Explicit channel conductance: can it be computed?
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Bacterial Toxins↗
Biomedical subjects
Andriy Anishkin
Publications and source records attributed to Andriy Anishkin.
The "dashpot" mechanism of stretch-dependent gating in MscS.
The crystal structure of the small conductance mechanosensitive channel (MscS) has been an invaluable tool in the search for the gating mechanism, however many functional aspects of the channel remain unsettled. Here we characterized the gating of MscS in Escherichia coli spheroplasts in a triple mutant (mscL-, mscS-, mscK-) background. We used a pressure clamp apparatus along with software developed in-lab to generate dose-response curves directly from two-channel recordings of current and pressure. In contrast to previous publications, we found that MscS exhibits essentially voltage-independent activation by tension, but at the same time strong voltage-dependent inactivation under depolarizing conditions. The MscS activation curves obtained under saturating ramps of pressure, at different voltages, gave estimates for the energy, area, and gating charge for the closed-to-open transition as 24 kT, 18 nm2, and +0.8, respectively. The character of activation and inactivation was similar in both K+ and Na+ buffers. Perhaps the most salient and intriguing property of MscS gating was a strong dependence on the rate of pressure application. Patches subjected to various pressure ramps from 2.7 to 240 mmHg/s revealed a midpoint of activation almost independent of rate. However, the resultant channel activity was dramatically lower when pressure was applied slowly, especially at depolarizing pipette voltages. It appears that MscS prefers to respond in full to abrupt stimuli but manages to ignore those applied slowly, as if the gate were connected to the tension-transmitting element via a velocity-sensitive "dashpot." With slower ramps, channels inactivate during the passage through a narrow region of pressures below the activation midpoint. This property of "dumping" a slowly applied force may be important in environmental situations where rehydration of cells occurs gradually and release of osmolytes is not desirable. MscS often enters the inactivated state through subconducting states favored by depolarizing voltage. The inactivation rate increases exponentially with depolarization. Based on these results we propose a kinetic scheme and gating mechanism to account for the observed phenomenology in the framework of available structural information.
Electrophysiology↗
Microbial mechanosensation.
Because bacterial mechanosensitive channels have been cloned, purified, crystallized and subjected to a genetic, biochemical and physical scrutiny, they have become the current structural models of mechanosensation to atomic detail. The key observation, supported by recent mutagenesis studies, is that these channels receive stretch force directly through the lipid bilayer at the interface levels bearing highest tension. Indeed, simulations of mechanosensitive channels steered by strategically applied bilayer stretch forces show channel opening. Our understanding of the gating energetics and trajectory are continually being refined by the combination of approaches applied. In addition, new microbial mechanosensitive channels from the TRP family have been characterized in yeasts. Unified by fundamental biophysical principles of gating, mechanosensitive channels provide broad insight into protein-membrane interactions and the role of hydrophobic hydration in gating.
Animals↗
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.
Electrophysiology↗
Mechanosensitive channels: what can we learn from 'simple' model systems?
Mechanosensitive ion channels, which convert external mechanical forces into electrical and chemical signals in cells, are diverse. Presently, there is no known common sequence 'signature' that identifies mechanosensitivity. Bacterial mechanosensitive channels gated by membrane tension represent convenient models allowing us to combine structural information with the insights gained from biophysical analysis, biochemistry, genetic screens, bacterial physiology and molecular computation. Here, the conformational transition driven by membrane tension in the bacterial channel MscL is discussed. The predicted pathway suggests roles for distinct protein domains, surrounding lipids and water in the gating process. MscL, a simple system, thus helps us obtain a coherent picture of molecular events, and build concepts and strategies that can be applied to more elaborate mechanosensory systems in the near future.
Amino Acid Sequence↗
Gating of the large mechanosensitive channel in situ: estimation of the spatial scale of the transition from channel population responses.
Physical expansion associated with the opening of a tension-sensitive channel has the same meaning as gating charge for a voltage-gated channel. Despite increasing evidence for the open-state conformation of MscL, the energetic description of its complex gating remains incomplete. The previously estimated in-plane expansion of MscL is considerably smaller than predicted by molecular models. To resolve this discrepancy, we conducted a systematic study of currents and dose-response curves for wild-type MscL in Escherichia coli giant spheroplasts. Using the all-point histogram method and calibrating tension against the threshold for the small mechanosensitive channel (MscS) in each patch, we found that the distribution of channels among the subconducting states is significantly less dependent on tension than the distribution between the closed and conducting states. At -20 mV, all substates together occupy approximately 30% of the open time and reduce the mean integral current by approximately 6%, essentially independent of tension or P(o). This is consistent with the gating scheme in which the major rate-limiting step is the transition between the closed state and a low-conducting substate, and validates both the use of the integral current as a measure of P(o), and treatment of dose-response curves in the two-state approximation. The apparent energy and area differences between the states deltaE and deltaA, extracted from 29 independent dose-response curves, varied in a linearly correlated manner whereas the midpoint tension stayed at approximately 10.4 mN/m. Statistical modeling suggests slight variability of gating parameters among channels in each patch, causing a strong reduction and correlated spread of apparent deltaE and deltaA. The slope of initial parts of activation curves, with a few channels being active, gave estimates of deltaE = 51 +/- 13 kT and deltaA = 20.4 +/- 4.8 nm(2), the latter being consistent with structural models of MscL, which predict deltaA = 23 nm(2).
Biophysics↗
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.
Chlorine↗
On the conformation of the COOH-terminal domain of the large mechanosensitive channel MscL.
COOH-terminal (S3) domains are conserved within the MscL family of bacterial mechanosensitive channels, but their function remains unclear. The X-ray structure of MscL from Mycobacterium tuberculosis (TbMscL) revealed cytoplasmic domains forming a pentameric bundle (Chang, G., R.H. Spencer, A.T. Lee, M.T. Barclay, and D.C. Rees. 1998. SCIENCE: 282:2220-2226). The helices, however, have an unusual orientation in which hydrophobic sidechains face outside while charged residues face inside, possibly due to specific crystallization conditions. Based on the structure of pentameric cartilage protein, we modeled the COOH-terminal region of E. coli MscL to better satisfy the hydrophobicity criteria, with sidechains of conserved aliphatic residues all inside the bundle. Molecular dynamic simulations predicted higher stability for this conformation compared with one modeled after the crystal structure of TbMscL, and suggested distances for disulfide trapping experiments. The single cysteine mutants L121C and I125C formed dimers under ambient conditions and more so in the presence of an oxidant. The double-cysteine mutants, L121C/L122C and L128C/L129C, often cross-link into tetrameric and pentameric structures, consistent with the new model. Patch-clamp examination of these double mutants under moderately oxidizing or reducing conditions indicated that the bundle cross-linking neither prevents the channel from opening nor changes thermodynamic parameters of gating. Destabilization of the bundle by replacing conservative leucines with small polar residues, or complete removal of COOH-terminal domain (Delta110-136 mutation), increased the occupancy of subconducting states but did not change gating parameters substantially. The Delta110-136 truncation mutant was functional in in vivo osmotic shock assays; however, the amount of ATP released into the shock medium was considerably larger than in controls. The data strongly suggest that in contrast to previous gating models (Sukharev, S., M. Betanzos, C.S. Chiang, and H.R. Guy. 2001a. NATURE: 409:720-724.), S3 domains are stably associated in both closed and open conformations. The bundle-like assembly of cytoplasmic helices provides stability to the open conformation, and may function as a size-exclusion filter at the cytoplasmic entrance to the MscL pore, preventing loss of essential metabolites.
Amino Acid Sequence↗