Lipid Bilayers Manipulated through Monolayer Technologies for Studies of Channel-Membrane Interplay

Abstract

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.

Hysteresis of a Tension-Sensitive K+ Channel Revealed by Time-Lapse Tension Measurements

“…In prevalent bilayer formation methods, lipid-lined water-in-oil droplets (DIB) or bubbles (CBB) are docked to form a bilayer. The CBB method has advantages such that each monolayer-lined bubble is maintained by steadily applying an intrabubble holding pressure by manipulating a pneumatic injector , (Figure ). Thus, the membrane tension is readily controlled by dynamic handling of the intrabubble pressure via the Young–Laplace principle: where γ mo is the monolayer tension and R 1 and R 2 are the principal radii of the bubble curvature) (pressure manipulation → tension change). , In turn, measurements of the intrabubble pressure enable quantitative evaluation of the tension also via the Young–Laplace principle (pressure measurement → tension evaluation).…”

Conductance selectivity of Na + across the K + channel via Na + trapped in a tortuous trajectory

“…The single-channel current of the KcsA channel in the presence of Na + in the intracellular solution was examined. The KcsA channel was reconstituted into a lipid bilayer using the contact bubble bilayer method (44)(45)(46). The acidic pH of one of the compartments (intracellular side) allowed current measurement from functionally oriented channels (see SI Appendix, Supplementary Information Methods).…”

Geometrical and electrophysiological data of the moving membrane method for the osmotic water permeability of a lipid bilayer

“…Bilayer tension measurements: The method for evaluating the bilayer tension was described in the previous paper [6] . The monolayer tension was evaluated by a conventional method using the Young-Lippmann principle [ 6 , 12 , 13 ], where C bi is the bilayer capacitance, V m is the membrane potential, and θ 0 and θ Vm are the contact angles at 0 mV and V m mV, respectively. Step voltages (+50 to +200 mV with an increment of 50 mV) were applied successively; each time after relaxation, the contact angle between the monolayer in the membrane torus and the bilayer was measured ( Fig.…”