Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels

Delcour, Anne H.; Martinac, Boris; Adler, Julius; Kung, Ching

doi:10.1016/s0006-3495(89)82710-9

Cited by 215 publications

(150 citation statements)

References 23 publications

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“…Fifty microliters of a 10 mg͞ml solution of azolectin (Sigma-Aldrich) in chloroform was air-dried on Teflon for 15 min, redissolved with 5 l of a 2 mg͞ml solution of synthetic channel protein in TFE (19), and allowed to dry for 15 min. The resultant film of material was desiccated under house vacuum for 1 h and then rehydrated for 1 h with 5 l of 250 mM KCl͞5 mM Hepes͞0.1 mM EDTA (20). One microliter of this rehydrated lipid was applied to the glass coverslip of the recording chamber and allowed to air dry for 10 min, and 0.5 l of 1 M MgCl 2 was spread on the coverslip and dried for 10 min.…”

Section: Methodsmentioning

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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Section: Methodsmentioning

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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“…Purified porin in Triton X-100 was incubated with small unilamellar vesicles of asolectin (type IV S; Sigma) at a protein-to-lipid ratio of 1:2,000 to 1:4,000 (wt/wt) for 30 min at room temperature before the addition of Bio-Beads (80 mg/ml; Bio-Rad, Ivry sur Seine, France) to get rid of detergent. After 4 h of incubation at room temperature, the beads were discarded, and the suspension was centrifuged for 20 min at 337,000 ϫ g. The pellet was resuspended in 30 l of 10 mM HEPES (pH 7.4), and aliquots of the suspension were subjected to a dehydration-rehydration procedure to obtain giant liposomes (11). For each experiment 1 to 2 l of proteoliposomes was placed in a 1-ml chamber containing recording solution (200 mM KCl, 10 mM HEPES, pH 7.4).…”

Section: Methodsmentioning

confidence: 99%

Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

Bornet

Saint

Fetnaci

et al. 2004

Antimicrob Agents Chemother

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In Enterobacter aerogenes, ␤-lactam resistance often involves a decrease in outer membrane permeability induced by modifications of porin synthesis. In ATCC 15038 strain, we observed a different pattern of porin production associated with a variable antibiotic susceptibility. We purified Omp35, which is expressed under conditions of low osmolality and analyzed its pore-forming properties in artificial membranes. This porin was found to be an OmpF-like protein with high conductance values. It showed a noticeably higher conductance compared to Omp36 and a specific location of WNYT residues in the L3 loop. The importance of the constriction region in the porin function suggests that this organization is involved in the level of susceptibility to negative large cephalosporins such as ceftriaxone by bacteria producing the Omp35 porin subfamily.The bacterial porins are major outer membrane proteins that form water-filled channels, allowing the diffusion across the outer membrane of small polar molecules, amino acids, and nutrients (14,18,23). General nonspecific porins, such as OmpF and OmpC from Escherichia coli, form homotrimers in the outer membrane. These porin trimers are constituted by large ␤-barrel monomer subunits which are constricted about halfway through the membrane by an internal loop, loop 3 (L3) (18). Resolution of the three-dimensional structure of the E. coli OmpF and Klebsiella pneumoniae OmpK36 has led to identification of the functional domains of the enterobacterial channels (5,8,13,17).Enterobacter aerogenes is one of the most frequently described gram-negative pathogens responsible for nosocomial respiratory tract infections in France and Belgium (2, 6, 10). This bacterial pathogen harbors a variety of antibiotic resistance mechanisms (7,9,19,24,30). Among them, the modification of outer membrane permeability, a porin deficiency associated with the expression of cephalosporinase activity, is frequently detected in clinical resistant isolates (7,19). We recently isolated a strain with an unusual porin phenotype: this EA3 strain synthesized a mutated Omp36 porin containing a G112D substitution in the L3 domain (19). Despite this prominent role of porins in drug susceptibility, only one porin, Omp36, is today functionally characterized in E. aerogenes (19).The aim of this work was to investigate the structural and functional properties of the second major pore-forming protein produced by the E. aerogenes strain. The sequence and the channel properties of the new porin Omp35 were determined and agree with the biological differences concerning the ␤-lactam susceptibility, observed between the bacteria producing either Omp35 or Omp36. MATERIALS AND METHODSBacterial strains, growth conditions, and antibiotic susceptibility tests. The E. aerogenes ATCC 15038 strain was used as the standard strain. E. aerogenes EAEP289 is a Kan s derivative of the previously described clinical strain EA27 (24). Bacteria were routinely grown in Luria-Bertani (LB) medium or nutrient broth (NB). For the determination of MI...

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“…Outer membrane fractions were purified by sucrose gradient centrifugation as described (21) and stored at Ϫ80°C. The bicinchonninic acid method (Pierce) was used for determination of protein concentrations.…”

Section: Methodsmentioning

confidence: 99%

“…An aliquot of native membrane was mixed with sonicated azolectin (10 mg/ml) at a protein:lipid ratio of ϳ1:1,700 (w/w) and reconstituted according to a dehydration-rehydration protocol (21). Electrophysiological experiments were performed on liposome blisters, as described (21).…”

Section: Methodsmentioning

confidence: 99%

Complex Inhibition of OmpF and OmpC Bacterial Porins by Polyamines

Iyer

Delcour

1997

Journal of Biological Chemistry

105

108

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The effects of four polyamines (putrescine, cadaverine, spermidine, and spermine) on the activity of bacterial porins OmpC and OmpF were investigated by electrophysiology. Membrane vesicles made from the outer membrane of Escherichia coli strains expressing only OmpC or OmpF were reconstituted into liposomes probed by patch clamp. The channel activity was recorded in control solutions and in the presence of increasing concentrations of a specific polyamine. In all cases, concentration-and voltage-dependent inhibitory effects were observed. They include both the suppression of channel openings and the enhancement of channel closures as well as the promotion of blocked or inactivated states. OmpF and OmpC, although highly homologous, have distinct sensitivities to modulation, especially by spermine. This compound inhibits OmpF in the nanomolar range, which is in agreement with its potency on eukaryotic channels. Putrescine was the least effective (upper millimolar range) and also had inhibitory effects qualitatively distinct from those exerted by the other polyamines. The compounds appear to bind to at least two distinct binding sites, one of which resides within the pore. The potencies to this site are lower when the polyamines are applied from the extracellular side than from the periplasmic side, suggesting an asymmetric binding site.Polyamines are a class of naturally occurring polycationic molecules produced through complex pathways involving decarboxylations of ornithine, arginine, or lysine (1, 2). The most ubiquitous are spermine, spermidine, cadaverine and putrescine. With the exception of spermine, which is associated exclusively with eukaryotes, the other three are endogenous to both eukaryotic and prokaryotic cell types. Polyamines have been implicated in a wide range of biological phenomena (1, 2). One of the most intriguing forms of polyamine action is the recently discovered modulation of ion channels of heart, muscles, and neurons (3-10).The cytoplasmic membrane of Escherichia coli cells is surrounded by an additional external membrane, the outer membrane, whose outer leaflet is made of highly negatively charged lipopolysaccharides. Polyamines are associated with the outer membrane of E. coli, possibly through their interactions with the lipopolysaccharides (11). Although polyamines have not been measured directly in the periplasmic space between the outer and cytoplasmic membranes, they are likely to accumulate in this compartment during their synthesis and transport (12)(13)(14). An arginine decarboxylase involved in the production of putrescine is located in the inner periplasmic space (12), and a lysine-cadaverine exchanger of the cytoplasmic membrane participates in the extrusion of cadaverine (13). Thus, polyamines appear to reside in the vicinity of the major poreforming proteins of the outer membrane, the porins. Porins are trimeric channels characterized extensively at the biochemical, structural, and genetic levels (15). They are the only ion channels whose structure is known at atomic re...

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Modified reconstitution method used in patch-clamp studies of Escherichia coli ion channels

Cited by 215 publications

References 23 publications

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

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

Omp35, a New Enterobacter aerogenes Porin Involved in Selective Susceptibility to Cephalosporins

Complex Inhibition of OmpF and OmpC Bacterial Porins by Polyamines

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