A nonenzymatic preparation of epithelial basolateral membrane for patch clamp

Abstract

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.

Permeability and Mg2+ blockade of histamine‐operated cation channel in endothelial cells of rat intrapulmonary artery.

“…Such results can be explained if endothelial cells have a polarity and if the K+ channels are mainly located in the basolateral membrane, since experiments showing K+ channels were carried out using isolated cells. Experiments on the basolateral membrane developed recently by Wehner, Garretson, Dawson, Segal & Reuss (1990) would be required to determine such polarity of endothelial cell membrane.…”

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

“…In basolateral membranes of the canine tracheal epithelium (Welsh and McCann, 1985) and apical thick ascending limb of rabbit kidney (Wang, White, Geibel, and Giebisch, 1990) the K channel conductance was only 20-25 pS. The conduction and/or kinetics characteristics differ, however, in some other basolateral K channels: rabbit urinary bladder and distal convoluted renal tubule (Taniguchi, Yoshitomi, and Imai, 1989), Necturus proximal tubule (Hunter, Kawahara, and Giebisch, 1986;Kawahara, Hunter, and Giebisch, 1987;Sackin and Palmer, 1987), intestine (Sheppard, Gir~ldez, and Sept~lveda, 1988) and gallbladder (Wehner, Garretson, Dawson, Segal, and Reuss, 1990), and human nasal airway epithelium (Kunzelmann, Pavenst~dt, Beck, Unal, Emmrich, Arndt, and Greger, 1989a).…”