AJP - Cell Physiology, Vol 258, Issue 6 C1159-C1164, Copyright © 1990 by American Physiological Society
A nonenzymatic preparation of epithelial basolateral membrane for patch clamp
F. Wehner, L. Garretson, K. Dawson, Y. Segal and L. Reuss
Department of Physiology and Biophysics, University of Texas Medical Branch, Galveston 77550.
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.
