and small solutes when gated by membrane tension under challenging osmotic conditions. The influence of voltage on channel activation and the functional state depicted by the available crystal structure of MscS remain debated. Therefore, in an effort to relate electrophysiological measurements on MscS and properties of the MscS crystal conformation, we report here MscS's response to voltage and pressure as determined by patch-clamp experiments, as well as MscS electrostatics and transport properties as determined through all-atom molecular dynamics simulations of the protein embedded in a lipid bilayer, a 224,000-atom system. The experiments reveal that MscS is a slightly anion-selective channel with a conductance of approximately 1 ns, activated by pressure and inactivated in a voltage-dependent manner. On the other hand, the simulations, covering over 200 ns and including biasing electrostatic potentials, show that MscS restrained to the crystal conformation exhibits low conductance; unrestrained it increases the channel radius upon application of a large electrostatic bias and exhibits then ion conduction that matches experimentally determined conductances. The simulated conductance stems mainly from Cl- ions.     

Molecular Analysis of Potential Hinge Residues in the Inactivation Gate of Brain Type IIA Na+ Channels

During inactivation of Na⁺ channels, the intracellular loop connecting domains III and IV is thought to fold into the channel protein and occlude the pore through interaction of the hydrophobic motif isoleucine-phenylalanine-methionine (IFM) with a receptor site. We have searched for amino acid residues flanking the IFM motif which may contribute to formation of molecular hinges that allow this motion of the inactivation gate. Site-directed mutagenesis of proline and glycine residues, which often are components of molecular hinges in proteins, revealed that G1484, G1485, P1512, P1514, and P1516 are required for normal fast inactivation. Mutations of these residues slow the time course of macroscopic inactivation. Single channel analysis of mutations G1484A, G1485A, and P1512A showed that the slowing of macroscopic inactivation is produced by increases in open duration and latency to first opening. These mutant channels also show a higher probability of entering a slow gating mode in which their inactivation is further impaired. The effects on gating transitions in the pathway to open Na⁺ channels indicate conformational coupling of activation to transitions in the inactivation gate. The results are consistent with the hypothesis that these glycine and proline residues contribute to hinge regions which allow movement of the inactivation gate during the inactivation process of Na⁺ channels.     

A competition smFRET assay to study ligand-induced conformational changes of the dengue virus protease

Ligand binding to proteins often is accompanied by conformational transitions. Here, we describe a competition assay based on single molecule Förster resonance energy transfer (smFRET) to investigate the ligand-induced conformational changes of the dengue virus (DENV) NS2B-NS3 protease, which can adopt at least two different conformations. First, a competitive ligand was used to stabilize the closed conformation of the protease. Subsequent addition of the allosteric inhibitor reduced the fraction of the closed conformation and simultaneously increased the fraction of the open conformation, demonstrating that the allosteric inhibitor stabilizes the open conformation. In addition, the proportions of open and closed conformations at different concentrations of the allosteric inhibitor were used to determine its binding affinity to the protease. The K_D value observed is in accordance with the IC₅₀ determined in the fluorometric assay. Our novel approach appears to be a valuable tool to study conformational transitions of other proteases and enzymes.     

A Gating Model for the Archeal Voltage-Dependent K Channel KvAP in DPhPC and POPE:POPG Decane Lipid Bilayers

Voltage-dependent K⁺ (Kv) channels form the basis of the excitability of nerves and muscles. KvAP is a well-characterized archeal Kv channel that has been widely used to investigate many aspects of Kv channel biochemistry, biophysics, and structure. In this study, a minimal kinetic gating model for KvAP function in two different phospholipid decane bilayers is developed. In most aspects, KvAP gating is similar to the well-studied eukaryotic Shaker Kv channel: conformational changes occur within four voltage sensors, followed by pore opening. Unlike the Shaker Kv channel, KvAP possesses an inactivated state that is accessible from the pre-open state of the channel. Changing the lipid composition of the membrane influences multiple gating transitions in the model, but, most dramatically, the rate of recovery from inactivation. Inhibition by the voltage sensor toxin VSTx1 is most easily explained if VSTx1 binds only to the depolarized conformation of the voltage sensor. By delaying the voltage sensor's return to the hyperpolarized conformation, VSTx1 favors the inactivated state of KvAP.     

Near-critical fluctuations and cytoskeleton-assisted phase separation lead to subdiffusion in cell membranes

Mechanosensitive channels, inner membrane proteins of bacteria, open and close in response to mechanical stimuli such as changes in membrane tension during osmotic stress. In bacteria, these channels act as safety valves preventing cell lysis upon hypoosmotic cell swelling: the channels open under membrane tension to release osmolytes along with water. The mechanosensitive channel of small conductance, MscS, consists, in addition to the transmembrane channel, of a large cytoplasmic domain (CD) that features a balloon-like, water filled chamber opening to the cytoplasm through seven side pores and a small distal pore. The CD is apparently a molecular sieve covering the channel that optimizes loss of osmolytes during osmoadaptation. We employ diffusion theory and molecular dynamics simulations to explore the transport kinetics of Glu⁻ and K⁺ as representative osmolytes. We suggest that the CD indeed acts as a filter that actually balances passage of Glu⁻ and K⁺, and possibly other positive and negative osmolytes, to yield a largely neutral efflux and, thereby, reduce cell depolarization in the open state and conserve to a large degree the essential metabolite Glu⁻.     

Molecular dynamics study of gating in the mechanosensitive channel of small conductance MscS

Mechanosensitive channels are a class of ubiquitous membrane proteins gated by mechanical strain in the cellular membrane. MscS, the mechanosensitive channel of small conductance, is found in the inner membrane of Escherichia coli and its crystallographic structure in an open form has been recently solved. By means of molecular dynamics simulations we studied the stability of the channel conformation suggested by crystallography in a fully solvated lipid (POPC) bilayer, the combined system encompassing 224,340 atoms. When restraining the backbone of the protein, the channel remained in the open form and the simulation revealed intermittent permeation of water molecules through the channel. Abolishing the restraints under constant pressure conditions led to spontaneous closure of the transmembrane channel, whereas abolishing the restraints when surface tension (20 dyn/cm) was applied led to channel widening. The large balloon-shaped cytoplasmic domain of MscS exhibited spontaneous diffusion of ions through its side openings. Interaction between the transmembrane domain and the cytoplasmic domain of MscS was observed and involved formation of salt bridges between residues Asp62 and Arg128; this interaction may be essential for the gating of MscS. K+ and Cl- ions showed distinctively different distributions in and around the channel.     

An optimized purification and reconstitution method for the MscS channel: strategies for spectroscopical analysis

The mechanosensitive channel of small conductance (MscS) plays a critical role in the osmoregulation of prokaryotic cells. The crystal structure of MscS revealed a homoheptamer with three transmembrane segments and a large cytoplasmic domain. It has been suggested that the crystal structure depicts an open state, but its actual functional conformation remains controversial. In the pursuit of spectroscopical approaches to MscS gating, we determined that standard purification methods yield two forms of MscS, with a considerable amount of unfolded channel. Here, we present an improved high-yield purification method based on Escherichia coli expression and a biochemical characterization of the reconstituted channel, optimized to yield approximately 4 mg of a single monodisperse product. Upon reconstitution into lipid vesicles, MscS is unusually prone to lateral aggregation depending on the lipid composition, particularly after sample freezing. Strategies for minimizing MscS aggregation in two dimensions for spectroscopic analyses of gating have been developed.     

Mutations in a Conserved Domain of E. coli MscS to the Most Conserved Superfamily Residue Leads to Kinetic Changes

In Escherichia coli (E. coli) the mechanosensitive channel of small conductance, MscS, gates in response to membrane tension created from acute external hypoosmotic shock, thus rescuing the bacterium from cell lysis. E. coli MscS is the most well studied member of the MscS superfamily of channels, whose members are found throughout the bacterial and plant kingdoms. Homology to the pore lining helix and upper vestibule domain of E. coli MscS is required for inclusion into the superfamily. Although highly conserved, in the second half of the pore lining helix (TM3B), E. coli MscS has five residues significantly different from other members of the superfamily. In superfamilies such as this, it remains unclear why variations within such a homologous region occur: is it tolerance of alternate residues, or does it define functional variance within the superfamily? Point mutations (S114I/T, L118F, A120S, L123F, F127E/K/T) and patch clamp electrophysiology were used to study the effect of changing these residues in E. coli MscS on sensitivity and gating. The data indicate that variation at these locations do not consistently lead to wildtype channel phenotypes, nor do they define large changes in mechanosensation, but often appear to effect changes in the E. coli MscS channel gating kinetics.     

Lipid Bilayer Modules as Determinants of K+ Channel Gating

The crystal structure of the sensorless pore module of a voltage-gated K⁺ (Kv) channel showed that lipids occupy a crevice between subunits. We asked if individual lipid monolayers of the bilayer embody independent modules linked to channel gating modulation. Functional studies using single channel current recordings of the sensorless pore module reconstituted in symmetric and asymmetric lipid bilayers allowed us to establish the deterministic role of lipid headgroup on gating. We discovered that individual monolayers with headgroups that coat the bilayer-aqueous interface with hydroxyls stabilize the channel open conformation. The hydroxyl need not be at a terminal position and the effect is not dependent on the presence of phosphate or net charge on the lipid headgroup. Asymmetric lipid bilayers allowed us to determine that phosphoglycerides with glycerol or inositol on the extracellular facing monolayer stabilize the open conformation of the channel. This indirect effect is attributed to a change in water structure at the membrane interface. By contrast, inclusion of the positively charged lysyl-dioleoyl-phosphatidylglycerol exclusively on the cytoplasmic facing monolayer of the bilayer increases drastically the probability of finding the channel open. Such modulation is mediated by a π-cation interaction between Phe-19 of the pore module and the lysyl moiety anchored to the phosphatidylglycerol headgroup. The new findings imply that the specific chemistry of the lipid headgroup and its selective location in either monolayer of the bilayer dictate the stability of the open conformation of a Kv pore module in the absence of voltage-sensing modules.