On the Structure of the N-Terminal Domain of the MscL Channel: Helical Bundle or Membrane Interface

Department of Physiology, University of Texas, Southwestern Medical Center at Dallas, Dallas, Texas 75390-9040, USA.
Biophysical Journal (Impact Factor: 3.97). 06/2008; 95(5):2283-91. DOI: 10.1529/biophysj.107.127423
Source: PubMed


The mechanosensitive channel of large conductance, MscL, serves as a biological emergency release valve protecting bacteria from acute osmotic downshock and is to date the best characterized mechanosensitive channel. A well-recognized and supported model for Escherichia coli MscL gating proposes that the N-terminal 11 amino acids of this protein form a bundle of amphipathic helices in the closed state that functionally serves as a cytoplasmic second gate. However, a recently reexamined crystal structure of a closed state of the Mycobacterium tuberculosis MscL shows these helices running along the cytoplasmic surface of the membrane. Thus, it is unclear if one structural model is correct or if they both reflect valid closed states. Here, we have systematically reevaluated this region utilizing cysteine-scanning, in vivo functional characterization, in vivo SCAM, electrophysiological studies, and disulfide-trapping experiments. The disulfide-trapping pattern and functional studies do not support the helical bundle and second-gate hypothesis but correlate well with the proposed structure for M. tuberculosis MscL. We propose a functional model that is consistent with the collective data.

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    • "Here we have identified the cause of heterogeneous expression in the original MscL expression construct developed in 1994 (Sukharev et al. 1994; Häse et al. 1995) and still widely used today. Despite recent studies showing that the N-terminal sequence of MscL influences the functional properties of the channel (Iscla et al. 2008; Corry et al. 2010), we show that this heterogeneously expressing construct is functionally indistinguishable from a newly engineered , homogeneously expressing channel, in agreement with an earlier study showing that extending the length of Fig. 4 Representative patch clamp data for the original, heterogeneously expressing MscL construct (original MscL) and the new MscL M1A and MscL L−7Y construct. Both channels were activated at ~−60 mmHg, and have similar current amplitudes when open the N-terminal domain has no effect on the functional properties of MscL (Häse et al. 1997a). "
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    ABSTRACT: The mechanosensitive channel of large conductance MscL is a well-characterized mechanically gated non-selective ion channel, which often serves as a prototype mechanosensitive channel for mechanotransduction studies. However, there are some discrepancies between MscL constructs used in these studies, most notably unintended heterogeneous expression from some MscL expression constructs. In this study we investigate the possible cause of this expression pattern, and compare the original non-homogenously expressing constructs with our new homogeneously expressing one to confirm that there is little functional difference between them. In addition, a new MscL construct has been developed with an improved molar extinction coefficient at 280 nm, enabling more accurate protein quantification.
    Biophysics of Structure and Mechanism 08/2015; 44(7). DOI:10.1007/s00249-015-1062-5 · 2.22 Impact Factor
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    • "Membrane topology of E. coli MscL channel predicted the formation of transmembrane helices TM1 and TM2 (Sukharev et al., 1994, 1999; Blount et al., 1996a,b). MscL channel is a transmembrane protein which exposed N-terminal towards cytoplasmic interface of plasma membrane and C-terminal faces the cytoplasm (Steinbacher et al., 2007; Iscla et al., 2008). Similarly, the structure of E. coli MscS channel revealed that this protein is a homoheptamer with each subunit containing a membrane domain and three transmembrane regions of TM1, TM2 and TM3 helices. "
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    ABSTRACT: Mechanosensitive channels are membrane proteins that open and shut in response to mechanical forces produced by osmotic pressure, sound, touch and gravity. These channels are involved in multiple physiological functions including hypoosmotic pressure, pain, hearing, blood pressure and cell volume regulation. In plants, these channels play a major role in proprioception, gravity sensing and maintenance of plastid shape and size. In the present study, we identified the mechanosensitive channel of small conductance like (MscS) homologue gene family in rice and analyzed their structure, phylogenetic relationship, localization and expression pattern. Five MscS like genes of rice (OsMSL) were found to be distributed on four chromosomes and clustered into two major groups. Subcellular localization predictions of the OsMSL family revealed their localization to plasma membrane, plastid envelope and mitochondria. The predicted gene structure, bonafide conserved signature motif, domain and the presence of transmembrane regions in each OsMSL strongly supported their identity as members of MscS-like gene family. Furthermore, in silico expression analysis of OsMSL genes revealed differential regulation patterns in tissue specific and abiotic stress libraries. These findings indicate that the in silico approach used here successfully identified in a genome-wide context MscS like gene family in rice, and further suggest the functional importance of MscS-like genes in rice.
    01/2015; 1:8-17. DOI:10.1016/j.plgene.2014.12.001
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    • "The TM1 and TM2 helices are linked by a periplasmic loop. The COOH-terminal part of the protein (residues 101–110) adopts an -helical bundle conformation and reside in the cytoplasm (Sukharev et al. 2001a, b; Gullingsrud and Schulten 2003; Maurer et al. 2008), while the segment S1 at the N-terminus is in close proximity to the cytoplasmic interface (Iscla et al. 2008; Steinbacher et al. 2007). On the other hand, MS channel of small conductance (MscS) is a 0.8–1-nS channel opened by moderate pressure (Perozo 2006). "
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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.
    European Biophysics Journal 09/2013; 38:1013-1027. · 2.22 Impact Factor
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