Reconstitution of an epithelial chloride channel. Conservation of the channel from mudpuppy to man.

• DOI: 10.1085/jgp.98.4.723
• Publication Date: 2004-05-13T23:48:59Z
• Authors: L M Tsai; M Dillard; R L Rosenberg; R J Falk; M L Gaido; A L Finn
• Keywords: Chloride channel; Apical membrane

We have previously shown that monoclonal antibody E12 (MAb E12), one of several such antibodies raised against theophylline-treated Necturus gallbladder (NGB) epithelial cells, inhibits the chloride conductance in apical membrane tissue. Since channels are critical to secretory function epithelia many different animals, we used this determine whether conserved, and an immunoaffinity column isolate channel protein. now demonstrate MAb cross-reacts with detergent-solubilized extracts tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows recognizes proteins Mr 219,000 NGB, toad gallbladder, urinary bladder, small intestine, A6 rat colon, rabbit gastric mucosa, human lymphocytes, nasal epithelium. Detergent-solubilized protein eluted then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into planar lipid bilayer. There it behaves as chloride-selective (PCl/PNa = 20.2 150/50 mM trans-bilayer NaCl gradient) whose unit is 62.4 +/- 4.6 pS, blocked bilayer antibody. The gating characteristics indicate can exist aggregates or independent single channels, interferes aggregates, leaving unchanged. From these data conclude recognized important component channel, conserved across wide range animal species.