Reconstitution of an epithelial chloride channel. Conservation of the channel from mudpuppy to man.
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 the apical membrane of that tissue. Since chloride channels are critical to the secretory function of epithelia in many different animals, we have used this antibody to determine whether the channels are conserved, and in an immunoaffinity column to isolate the channel protein. We now demonstrate that MAb E12 cross-reacts with detergent-solubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of Mr 219,000 in NGB, toad gallbladder, urinary bladder, and small intestine, A6 cells, rat colon, rabbit gastric mucosa, human lymphocytes, and human nasal epithelial cells, and inhibits the chloride conductance in toad gallbladder, rat colon, and human nasal epithelium. Detergent-solubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (Mr 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PCl/PNa = 20.2 in a 150/50 mM trans-bilayer NaCl gradient) whose unit conductance is 62.4 +/- 4.6 pS, and which is blocked in the bilayer by the antibody. The gating characteristics of this channel indicate that it can exist as aggregates or as independent single channels, and that the antibody interferes with gating of the aggregates, leaving the unit channels unchanged. From these data we conclude that the protein of Mr 219,000 recognized by this monoclonal antibody is an important component of an epithelial chloride channel, and that this channel is conserved across a wide range of animal species.
Tsai
L M
LM
Department of Physiology, School of Medicine, University of North Carolina, Chapel Hill 27599.
Dillard
M
M
Rosenberg
R L
RL
Falk
R J
RJ
Gaido
M L
ML
Finn
A L
AL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D015153:Blotting, Western; D002024:Bufo marinus / Q000502:physiology; D002460:Cell Line / Q000502:physiology; D018118:Chloride Channels; D002846:Chromatography, Affinity; D003429:Cross Reactions; D004553:Electric Conductivity / Q000502:physiology; D004847:Epithelial Cells; D004848:Epithelium / Q000502:physiology*; D006801:Humans; D016285:Iloprost / Q000494:pharmacology; D015640:Ion Channel Gating / Q000502:physiology*; D008214:Lymphocytes / Q000502:physiology; D008565:Membrane Proteins / Q000187:drug effects / Q000302:isolation & purification / Q000502:physiology*; D008567:Membranes, Artificial; D009337:Necturus / Q000502:physiology*; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains / Q000502:physiology; D013806:Theophylline / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D018118:Chloride Channels; D008565:Membrane Proteins; D008567:Membranes, Artificial; D013806:Theophylline; D016285:Iloprost