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The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo.

ABSTRACT: The mechanosensitive channel of large conductance (MscL) from E. coli serves as an emergency release valve allowing the cell to survive acute osmotic downshock. It is one of the best studied mechanosensitive channels and serves as a paradigm for how a protein can sense and respond to membrane tension. Two MscL crystal structures of the orthologs M. tuberculosis and S. aureus have been solved showing pentameric and tetrameric structures, respectively. Several studies followed to understand whether the discrepancy in their stoichiometry was a species difference or a consequence of the protein manipulation for crystallization. Two independent studies now agree that the full-length S. aureus MscL is actually a pentamer, not tetramer. While detergents appear to play a role in modifying the oligomeric state of the protein, a cytoplasmic helical bundle has also been implicated. Here, we evaluate the role of the C-terminal region of S. aureus MscL in the oligomerization of the channel in native membranes by using an in vivo disulfide-trapping technique. We find that the oligomeric state of S. aureus MscLs with different C-terminal truncations, including the one used to obtain the tetrameric S. aureus MscL crystal structure, are pentamers in vivo. Thus, the C-terminal domain of the S. aureus protein only plays a critical role in the oligomeric state of the SaMscL protein when it is solubilized in detergent.

SUBMITTER: Iscla I

PROVIDER: S-EPMC3190158 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo.

Iscla Irene I Wray Robin R Blount Paul P

Protein science : a publication of the Protein Society 20110719 9

The mechanosensitive channel of large conductance (MscL) from E. coli serves as an emergency release valve allowing the cell to survive acute osmotic downshock. It is one of the best studied mechanosensitive channels and serves as a paradigm for how a protein can sense and respond to membrane tension. Two MscL crystal structures of the orthologs M. tuberculosis and S. aureus have been solved showing pentameric and tetrameric structures, respectively. Several studies followed to understand whethe ...[more]

PMID: 21739498

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Project description:Mechanosensitive channels of small conductance (MscS) are ubiquitous turgor pressure regulators found in many walled cells and some intracellular organelles. Escherichia coli MscS acting as a tension-activated osmolyte release valve shows a nonsaturable conductance (1.2 nS in a 39 mS/cm electrolyte) and weak preference for anions. Pursuing the transition pathways in this channel, we applied the extrapolated motion protocol (cycles of displacements, minimizations, and short simulations) to the previously generated compact resting conformation of MscS. We observed tilting and straightening of the kinked pore-forming TM3 helices during the barrel expansion. Extended all-atom simulations confirmed the stability of the open conformation in the bilayer. A 53 degrees spontaneous axial rotation of TM3s observed after equilibration increased the width and polarity of the pore allowing for stable voltage-independent hydration and presence of both cations and anions throughout the pore. The resultant open state, characterized by a pore 1.6 nm wide, satisfied the experimental conductance and in-plane expansion. Applied transmembrane electric field (+/-100 to +/-200 mV) in simulations produced a flow of both K(+) and Cl(-), with Cl(-) current dominating at higher voltages. Electroosmotic water flux strongly correlated with the chloride current (approximately 8 waters per Cl(-)). The selectivity and rectification were in agreement with the experimental measurements performed in the same range of voltages. Among the charged residues surrounding the pore, only K169 was found to contribute noticeably in the rectification. We conclude that (a) the barrel expansion involving tilting, straightening, and rotation of TM3s provides the geometry and electrostatics that accounts for the conductive properties of the open pore; (b) the observed regimen of ion passage through the pore is similar to electrodiffusion, thus macroscopic estimations closely approximate the experimental and molecular dynamics-simulated conductances; (c) increased interaction of the opposing ionic fluxes at higher voltages may result in selectivities stronger than measured near the reversal potential.

Project description:The mechanosensitive channel of large conductance (MscL) is capable of transducing mechanical stimuli such as membrane tension into an electrochemical response. MscL provides a widely-studied model system for mechanotransduction and, more generally, for how bilayer mechanical properties regulate protein conformational changes. Much effort has been expended on the detailed experimental characterization of the molecular structure and biological function of MscL. However, despite its central significance, even basic issues such as the physiologically relevant oligomeric states and molecular structures of MscL remain a matter of debate. In particular, tetrameric, pentameric, and hexameric oligomeric states of MscL have been proposed, together with a range of detailed molecular structures of MscL in the closed and open channel states. Previous theoretical work has shown that the basic phenomenology of MscL gating can be understood using an elastic model describing the energetic cost of the thickness deformations induced by MscL in the surrounding lipid bilayer. Here, we generalize this elastic model to account for the proposed oligomeric states and hydrophobic shapes of MscL. We find that the oligomeric state and hydrophobic shape of MscL are reflected in the energetic cost of lipid bilayer deformations. We make quantitative predictions pertaining to the gating characteristics associated with various structural models of MscL and, in particular, show that different oligomeric states and hydrophobic shapes of MscL yield distinct membrane contributions to the gating energy and gating tension. Thus, the functional properties of MscL provide a signature of the oligomeric state and hydrophobic shape of MscL. Our results suggest that, in addition to the hydrophobic mismatch between membrane proteins and the surrounding lipid bilayer, the symmetry and shape of the hydrophobic surfaces of membrane proteins play an important role in the regulation of protein function by bilayer membranes.

Project description:BackgroundPhysical exercise directly stretching the peripheral nerve promotes nerve regeneration; however, its action mechanism remains elusive. Our present study aimed to investigate the effects of mechanosensitive channel of large conductance (MscL) activated by mechanical stretching on the cultured Schwann cells (SCs) and explore the possible mechanism.MethodsPrimary SCs from neonatal mice at 3-5 days of age were derived and transfected with the lentivirus vector expressing a mutant version of MscL, MscL-G22S. We first detected the cell viability and calcium ion (Ca2+) influx in the MscL-G22S-expressing SCs with low-intensity mechanical stretching and the controls. Proteomic and energy metabolomics analyses were performed to investigate the comprehensive effects of MscL-G22S activation on SCs. Measurement of glycolysis- and oxidative phosphorylation-related molecules and ATP production were respectively performed to further validate the effects of MscL-G22S activation on SCs. Finally, the roles of phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway in the mechanism of energy metabolism modulation of SCs by MscL-G22S activation was investigated.ResultsMechanical stretching-induced MscL-G22S activation significantly increased the cell viability and Ca2+ influx into the SCs. Both the proteomic and targeted energy metabolomics analysis indicated the upregulation of energy metabolism as the main action mechanism of MscL-G22S-activation on SCs. MscL-G22S-activated SCs showed significant upregulation of glycolysis and oxidative phosphorylation when SCs with stretching alone had only mild upregulation of energy metabolism than those without stimuli. MscL-G22S activation caused significant phosphorylation of the PI3K/AKT/mTOR signaling pathway and upregulation of HIF-1α/c-Myc. Inhibition of PI3K abolished the MscL-G22S activation-induced upregulation of HIF-1α/c-Myc signaling in SCs and reduced the levels of glycolysis- and oxidative phosphorylation-related substrates and mitochondrial activity.ConclusionMechanical stretching activates MscL-G22S to significantly promote the energy metabolism of SCs and the production of energic substrates, which may be applied to enhance nerve regeneration via the glia-axonal metabolic coupling.

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The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo.

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The oligomeric state of the truncated mechanosensitive channel of large conductance shows no variance in vivo.

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