Nystatin-induced liposome fusion. A versatile approach to ion channel reconstitution into planar bilayers.
A simple method is described for promoting and detecting fusion of liposomes with planar bilayer membranes. Liposomes containing ergosterol are doped with the pore-forming antibiotic nystatin, and the planar bilayer is kept ergosterol-free. Under these conditions, when a transbilayer salt gradient is applied, liposomes added to the high-salt side of the bilayer elicit the appearance of abrupt conductance jumps of 5-300 pS. The increase in conductance is transient, decaying back to baseline on the order of 10 s. Each of these "spikes" represents the fusion of a single liposome with the bilayer, resulting in the simultaneous insertion of many nystatin channels. Relaxation of the conductance back to baseline occurs because ergosterol, required for the integrity of the nystatin pore, diffuses away into the sterol-free planar bilayer after liposome fusion. When Torpedo Cl- channels are reconstituted into liposomes containing ergosterol and nystatin, fusion spikes are observed simultaneously with the appearance of Cl- channels. This method allows the calculation of the density of functional ion channels in a preparation of proteoliposomes containing reconstituted channel protein.
Woodbury
D J
DJ
Howard Hughes Medical Institute, Graduate Department of Biochemistry, Brandeis University, Waltham, Massachusetts 02254.
Miller
C
C
D055592:Biophysical Phenomena; D001703:Biophysics; D018118:Chloride Channels; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008561:Membrane Fusion; D008565:Membrane Proteins; D009761:Nystatin
D018118:Chloride Channels; D007473:Ion Channels; D008051:Lipid Bilayers; D008081:Liposomes; D008565:Membrane Proteins; D009761:Nystatin