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.
A simple theoretical model is formulated which describes dynamical features of the interaction of two-level atoms with a continuum of radiation oscillators. In particular, this paper is concerned with resonance scattering by single atoms in the presence of an external classical field, and spontaneous emission by a system of identical atoms. In the case of resonance scattering, the response of an atom to rapid changes of the amplitude of the external field is studied. The model for spontaneous emission by a system of atoms is based on the assumptions that all atoms have the same resonance frequency, and that the atoms are contained in a volume whose extension is small compared with the wavelength of the emitted radiation. The complete evolution of a system of m atoms is determined by a set of coupled first-order linear differential equations, and explicit solutions are presented for the values m = 1, 2, 3, 4, and 8. Pair correlations of the atoms and photon-number fluctuations are discussed. An approximation for resonance fluorescence by a system of atoms with a broad distribution of resonance frequencies is included in an Appendix.
A native chloride channel in Necturus gallbladder epithelial cells is opened by a theophylline-induced rise in cellular cyclic AMP and is closed by removal of theophylline or by addition of specific antibody; however, it does not close if okadaic acid, an inhibitor of protein phosphatases 1 and 2A, is added. The purified channel reconstituted into lipid bilayers closes upon the addition of protein phosphatase 2A and is reopened by the addition of Mg-ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. These results indicate that the channel protein is purified in a phosphorylated state and that its functional characteristics are at least partly controlled by direct phosphorylation and dephosphorylation.
We have purified a protein from Necturus maculosus gallbladder cells that forms chloride channels in an artificial membrane. The same protein apparently can form channels that are highly selective for chloride but can have conductances varying from 9 to about 150 pS. The highconductance channels are blocked by the monoclonal antibody used to purify the protein, but this antibody has no effect on the 9-pS channels. The observation that gating of the low-and high-conductance states is independent and that the antibody affects only the latter has implications regarding the control of chloride conductance in cell membranes and the different types of channels described in those cells.
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