Controlled delivery of membrane proteins to artificial lipid bilayers by nystatin–ergosterol modulated vesicle fusion

Abstract

The study of ion channels and other membrane proteins and their potential use as biosensors and drug screening targets require their reconstitution in an artificial membrane. These applications would greatly benefit from microfabricated devices in which stable artificial lipid bilayers can be rapidly and reliably formed. However, the amount of protein delivered to the bilayer must be carefully controlled. A vesicle fusion technique is investigated where composite ion channels of the polyene antibiotic nystatin and the sterol ergosterol are employed to render proteincarrying vesicles fusogenic. After fusion with an ergosterol-free artificial bilayer, the nystatinergosterol channels do not dissociate immediately and thus cause a transient current signal that marks the vesicle fusion event. Experimental pitfalls of this method were identified, the influence of the nystatin and ergosterol concentration on the fusion rate and the shape of the fusion event marker was explored, and the number of different lipid species was reduced. Under these conditions, the b-amyloid peptide could be delivered in a controlled manner to a standard planar bilayer. Additionally, electrical recordings were obtained of vesicles fusing with a planar lipid bilayer in a microfabricated device, demonstrating the suitability of nystatin-ergosterol modulated vesicle fusion for protein delivery within microsystems.

Drunken Membranes: Short-Chain Alcohols Alter Fusion of Liposomes to Planar Lipid Bilayers

“…This assay has been used continuously in a variety of labs for fusion/reconstruction studies (48)(49)(50)(51)(54)(55)(56)(57)(58)(59)(60). Liposome fusion to planar bilayers was measured using standard electrophysiological equipment (Bilayer Clamp Amplifier model BC-535 or BC-525C, Warner Instruments, Hamden CT).…”

Shaped Apertures in Photoresist Films Enhance the Lifetime and Mechanical Stability of Suspended Lipid Bilayers

“…Electrical measurements of ion permeation are the most powerful methods to characterize ion channel function, especially at the single-channel level, and its modulation by agonists and antagonists, including pharmacological drugs (1,2). Of the two main electrophysiology variations, patch clamping of natural cell membranes relies on cellular expression and membrane incorporation of the channel of interest, at a low background of endogenous channels (3,4), whereas bilayer lipid membrane (BLM) electrophysiology typically involves incorporation, by proteoliposome fusion, of a purified ion channel into a planar bilayer of synthetic phospholipids (5)(6)(7)(8). Although the BLM method has some distinct advantages, most notably the possibility to obtain the channel from any desired expression system and control over the lipid composition of a bilayer that does not contain any other ion channels, it suffers from low throughput due to the fragility of the planar bilayers.…”

Microfluidic array platform for simultaneous lipid bilayer membrane formation

“…difficult to target single channels, limited or no possible exchange of internal medium composition). Suspended and supported BLM platforms can allow for the quick and reliable formation of BLMs (Le Pioufle et al, 2008;Sandison et al, 2007a,b), but generally more complex protocols than the ones used in tethered bilayer systems are required for the delivery of large, non-spontaneously inserting proteins (Zagnoni et al, 2007;De Planque et al, 2006).…”