lipid-bilayer-experiments-with-contact-bubble-bilayers-for-patch

Lipid Bilayer Experiments with Contact Bubble Bilayers for Patch-Clampers

Lipid bilayers provide a unique experimental platform for functional studies of ion channels, allowing the examination of channel-membrane interactions ...

University of Fukui Faculty of Medical Sciences

In fluidic biomembranes, lipids and membrane proteins diffuse restlessly, and lipid compositions change steadily. To mimic dynamic behavior of the biomembranes, a method for introducing rapid changes in the constituents in the lipid bilayer was developed. In contact bubble bilayers (CBB), as a water-in-oil droplet bilayer system, the bilayer hydrophobic interior is contiguous with the bulk oil phase. Making use of this geometrical feature as an access route, hydrophobic substances were administered into the bilayer. Polytheonamide B, a cytotoxic hydrophobic peptide, was applied, and oriented incorporation and relevant single-channel current recordings were enabled. Nystatin was pre-loaded in the CBB, and sterol perfusion exhibited slow development of the macroscopic current. On the contrary, the reconstituted KcsA potassium channels immediately attenuate the channel activity when cholesterol was applied. This oil-phase route in the CBB allows rapid perfusion of hydrophobic substances around the bilayer-embedded channels during continuous recordings of channel currents.

Membrane Perfusion of Hydrophobic Substances Around Channels Embedded in the Contact Bubble Bilayer.

Amphidinol 3 (AM3) is an anti-fungal polyene extracted from a marine dinoflagellate. Here, we examined the ion channel activity and membrane-embedded structure of AM3 using a lipid bilayer method and atomic force microscopy (AFM). AM3 exhibited large-conductance (~1 nS) and non-selective single-channel activity only when sterols were present in the membrane leaflet of the AM3-added side. The variable conductance suggests the formation of a multimeric barrel-stave pore. At high AM3 concentrations, giant-conductance "jumbo" channels (~40 nS) emerged. AFM revealed a thicker raft-like membrane phase with the appearance of a wrinkled surface, in which phase pores (diameter: ~10 nm) were observed. The flip-flop of ergosterol occurred only after the appearance of the jumbo channel, indicating that the jumbo channel induced a continuity between the outer and inner leaflets of the membrane: a feature characteristic of toroidal-like pores. Thus, AM3 forms different types of sterol-aided polymorphic channels in a concentration dependent manner.

Channel Formation and Membrane Deformation via Sterol-Aided Polymorphism of Amphidinol 3.

Molecular mechanisms underlying channel-membrane interplay have been extensively studied. Cholesterol, as a major component of the cell membrane, participates either in specific binding to channels or via modification of membrane physical features. Here, we examined the action of various sterols (cholesterol, epicholesterol, etc.) on a prototypical potassium channel (KcsA). Single-channel current recordings of the KcsA channel were performed in a water-in-oil droplet bilayer (contact bubble bilayer) with a mixed phospholipid composition (azolectin). Upon membrane perfusion of sterols, the activated gate at acidic pH closed immediately, irrespective of the sterol species. During perfusion, we found that the contacting bubbles changed their shapes, indicating alterations in membrane physical features. Absolute bilayer tension was measured according to the principle of surface chemistry, and inherent bilayer tension was ∼5 mN/m. All tested sterols decreased the tension, and the nonspecific sterol action to the channel was likely mediated by the bilayer tension. Purely mechanical manipulation that reduced bilayer tension also closed the gate, whereas the resting channel at neutral pH never activated upon increased tension. Thus, rather than conventional stretch activation, the channel, once ready to activate by acidic pH, changes the open probability through the action of bilayer tension. This constitutes a channel regulating modality by two successive stimuli. In the contact bubble bilayer, inherent bilayer tension was high, and the channel remained boosted. In the cell membrane, resting tension is low, and it is anticipated that the ready-to-activate channel remains closed until bilayer tension reaches a few millinewton/meter during physiological and pathological cellular activities.

Article	Year
Membrane Perfusion of Hydrophobic Substances Around Channels Embedded in the Contact Bubble Bilayer. Scientific reports\| PubMed ID: 28761089	2017
Channel Formation and Membrane Deformation via Sterol-Aided Polymorphism of Amphidinol 3. Scientific reports\| PubMed ID: 28883505	2017
Constitutive boost of a K channel via inherent bilayer tension and a unique tension-dependent modality. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 30509986	2018

Shigetoshi Oiki

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