The mechanosensitive channel of large conductance, MscL, is a ubiquitous membrane-embedded valve involved in turgor regulation in bacteria. The crystal structure of MscL from Mycobacterium tuberculosis provides a starting point for analysing molecular mechanisms of tension-dependent channel gating. Here we develop structural models in which a cytoplasmic gate is formed by a bundle of five amino-terminal helices (S1), previously unresolved in the crystal structure. When membrane tension is applied, the transmembrane barrel expands and pulls the gate apart through the S1-M1 linker. We tested these models by substituting cysteines for residues predicted to be near each other only in either the closed or open conformation. Our results demonstrate that S1 segments form the bundle when the channel is closed, and crosslinking between S1 segments prevents opening. S1 segments interact with M2 when the channel is open, and crosslinking of S1 to M2 impedes channel closing. Gating is affected by the length of the S1-M1 linker in a manner consistent with the model, revealing critical spatial relationships between the domains that transmit force from the lipid bilayer to the channel gate.

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).

MscL, a bacterial mechanosensitive channel of large conductance, is the first structurally characterized mechanosensor protein. Molecular models of its gating mechanisms are tested here. Disulfide crosslinking shows that M1 transmembrane alpha-helices in MscL of resting Escherichia coli are arranged similarly to those in the crystal structure of MscL from Mycobacterium tuberculosis. An expanded conformation was trapped in osmotically shocked cells by the specific bridging between Cys 20 and Cys 36 of adjacent M1 helices. These bridges stabilized the open channel. Disulfide bonds engineered between the M1 and M2 helices of adjacent subunits (Cys 32-Cys 81) do not prevent channel gating. These findings support gating models in which interactions between M1 and M2 of adjacent subunits remain unaltered while their tilts simultaneously increase. The MscL barrel, therefore, undergoes a large concerted iris-like expansion and flattening when perturbed by membrane tension.

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 (ΔA) and energy (ΔE) changes between C and S states commensurably with the severity of mutation. GOF mutants also exhibited a considerably larger ΔE associated with the second (S→O) subtransition, but a ΔA 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.

Abstract-Voltage-gated T-type Ca 2ϩ channels (T-channels) are normally expressed during embryonic development in ventricular myocytes but are undetectable in adult ventricular myocytes. Interestingly, T-channels are reexpressed in hypertrophied or failing hearts. It is unclear whether T-channels play a role in the pathogenesis of cardiomyopathy and what the mechanism might be. Here we show that the ␣ 1H voltage-gated T-type Ca 2ϩ channel (Ca v 3.2) is involved in the pathogenesis of cardiac hypertrophy via the activation of calcineurin/nuclear factor of activated T cells (NFAT) pathway. Specifically, pressure overload-induced hypertrophy was severely suppressed in mice deficient for Ca v 3.2 (Ca v 3.2 Ϫ/Ϫ ) but not in mice deficient for Ca v 3.1 (Ca v 3.1 Ϫ/Ϫ ). Angiotensin II-induced cardiac hypertrophy was also suppressed in Ca v 3.2 Ϫ/Ϫ mice. Consistent with these findings, cultured neonatal myocytes isolated from Ca v 3.2 Ϫ/Ϫ mice fail to respond hypertrophic stimulation by treatment with angiotensin II. Together, these results demonstrate the importance of Ca v 3.2 in the development of cardiac hypertrophy both in vitro and in vivo. To test whether Ca v 3.2 mediates the hypertrophic response through the calcineurin/NFAT pathway, we generated Ca v 3.2 Ϫ/Ϫ , NFAT-luciferase reporter mice and showed that NFAT-luciferase reporter activity failed to increase after pressure overload in the Ca v 3.2 Ϫ/Ϫ /NFAT-Luc mice. Our results provide strong genetic evidence that Ca v 3.2 indeed plays a pivotal role in the induction of calcineurin/NFAT hypertrophic signaling and is crucial for the activation of pathological cardiac hypertrophy. H ypertrophic growth and remodeling of the adult heart begin as normal compensatory responses to a variety of physiological and pathological stimuli including exercise, pregnancy, pressure overload, hypertension, myocardial infarction, and primary genetic abnormalities. [1][2][3] Prolonged hypertrophic response may eventually lead to heart failure and death. Pathological hypertrophy is often associated with structural and functional remodeling of the heart. Profound changes in gene expression during pathological cardiac hypertrophy are common, particularly in the case of genes that code for proteins involved in regulating contraction ion channels, for example. 4 Alterations of intracellular Ca 2ϩ handling could lead to abnormal Ca 2ϩ signaling cascades, a phenomenon that has been shown to contribute to the pathogenesis of cardiac hypertrophy and heart failure. 5,6 However, the detailed mechanism of how the cardiac myocytes distinguish Ca 2ϩ transients that occur at every heartbeat from those meant to trigger intracellular hypertrophic signaling remains largely unknown. Intracellular Ca 2ϩ levels could be altered because of either changes in Ca 2ϩ release from intracellular organelles or the influx of extracellular Ca 2ϩ , and in both cases, this could be attributable to the activities of either ligand-or voltage-activated Ca 2ϩ channels. Indeed, ligand-activated Ca 2ϩ channels, such as ...