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

Abstract

We have previously shown that monoclonal antibody El2 (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 El2 cross-reacts with detergentsolubilized extracts of different tissues from various species by enzyme-linked immunosorbent assay (ELISA). Western blot analysis shows that this monoclonal antibody recognizes proteins of M r 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. Detergentsolubilized protein eluted from an immunoaffinity column and then further purified via FPLC yields a fraction (M, 200,000-220,000) which has been reconstituted into a planar lipid bilayer. There it behaves as a chloride-selective channel (PcJPNa = 20.2 in a 150/50 mM trans-bilayer NaCI 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 M r 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.

cAMP-activated apical membrane chloride channels in Necturus gallbladder epithelium. Conductance, selectivity, and block.

“…The opposite effect, namely, disappearance of C1-channel rectification by dephosphorylation, has been observed in A6 cells (Marunaka and Eaton, 1990). The ~ 62-pS channel purified by Finn and co-workers (Finn et al, 1989;Tsai et al, 1991) was never observed in our patch-clamp experiments.…”

Sucrase-isomaltase is an adenosine 3',5'-cyclic monophosphate–dependent epithelial chloride channel

“…We have previously shown that mAb E12 blocks all cAMP-dependent current in mudpuppy and toad gallbladder, rat colon, cultured human nasal epithelial cells, 6 and mouse colon (Finn AL, unpublished observations), suggesting that this chloride channel is also quite abundant. Indeed, in the mudpuppy gallbladder, this channel must make up a sizable fraction of total apical membrane protein because an increase in cellular cAMP can increase total membrane conductance up to 200-fold, all of which is caused by the appearance of a chloride current that is fully blocked by mAb E12.…”

“…6 -8 These channels have identical kinetic characteristics despite a 5-10-fold dilution of the protein, reducing the likelihood that an undetectable impurity might be the channel molecule. Second, the channel is blocked by the antibody, whether it is reconstituted in lipid bilayers 6,8 or expressed in Xenopus oocytes, whereas CFTR channels are not blocked by the antibody. It is also unlikely that the expression of SI in the heterologous systems described above could be accounted for by "piggybacking" of another protein on SI because at least 2 major characteristics of the expression seen in Figures 3 and 4 (the requirement of phosphorylation and the blockade by mAb E12) are similar to those seen in the bilayer expression system.…”

“…We then obtained and purified a monoclonal antibody (mAb E12) that fully blocks such chloride currents in epithelial cells from mudpuppies to humans and recognizes a protein of approximately 200 kilodaltons (p200). 6 Reconstitution of the purified protein in lipid bilayers yields a 9-pS chloride channel that requires phosphorylation for activity and is blocked by the antibody. 6 -8 In the present study, we used the antibody to purify p200.…”

“…We scraped the cells from the colons of 100 mudpuppies (we have previously shown that this protein is present in colon cells 6 ) into ice-cold Necturus Ringer's solution containing protease inhibitors (1 mmol/L phenylmethylsulfonyl fluoride, 10 mg/mL aprotinin, 0.1 mmol/L leupeptin, and 1 mmol/L pepstatin), homogenized for 1 minute in a VirTis homogenizer (VirTis, Gardiner, NY) and centrifuged at 40,000g for 20 minutes. The supernatant was rotated overnight at 4°C in 10 mmol/L CHAPS and separated into fractions on Sephacryl S-200-HR (Sigma, St. Louis, MO).…”