A nonenzymatic preparation of epithelial basolateral membrane for patch clamp.
A preparation has been developed that permits patch clamping of the basolateral membrane of Necturus gallbladder epithelial cells with a high success rate. The epithelium is separated from the underlying tissues mechanically, without enzymatic treatment. Its apical surface is attached to a plastic cover slip, and the basolateral surface, facing up, is cleaned with a suction pipette under microscopic observation. With this cleaning procedure, the success rate in obtaining gigaohm seals increases from less than 1% to approximately 10% of the attempts. The cells appear to retain their structural and functional integrity, as evidenced by electron-microscopic appearance and magnitude of cell membrane voltages. Major advantages of the preparation are that the basolateral membrane domain is preserved and that enzymatic treatment, which could potentially alter membrane proteins, is not necessary.
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
F
F
Wehner
L
L
Garretson
K
K
Dawson
Y
Y
Segal
L
L
Reuss
D000818:Animals; D002462:Cell Membrane / Q000502:physiology* / Q000648:ultrastructure; D004594:Electrophysiology / Q000379:methods; D004848:Epithelium / Q000502:physiology* / Q000648:ultrastructure; D005704:Gallbladder / Q000502:physiology; D066298:In Vitro Techniques; D007473:Ion Channels / Q000502:physiology; D008564:Membrane Potentials; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D009337:Necturus
D007473:Ion Channels