Purification and Functional Reconstitution of the Recombinant Large Mechanosensitive Ion Channel (MscL) of Escherichia coli

Häse, Claudia C.; Dain, Alexander C. Le; Martinac, Boris

doi:10.1074/jbc.270.31.18329

Cited by 195 publications

(195 citation statements)

References 15 publications

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“…The negative lateral pressure in the lipid bilayer is considered to mimic the stretched membrane used in patch-clamp experiments. 6,37 Calculation of transmembrane pressure profile. In order to determine whether this method for applying negative pressure to the membrane retains the original features without the intrusion of any fatal artifacts, we calculated a pressure profile of the membrane with the method proposed in an earlier work.…”

Section: Discussionmentioning

confidence: 99%

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

2012

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Section: Discussionmentioning

confidence: 99%

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

2012

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“…Some of these bacterial channel proteins are smaller and more amenable to expression and purification than mammalian channels. Importantly, bacterial channels can often be reconstituted into vesicles and other synthetic membrane systems, allowing detailed functional characterization (4)(5)(6). Furthermore, several bacterial channels have been described at atomic resolution by x-ray crystallography (7)(8)(9)(10)(11), providing opportunities for correlating structure and function in a key class of membrane protein.…”

mentioning

confidence: 99%

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Clayton

Shapovalov

Maurer

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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Total chemical protein synthesis was used to generate multimilligram quantities of the mechanosensitive channel of large conductance from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (Tb-MscL). Cysteine residues introduced to allow chemical ligation were masked with cysteine-reactive molecules, resulting in side chain functional groups similar to those of the wild-type protein. Synthetic channel proteins were transferred to 2,2,2-trifluoroethanol and reconstituted into vesicle membranes. Fluorescent imaging of vesicles showed that channel proteins were membrane-localized. Single-channel recordings showed that reconstituted synthetic Ec-MscL has conductance, pressure dependence, and substate distribution similar to those of the recombinant channel. Reconstituted synthetic Tb-MscL also displayed conductance and pressure dependence similar to that of the recombinant protein. Possibilities for the incorporation of unnatural amino acids and biophysical probes, and applications of such synthetic ion channel analogs, are discussed. R ecently, ion channel research has been aided by the discovery of bacterial ion channels that share many key features of mammalian channels (1-3). Some of these bacterial channel proteins are smaller and more amenable to expression and purification than mammalian channels. Importantly, bacterial channels can often be reconstituted into vesicles and other synthetic membrane systems, allowing detailed functional characterization (4-6). Furthermore, several bacterial channels have been described at atomic resolution by x-ray crystallography (7-11), providing opportunities for correlating structure and function in a key class of membrane protein.Although the preparation of many medium-sized watersoluble proteins by chemical ligation is now a routine procedure (12)(13)(14), the synthesis of ion channels and other membrane proteins presents unique challenges. Many hydrophobic peptides have sequences that couple inefficiently, meaning that the synthesis of each segment must be optimized. Hydrophobic peptides also have limited solubility and tend to aggregate. Furthermore, after synthesis is complete the native oligomeric protein must be formed from unfolded monomers. However, recent advances in chemical ligation methodologies have permitted the semisynthesis (15) and the total chemical synthesis of membrane proteins, including the 97-residue type III (single membrane-spanning segment) M2 protein of the influenza virus (16). The synthetic M2 protein assembled into the native tetrameric form on reconstitution into dodecylphosphocholine micelles.Here, we describe the chemical synthesis of two polytopic membrane proteins, the mechanosensitive ion channels from Escherichia coli (Ec-MscL) and Mycobacterium tuberculosis (TbMscL), and the vesicle reconstitution of these channels into a form functionally similar to that of the recombinant protein.Multimilligram quantities of Ec-MscL and Tb-MscL were generated by optimizing synthesis, purification, and ligation protocols previously developed fo...

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“…tension change required to cause an e-fold increase in relative channel activity. For our purposes, we performed parametric simulations over a range of values for , and T 1/2 corresponding to a range of parameter values reported in the SAC classification literature (24,29,30,41,49,50). Model-generated membrane tension was used to calculate a P o "wave" via Eq.…”

Section: Resultsmentioning

confidence: 99%

“…The gray arrow in Fig. 1 represents a biophysics theory, which states that SAC opening probability is described by a Boltzmann distribution dependent on tension over the SAC (22,24,29,30,32,41). The dashed black arrows represent relationships between stretch and plasma membrane tension, including a negative feedback mechanism created by tension-reducing lipid insertion, all of which we wish to predict with a mathematical model fit to data derived from the appropriate literature.…”

mentioning

confidence: 99%

Modeling the effect of stretch and plasma membrane tension on Na⁺-K⁺-ATPase activity in alveolar epithelial cells

Fisher¹,

Margulies²

2007

American Journal of Physiology-Lung Cellular and Molecular Phys

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Fisher, Jacob L., and Susan S. Margulies. Modeling the effect of stretch and plasma membrane tension on Na ϩ -K ϩ -ATPase activity in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 292: L40-L53, 2007. First published August 4, 2006; doi:10.1152/ajplung.00425.2005.-While a number of whole cell mechanical models have been proposed, few, if any, have focused on the relationship among plasma membrane tension, plasma membrane unfolding, and plasma membrane expansion and relaxation via lipid insertion. The goal of this communication is to develop such a model to better understand how plasma membrane tension, which we propose stimulates Na ϩ -K ϩ -ATPase activity but possibly also causes cell injury, may be generated in alveolar epithelial cells during mechanical ventilation. Assuming basic relationships between plasma membrane unfolding and tension and lipid insertion as the result of tension, we have captured plasma membrane mechanical responses observed in alveolar epithelial cells: fast deformation during fast cyclic stretch, slower, time-dependent deformation via lipid insertion during tonic stretch, and cell recovery after release from stretch. The model estimates plasma membrane tension and predicts Na ϩ -K ϩ -ATPase activation for a specified cell deformation time course. Model parameters were fit to plasma membrane tension, whole cell capacitance, and plasma membrane area data collected from the literature for osmotically swollen and shrunken cells. Predictions of membrane tension and stretch-stimulated Na ϩ -K ϩ -ATPase activity were validated with measurements from previous studies. As a proof of concept, we demonstrate experimentally that tonic stretch and consequent plasma membrane recruitment can be exploited to condition cells against subsequent cyclic stretch and hence mitigate stretchinduced responses, including stretch-induced cell death and stretch-induced modulation of Na ϩ -K ϩ -ATPase activity. Finally, the model was exercised to evaluate plasma membrane tension and potential Na ϩ -K ϩ -ATPase stimulation for an assortment of traditional and novel ventilation techniques.ventilator-induced lung injury; lipid trafficking; edema recovery INVESTIGATORS HAVE FOUND alveolar epithelial stretch magnitude to be related to cell injury in vitro (55, 56) and tidal volume to be related to ventilator-induced lung injury (VILI) in animal models (12-15). Studies have revealed that the frequency of epithelial stretch or ventilation can also be an important factor in cell injury (56, 60). The model developed in this study examines how combinations of amplitude and frequency in ventilation procedures affect Na ϩ -K ϩ -ATPase activity via hypothesized pathways including alveolar epithelial cell plasma membrane tension and stretch-activated channel (SAC) stimulation.To date, numerous models have been proposed to describe cellular mechanical behavior, from early continuum viscoelastic representations for erythrocytes and leukocytes (11,27,65) to more sophisticated variations that modeled cell membrane an...

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Purification and Functional Reconstitution of the Recombinant Large Mechanosensitive Ion Channel (MscL) of Escherichia coli

Cited by 195 publications

References 15 publications

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Modeling the effect of stretch and plasma membrane tension on Na⁺-K⁺-ATPase activity in alveolar epithelial cells

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Purification and Functional Reconstitution of the Recombinant Large Mechanosensitive Ion Channel (MscL) of Escherichia coli

Cited by 195 publications

References 15 publications

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

The gating mechanism of the bacterial mechanosensitive channel MscL revealed by molecular dynamics simulations

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

Modeling the effect of stretch and plasma membrane tension on Na+-K+-ATPase activity in alveolar epithelial cells

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Modeling the effect of stretch and plasma membrane tension on Na⁺-K⁺-ATPase activity in alveolar epithelial cells