Single Channel Analysis of Membrane Proteins in Artificial Bilayer Membranes

Bartsch, Philipp; Harsman, Anke; Wagner, Richard

doi:10.1007/978-1-62703-487-6_22

Cited by 12 publications

(17 citation statements)

References 36 publications

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“…The vertical lipid bilayer experiments were performed on a setup from IonoVation, which is described in detail elsewhere ( Bartsch et al, 2013 ). Two Teflon chambers, with a volume of 2.5 ml each, were separated by a 25-µm-thick Teflon foil with a central 100-µm large hole.…”

Section: Methodsmentioning

confidence: 99%

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Winterstein

Kukovetz

Rauh

et al. 2018

Journal of General Physiology

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Section: Methodsmentioning

confidence: 99%

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Winterstein

Kukovetz

Rauh

et al. 2018

Journal of General Physiology

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“…Single Channel Recordings from Planar Lipid Bilayers. Planar lipid bilayer measurements of reconstituted rATOM40 were essentially performed as described previously (49,61). Briefly, single-channel currents were measured in standard buffer conditions (250 mM KCl, 10 mM Mops/Tris pH 7) in the absence and presence of 150 μM tRNA (bulk tRNA from baker's yeast; Roche) on both sides of the bilayer using a GeneClamp 500 amplifier and the CV-5-1G headstage (Axon Instruments).…”

Section: Methodsmentioning

confidence: 99%

tRNAs and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes

Niemann

Harsman

Mani

et al. 2017

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

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Mitochondrial tRNA import is widespread, but the mechanism by which tRNAs are imported remains largely unknown. The mitochondrion of the parasitic protozoan lacks tRNA genes, and thus imports all tRNAs from the cytosol. Here we show that in in vivo import of tRNAs requires four subunits of the mitochondrial outer membrane protein translocase but not the two receptor subunits, one of which is essential for protein import. The latter shows that it is possible to uncouple mitochondrial tRNA import from protein import. Ablation of the intermembrane space domain of the translocase subunit, archaic translocase of the outer membrane (ATOM)14, on the other hand, while not affecting the architecture of the translocase, impedes both protein and tRNA import. A protein import intermediate arrested in the translocation channel prevents both protein and tRNA import. In the presence of tRNA, blocking events of single-channel currents through the pore formed by recombinant ATOM40 were detected in electrophysiological recordings. These results indicate that both types of macromolecules use the same import channel across the outer membrane. However, while tRNA import depends on the core subunits of the protein import translocase, it does not require the protein import receptors, indicating that the two processes are not mechanistically linked.

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“…However, this can be ruled out from both theoretical and experimental considerations. We are using agar protected Ag+/AgCl electrodes ( R typ ≌ 10 k Ω), which are carefully checked for electrode offset , which then is corrected automatically by the amplifier, at the beginning of each bilayer recording (30). In none of the five control experiments with control bilayers, the addition of 1.2 μl 330 μM R6G in EtOH had any effect on the electrode potentials.…”

Section: Resultsmentioning

confidence: 99%

A new twist in ABC transporter mediated multidrug resistance – Pdr5 is a drug/proton co-transporter

Wagner

Blum

Raschka

et al. 2021

Preprint

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The two major efflux pump systems that are involved in multidrug resistance (MDR) are (i) ATP binding cassette (ABC) transporters and (ii) secondary transporters. While the former use binding and hydrolysis of ATP to facilitate export of cytotoxic compounds, the latter utilize electrochemical gradients to expel their substrates. Pdr5 from Saccharomyces cerevisiae is a prominent member of eukaryotic ATP binding cassette (ABC) transporters that are involved in multidrug resistance (MDR) and used as a frequently studied model system. Although investigated for decades, the underlying molecular mechanisms of drug transport and substrate specificity remain elusive. Here, we provide electrophysiological data on the reconstituted Pdr5 demonstrating that this MDR efflux pump does not only actively translocate its substrates across the lipid bilayer, but at the same time generates a proton motif force in the presence of Mg2+-ATP and substrates by acting as a proton/drug co-transporter. Similar observations have not yet been reported for any other MDR efflux pump. We conclude from these results that the mechanism of MDR conferred by Pdr5 and likely other transporters is more complex than the sole extrusion of cytotoxic compounds and involves secondary coupled processes suitable to increase the effectiveness.

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Single Channel Analysis of Membrane Proteins in Artificial Bilayer Membranes

Cited by 12 publications

References 36 publications

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

tRNAs and proteins use the same import channel for translocation across the mitochondrial outer membrane of trypanosomes

A new twist in ABC transporter mediated multidrug resistance – Pdr5 is a drug/proton co-transporter

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