An in vivo assay identifies changes in residue accessibility on mechanosensitive channel gating

Department of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75390-9040, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 08/2004; 101(27):10161-5. DOI: 10.1073/pnas.0402040101
Source: PubMed


MscL is a mechanosensitive channel of large conductance that functions as an "emergency release valve," allowing bacteria to survive acute hypoosmotic stress. Although Escherichia coli MscL is the best-studied mechanosensitive channel, structural rearrangements occurring during gating remain disputed. Introduction of a charged residue into the pore of MscL was shown to result in a reduced-viability phenotype. Here, we probe for residues in the transmembrane domains that are exposed to the aqueous environment in the presence and absence of hypoosmotic shock by reacting a charged sulfhydryl reagent with substituted cysteines. Subsequent analysis of cell viability allows for an assessment of residues exposed in the closed and opening states in vivo. The results suggest that the crystal structure of MscL derived from the Mycobacterium tuberculosis orthologue may reflect a nearly closed rather than fully closed state and support a clockwise rotation of the pore-forming first transmembrane domain on gating.

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    • "8 ) , Batiza et al . ( 2002 ) and Bartlett et al . ( 2004 ) A20 ( 0 . 98 ) S ( <0 . 01 ) A20C / V Maurer and Dougherty ( 2003 ) and Bartlett et al . ( 2004 ) V21 ( 0 . 86 ) I ( 0 . 11 ) V21C Bartlett et al . ( 2004 ) and Levin and Blount ( 2004 ) G22 ( 0 . 75 ) A ( 0 . 24 ) G22C / D / I / N / S Ou et al . ( 1998 ) , Maurer and Dougherty ( 2001 ) , Bartlett et al . ( 2004 ) , Levin and Blount ( 2004 ) , Anishkin et al . ( 2005 ) , Powl et al . ( 2008a , b ) and Yoshimura et al . ( 2008 ) V23 ( 0 . 76 ) I ( 0 . 19 ) V23A / C / D / G / I / T Ou et al . ( 1998 ) , Maurer and Dougherty ( 2003 ) , Bartlett et al . ( 2004 ) , Levin and Blount ( 2004 ) and Anishkin et al . ( 2005 ) I24 ( 0 . 71 ) V ( 0 . 28 ) I"
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    ABSTRACT: Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their function, it is important to iden-tify the conserved motifs using sequence analysis methods. In this study, the sequence conservation was investigated by an in silico analysis to generate sequence logos. We have identiWed new conserved motifs in the domains TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identiWed new motifs for the TM3 and the cytoplasmic car-boxy-terminal domain from 309 homologs of MS channel of small conductance (MscS). We found that the conserva-tion in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in MscS homologs is high only for TM3 in Bacteria and Archaea.
    Full-text · Article · Sep 2013 · European Biophysics Journal
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    • "The predictions of the relative location of specific residues in the closed structure have been supported by electron paramagnetic resonance (EPR) spectroscopy in combination with site-directed spin labeling (SDSL) (Perozo et al., 2001). However, one should note that some evidence suggests a slightly altered model around the constriction point of the pore for the " fully closed " E. coli MscL channel (Bartlett et al., 2004; Iscla et al., 2004; Levin and Blount, 2004; Li et al., 2004; Bartlett et al., 2006). Several experiments using relatively independent approaches performed with the E. coli MscL channel now suggest that the crystal structure does not represent the fully closed state found in membranes (see (Blount et al., 2007) for a full discussion). "
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    ABSTRACT: What are the stimuli that are sensed by mechanoreceptors? Researchers have now begun to address this question and determine the molecular mechanisms underlying channels that are gated by mechanical forces. Two quite different models now exist. The first is that the channels are ‘tethered’ to cytoskeleton and or extracellular components, which thus exert forces on the channel that lead to gating. The second model predicts that the channel protein directly senses biophysical changes that occur within the membrane when it is under tension. Several lines of evidence indicate that many putative mechanosensitive channels are indeed tethered by other proteins, however in many instances the exact role this tethering plays in mechanosensing has yet to be fully clarified. On the other hand, the cloning and study of bacterial mechanosensitive channels demonstrated that channels can directly sense tension within the membrane. Evidence obtained from several of the more complex eukaryotic mechanosensory systems suggests that a number of eukaryotic channels from divergent families similarly sense tension within the membrane.
    Full-text · Chapter · Dec 2007
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    • "Together, these data strongly support the hypothesis that G26, not V23, is the constriction point from the periplasmic side. A clockwise rotation of TM1 has been previously proposed to be coupled with channel opening (Perozo et al., 2002; Bartlett et al., 2004). Here we find that metal binding to G22H, which is clockwise to G26, shows one of the largest metalinduced inhibitions, whereas V23H and I24H, which are counterclockwise, conferred less inhibition of gating (Fig. 4). "
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    ABSTRACT: The mechanosensitive channel of large conductance, MscL, of Escherichia coli is one of the best-studied mechanosensitive proteins. Although the structure of the closed or "nearly-closed" state of the Mycobacterium tuberculosis ortholog has been solved and mechanisms of gating have been proposed, the transition from the closed to the open states remains controversial. Here, we probe the relative position of specific residues predicted to line the pore of MscL in either the closed state or during the closed-to-open transition by engineering single-site histidine substitutions and assessing the ability of Ni2+, Cd2+ or Zn2+ ions to affect channel activity. All residues predicted to be within the pore led to a change in channel threshold pressure, although the direction and extent of this change were dependent upon the mutation and metal used. One of the MscL mutants, L19H, exhibited gating that was inhibited by Cd2+ but stimulated by Ni2+, suggesting that these metals bind to and influence different states of the channel. Together, the results derived from this study support the hypotheses that the crystal structure depicts a "nearly closed" rather than a "fully closed" state of MscL, and that a clockwise rotation of transmembrane domain 1 occurs early in the gating process.
    Full-text · Article · Dec 2004 · Biophysical Journal
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