Biophysics and Structure of the Patch and the Gigaseal

Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ comp...

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Bibliographic Details
Published in:Biophysical journal 2009-08, Vol.97 (3), p.738-747
Main Authors: Suchyna, Thomas M., Markin, Vladislav S., Sachs, Frederick
Format: Article
Language:English
Subjects:
Animals
Astrocytes - physiology
Biophysics
Cations
Cell Line
Cell Membrane - physiology
Cells
Cells, Cultured
Cellular Biophysics and Electrophysiology
Channels
Creep (materials)
Cytoskeleton - physiology
Elasticity - physiology
Electric potential
Electroosmosis
Humans
Hydrogen-Ion Concentration
Ions
Kinetics
Lipids
Membrane Potentials - physiology
Membrane Proteins - metabolism
Membranes
Mice
Muscle Fibers, Skeletal - physiology
Patch-Clamp Techniques - methods
Pipettes
Potassium Chloride - metabolism
Proteins
Rats
Saline
Seals
Voltage
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Summary:Interpreting channel behavior in patches requires an understanding of patch structure and dynamics, especially in studies of mechanosensitive channels. High resolution optical studies show that patch formation occurs via blebbing that disrupts normal membrane structure and redistributes in situ components including ion channels. There is a 1–2 μm region of the seal below the patch where proteins are excluded and this may consist of extracted lipids that form the gigaseal. Patch domes often have complex geometries with inhomogeneous stresses due to the membrane-glass adhesion energy (Ea), cytoskeletal forces, and possible lipid subdomains. The resting tension in the patch dome ranges from 1–4 mN/m, a significant fraction of the lytic tension of a bilayer (∼10 mN/m). Thus, all patch experiments are conducted under substantial, and uneven, resting tension that may alter the kinetics of many channels. Ea seems dominated by van der Waals attraction overlaid with a normally repulsive Coulombic force. High ionic strength pipette saline increased Ea and, surprisingly, increased cytoskeletal rigidity in cell-attached patches. Low pH pipette saline also increased Ea and reduced the seal selectivity for cations, presumably by neutralizing the membrane surface charge. The seal is a negatively charged, cation selective, space with a resistance of ∼7 gigohm/μm in 100 mM KCl, and the high resistivity of the space may result from the presence of high viscosity glycoproteins. Patches creep up the pipette over time with voltage independent and voltage dependent components. Voltage-independent creep is expected from the capillary attraction of Ea and the flow of fresh lipids from the cell. Voltage-dependent creep seems to arise from electroosmosis in the seal. Neutralization of negative charges on the seal membrane with low pH decreased the creep rate and reversed the direction of creep at positive pipette potentials.
ISSN:0006-3495
1542-0086
DOI:10.1016/j.bpj.2009.05.018