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

Outward currents through Kir2.1 channels regulate the electrical properties of excitable cells. These currents are subject to voltage-dependent attenuation by the binding of polyamines to high- and low-affinity sites, which leads to inward rectification, thereby controlling cell excitability. To examine the effects of positive charges at the low-affinity site in the cytoplasmic pore on inward rectification, we studied a mutant Kir channel (E224K/H226E) and measured single-channel currents and streaming potentials (Vstream), the latter provide the ratio of water to ions queued in a single-file permeation process in the selectivity filter. The water-ion coupling ratio was near one at a high K(+) concentration ([K(+)]) for the wild-type channel and increased substantially as [K(+)] decreased. On the other hand, fewer ions occupied the selectivity filter in the mutant at all [K(+)]. A model for the Kir channel involving a K(+) binding site in the wide pore was introduced. Model analyses revealed that the rate constants associated with the binding and release to and from the wide-pore K(+) binding site was modified in the mutant. These effects lead to the reduced contribution of a conventional two-ion permeation mode to total conductance, especially at positive potentials, thereby inward rectification. 

Mechanism for attenuated outward conductance induced by mutations in the cytoplasmic pore of Kir2.1 channels.

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.

Fluidity and mosaicity are two critical features of biomembranes, by which membrane proteins function through chemical and physical interactions within a bilayer. To understand this complex and dynamic system, artificial lipid bilayer membranes have served as unprecedented tools for experimental examination, in which some aspects of biomembrane features have been extracted, and to which various methodologies have been applied. Among the lipid bilayers involving liposomes, planar lipid bilayers and nanodiscs, recent developments of lipid bilayer methods and the results of our channel studies are reviewed herein. Principles and techniques of bilayer formation are summarized, which have been extended to the current techniques, where a bilayer is formed from lipid-coated water-in-oil droplets (water-in-oil bilayer). In our newly developed method, termed the contact bubble bilayer (CBB) method, a water bubble is blown from a pipette into a bulk oil phase, and monolayer-lined bubbles are docked to form a bilayer through manipulation by pipette. An asymmetric bilayer can be readily formed, and changes in composition in one leaflet were possible. Taking advantage of the topological configuration of the CBB, such that the membrane's hydrophobic interior is contiguous with the surrounding bulk organic phase, oil-dissolved substances such as cholesterol were delivered directly to the bilayer interior to perfuse around the membrane-embedded channels (membrane perfusion), and current recordings in the single-channel allowed detection of immediate changes in the channels' response to cholesterol. Chemical and mechanical manipulation in each monolayer (monolayer technology) allows the examination of dynamic channel-membrane interplay.

Article	Year
Mechanism for attenuated outward conductance induced by mutations in the cytoplasmic pore of Kir2.1 channels. Scientific reports\| PubMed ID: 26678093	2015
Membrane Perfusion of Hydrophobic Substances Around Channels Embedded in the Contact Bubble Bilayer. Scientific reports\| PubMed ID: 28761089	2017
Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay. Biological & pharmaceutical bulletin\| PubMed ID: 29491206	2018

Masayuki Iwamoto

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