Staphylococcus aureus alpha-toxin-induced pores: Channel-like behavior in lipid bilayers and patch clamped cells

Korchev, Yuri; Alder, G. M.; Bakhramov, A.; Bashford, C. L.; Joomun, B. S.; Sviderskaya, Elena V.; Usherwood, P.N.R.; Pasternak, C. A.

doi:10.1007/bf00234660

Cited by 45 publications

(35 citation statements)

References 35 publications

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“…The single channel conductance and voltage dependency determined from these current records also agreed with those measured by conventional techniques. [21][22][23] Gold is easily fabricated to fine shapes and modified by the formation of self-assembled monolayers using thiol-derivatives. On the other hand, potential differences between gold electrodes are easily induced by ion charging across the membrane.…”

Section: Resultsmentioning

confidence: 99%

A Simple Method for Ion Channel Recordings Using Fine Gold Electrode

et al. 2016

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The artificial bilayer single-channel recording technique is commonly used to observe detailed pharmacological properties of various ion channel proteins. It permits easy control of the solution and membrane lipid composition, and is also compatible with pharmacological screening devices. However, its use is limited due to low measurement efficiency. Here, we develop a novel artificial bilayer single-channel recording technique in which bilayers are made and channels are reconstituted into the membranes by contacting a gold electrode to the lipid-solution interface. Using this technique, we measured the single-channel currents of two channel-forming peptides, gramicidin and alamethicin, and a channelforming protein, α-hemolysin. This technique requires only one action, allowing the technique to potentially be combined with high-throughput screening devices.

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Section: Resultsmentioning

confidence: 99%

A Simple Method for Ion Channel Recordings Using Fine Gold Electrode

et al. 2016

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“…Planar bilayers were formed from DPhPC or Lettré cell plasma membrane (Schindler, 1980) as described by Korchev et al (1995). In one experiment, POPC/cholesterol (1/1 w/w) liposomes that had been used for a calcein leakage experiment in the presence of PEG 2,000 and ␣-toxin were pelleted and bilayers formed by the technique used for Lettré cell plasma membrane.…”

Section: Bilayer Experimentsmentioning

confidence: 99%

Pore Formation by S. aureus α-toxin in Liposomes and Planar Lipid Bilayers: Effects of Nonelectrolytes

Bashford

Alder

Fulford

et al. 1996

Journal of Membrane Biology

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Nonelectrolytes such as polyethylene glycols (PEG) and dextrans (i) promote the association of S. aureus alpha-toxin with liposomes (shown by Coomassie staining) and (ii) enhance the rate and extent of calcein leakage from calcein-loaded liposomes; such leakage is inhibited by H+, Zn2+ and Ca2+ to the same extent as that of nonPEG-treated liposomes. Incubation of liposomes treated with alpha-toxin in the presence of PEG with the hydrophobic photo-affinity probe 3-(trifluoromethyl)-3-m-[125I]iodophenyl) diazirine (125I-TID) labels monomeric and-predominantly-hexameric forms of liposome-associated alpha-toxin; in the absence of PEG little labeling is apparent. At high concentrations of H+ and Zn2+ but not of Ca(2+)-all of which inhibit calcein leakage-the distribution of label between hexamer and monomer is perturbed in favor of the latter. In alpha-toxin-treated planar lipid bilayers from which excess toxin has been washed away, PEGs and dextrans strongly promote the appearance of ion-conducting pores. The properties of such pores are similar in most regards to pores induced in the absence of nonelectrolytes; the differ only in being more sensitive to "closure" by voltage (as are pores induced in cells). In both systems, the stimulation by nonelectrolytes increase with concentration and with molecular mass up to a maximum around 2,000 Da. We conclude (i) that most of the alpha toxin that becomes associated with liposome or planar lipid bilayers does not form active pores and (ii) that the properties of alpha-toxin-induced pores in lipid bilayers can be modulated to resemble those in cells.

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“…5). The discovery of voltage gating in MspA as another ␤-barrel protein with no structural similarities to either porins of Gram-negative bacteria nor ␤-barrel toxins such as ␣-hemolysin of Staphylococcus aureus (30,31) supports the hypothesis that voltage gating might indeed be an intrinsic property of ␤-barrel proteins as first described by Lakey and co-workers (23). Hence, our finding favors a mechanism in which an externally applied voltage perturbs the electric field in ␤-barrel pores that is required for ion translocation (23).…”

Section: Discussionmentioning

confidence: 99%

Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis

Huff¹,

Pavlenok

Sukumaran³

et al. 2009

Journal of Biological Chemistry

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MspA is the major porin of Mycobacterium smegmatis and mediates diffusion of small and hydrophilic solutes across the outer membrane. The octameric structure of MspA, its sharply defined constriction zone, and a large periplasmic loop L6 represent novel structural features. L6 consists of 13 amino acids and is directly adjacent to the constriction zone. Deletion of 3, 5, 7, 9, and 11 amino acids of the L6 loop resulted in functional pores that restored glucose uptake and growth of a porin mutant of M. smegmatis. Lipid bilayer experiments revealed that all mutant channels were noisier than wild type (wt) MspA, indicating that L6 is required for pore stability in vitro. Voltage gating of the Escherichia coli porin OmpF was attributed to loops that collapse into the channel in response to a strong electrical field. Here, we show that deletion mutants ⌬7, ⌬9, and ⌬11 had critical voltages similar to wt MspA. This demonstrated that the L6 loop is not the primary voltage-dependent gating mechanism of MspA. Surprisingly, large deletions in L6 resulted in 3-6-fold less extractable pores, whereas small deletions did not alter expression levels of MspA. Pores with large deletions in L6 were more permissive for glucose than smaller deletion mutants, whereas their single channel conductance was similar to that of wt MspA. These results indicate that translocation of ions through the MspA pore is governed by different mechanisms than that of neutral solutes. This is the first study identifying a molecular determinant of solute translocation in a mycobacterial porin.Mycobacteria have a unique outer membrane composed of covalently attached mycolic acids and a variety of other loosely associated lipids. Visualized by cryo-electron tomography (1), the outer membrane is an efficient permeability barrier that renders mycobacteria intrinsically resistant to many drugs and toxic compounds. As is the case for Gramnegative bacteria, this permeability barrier must be functionalized by outer membrane proteins. Although over 60 outer membrane proteins are known to exist in Escherichia coli (41), very few have been described in mycobacteria to date.The major protein of Mycobacterium smegmatis is MspA (42), which is the primary member of the nearly identical Msp (M. smegmatis porin) proteins. Msp proteins account for ϳ80% of detergent-extractable proteins at 100°C (2) in wt 4 M. smegmatis mc 2 155. MspA represents the main pathway for small and hydrophilic solutes and nutrients across the outer membrane (3). One hallmark of MspA is its extremely high stability. Functional pores are detectable at temperatures up to 100°C, a pH range from 0 -14, and resist the action of denaturing agents such as 2% SDS (4), thus providing the utility of MspA for nanotechnological applications. However, it is unknown how M. smegmatis utilizes Msp porins to functionalize the outer membrane in varying conditions or what parts of the channel determine substrate translocation. It is our goal to understand the translocation determinants of the MspA channel.The cry...

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Staphylococcus aureus alpha-toxin-induced pores: Channel-like behavior in lipid bilayers and patch clamped cells

Cited by 45 publications

References 35 publications

A Simple Method for Ion Channel Recordings Using Fine Gold Electrode

A Simple Method for Ion Channel Recordings Using Fine Gold Electrode

Pore Formation by S. aureus α-toxin in Liposomes and Planar Lipid Bilayers: Effects of Nonelectrolytes

Functions of the Periplasmic Loop of the Porin MspA from Mycobacterium smegmatis

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