S. aureus MscL Is a Pentamer In Vivo but of Variable Stoichiometries In Vitro: Implications for Detergent-Solubilized Membrane Proteins

Detergent-induced rearrangements of membrane-protein subunits explain why two MscL channel stoichiometries have been resolved by X-ray crystallography - but S. aureus MscL is truly a pentamer in vivo

Single-Incision Laparoscopic Cholecystectomy Using the Marionette Transumbilical Approach Is Safe and Efficient with Careful Patient Selection: A Comparative Analysis with Conventional Multiport Laparoscopic Cholecystectomy

Objectives The “marionette technique” for transumbilical laparoscopic cholecystectomy (m-TLC) offers improved cosmesis and possibly shorter postoperative recovery for patient undergoing laparoscopic cholecystectomy versus the four-port conventional laparoscopic cholecystectomy (CLC). We compared the outcomes of m-TLC and CLC at a tertiary care facility in New York. Methods A retrospective chart review was conducted and data on patients who underwent m-TLC and CLC were retrieved. Hospital length of stay (LOS), operative time, and complications were compared between the two groups using linear and logistic regression, as appropriate. Results M-TLC group patients were significantly younger, predominantly females with lower body mass index. They were less likely to have previous abdominal surgery and more likely to have noninflammatory pathology (p < 0.05 for all). Nonadjusted LOS (1 vs. 3 days, p-value < 0.0001) and operative time (50 vs. 56 minutes, p-value = 0.007) were significantly lower among patients who underwent m-TLC; however, there was no significant difference on multivariate analysis. In multivariate analysis, there was no difference in the overall complication rate (odds ratio: 1.63; 95% confidence interval 0.02–2.39). Conclusion With careful patient selection, m-TLC offers better cosmesis with comparable safety outcomes. Level of evidence Level III

Manipulating the permeation of charged compounds through the MscL nanovalve

MscL is a bacterial mechanosensor that serves as a biological emergency release valve, releasing cytoplasmic solutes to the environment on osmotic downshock. Previous studies have recognized that this channel has properties that make it ideal for use as a triggered nanovalve for vesicular-based targeted drug-release devices. One can even change the modality of the sensor. Briefly, the introduction of charges into the MscL pore lumen gates the channel in the absence of membrane tension; thus, by inserting compounds that acquire a charge on exposure to an alternative stimulus, such as light or pH, into the pore of the channel, controllable nanoswitches that detect these alternative modalities have been engineered. However, a charge in the pore lumen could not only encourage actuation of the nanopore but also have a significant influence on the permeation of large charged compounds, which would thus have important implications for the efficiency of drug-release devices. In this study, we used in vivo and electrophysiological approaches to demonstrate that the introduction of a charge into pore lumen of MscL does indeed influence the permeation of charged molecules. These effects were more drastic for larger compounds and, surprisingly, were related to the orientation of the MscL channel in the membrane.—Yang, L-M, Blount, P. Manipulating the permeation of charged compounds through the MscL nanovalve

Molecular Sieve Mechanism of Selective Release of Cytoplasmic Proteins by Osmotically Shocked Escherichia coli

Escherichia coli cells, the outer membrane of which is permeabilized with EDTA, release a specific subset of cytoplasmic proteins upon a sudden drop in osmolarity in the surrounding medium. This subset includes EF-Tu, thioredoxin, and DnaK among other proteins, and comprises ∼10% of the total bacterial protein content. As we demonstrate here, the same proteins are released from electroporated E. coli cells pretreated with EDTA. Although known for several decades, the phenomenon of selective release of proteins has received no satisfactory explanation. Here we show that the subset of released proteins is almost identical to the subset of proteins that are able to pass through a 100-kDa-cutoff cellulose membrane upon molecular filtration of an E. coli homogenate. This finding indicates that in osmotically shocked or electroporated bacteria, proteins are strained through a molecular sieve formed by the transiently damaged bacterial envelope. As a result, proteins of small native sizes are selectively released, whereas large proteins and large protein complexes are retained by bacterial cells

The role of the periplasmic loop residue glutamine 65 for MscL mechanosensitivity

The periplasmic loop of MscL, the mechanosensitive channel of large conductance, acts as a spring resisting the opening of the channel. Recently, a high-throughput functional screening of a range of MscL structural mutants indicated that the substitution of residue glutamine (Q) 65 with arginine (R) or leucine (L) leads to a wild-type (WT)-like and a loss-of-function (LOF) phenotype, respectively (Maurer and Dougherty J. Biol. Chem. 278(23):21076-21082, 2003). We used electron paramagnetic resonance (EPR) spectroscopy, single-channel recording and in vivo experiments to investigate further the effect of R and L mutation of Q65 on the gating mechanism of MscL. Structural analysis of Q65R and Q65L was carried out by coupling the site-directed spin labeling (SDSL) with EPR spectroscopy. A SDSL cysteine mutant of the isoleucine 24 residue (124CSL) in the first transmembrane domain, TM1, of MscL served as a reporter residue in EPR experiments. This was due to its strong spin-spin interaction with the neighboring 124C-SL residues in the MscL channel pentamer (Perozo et al.Nature 418:942-948, 2002). The effects of bilayer incorporation of lysophosphatidylcholine on the MscL mutants were also investigated. Functional analysis was carried out using patch-clamp recordings from these mutants and WT MscL reconstituted into artificial liposomes. Although our data are largely in agreement with the high-throughput mutational analysis of Maurer and Dougherty, this study shows that Q65R and Q65L form functional channels and that these mutations lead to partial gain-of-function (GOF) and LOF mutation, respectively. Overall, our study confirms and advances the notion that the periplasmic loop plays a role in setting the channel mechanosensitivity