Giant lipid vesicles impaled with glass microelectrodes: GigaOhm seal by membrane spreading.
Giant unilamellar lipid vesicles could be perfect systems to study ion channels in the environment of lipid membranes with defined chemical and physical properties. Prerequisite for electrical measurements is an intravesicular electrical contact. We describe the impalement of giant lipid vesicles by glass micropipet electrodes with a tight seal. To avoid displacement or burst during impalement, the vesicles are immobilized in relaxed conditions by microscopic picket fences of polyimide. The outer surface of the pipets is selectively coated with silanes or polylysine. Structurally, the impalement is verified by ejecting a fluorescent solution out of the pipet. For electrical characterization, current pulses are applied to the pipet and voltage transients are recorded. The data are evaluated in terms of the capacitance and effective resistance of the membrane. Directly after impalement, we observe a seal resistance up to 1.2 GOmega that continuously decays within a period of up to 20 min until it suddenly disappears without burst of the vesicle. During impalement, a spreading of the vesicle membrane along the outer surface of the pipets is observed using a fluorescent membrane-bound dye. We assign the tight pipet-vesicle contact to spreading of the lipid bilayer by a rolling mechanism and the loss of resistance to micro- and macropores that are induced by the resulting membrane tension. Limitation of spreading is attempted with barriers on the pipet.
Reccius
Christian H
CH
Department of Membrane and Neurophysics, Max Planck Institute for Biochemistry, Martinsried/München, Germany 82152.
Fromherz
Peter
P
D017097:Electric Impedance; D004563:Electrochemistry; D005898:Glass / Q000737:chemistry; D007473:Ion Channels / Q000502:physiology; D008055:Lipids / Q000737:chemistry*; D008563:Membrane Lipids / Q000737:chemistry* / Q000502:physiology; D008839:Microelectrodes; D010316:Particle Size; D012825:Silicon / Q000737:chemistry; D013499:Surface Properties; D013997:Time Factors
D007473:Ion Channels; D008055:Lipids; D008563:Membrane Lipids; D012825:Silicon