Identification of a membrane protein from T84 cells using antibodies made against a DIDS-binding peptide.
The outwardly rectified chloride channel of secretory epithelial cells is inhibited by disulfonic stilbene (DS) compounds such as 4,4'-diisothiostilbene-2,2'-disulfonic acid (DIDS) [R. J. Bridges, R. T. Worrell, R. A. Frizzell, and D. J. Benos, Am. J. Physiol. 256 (Cell Physiol. 25): C902-C912, 1989]. A 13-amino acid peptide (P49) corresponding to the putative DS binding site region of the murine anion exchange protein was synthesized, and polyclonal antibodies were generated against it and then purified over a P49 affinity column. The resulting monospecific antibodies reacted on Western blots with a 95- to 100-kDa protein from human erythrocytes and a 55- to 60-kDa protein from the human colonic tumor cell line, T84. The reaction with T84 protein did not appear to represent recognition of an anion exchanger because anion efflux from T84 cells was independent of external Cl-. In addition, monoclonal antibodies raised against human band 3 recognized the band 3 protein in human red cell ghost preparations but recognized nothing in T84 cell membrane preparations. In T84 cells, DIDS protected the 60-kDa protein from antibody binding. The anti-P49 antibody blocked outwardly rectified Cl- channels incorporated into planar lipid bilayer membranes from rat colon. Immunocytochemical data reveal specific binding of the anti-P49 antibody to perinuclear cytoplasmic vesicles. Forskolin caused these antibody-labeled vesicles to migrate from the perinuclear region to the plasma membrane under conditions and with a time course identical to that seen for stimulation of Cl- transport in these cells. Our results suggest that the protein may be a part of a chloride channel complex of secretory epithelial cells.
Sorscher
E J
EJ
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Fuller
C M
CM
Bridges
R J
RJ
Tousson
A
A
Marchase
R B
RB
Brinkley
B R
BR
Frizzell
R A
RA
Benos
D J
DJ
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000378:metabolism; D000906:Antibodies; D001665:Binding Sites; D002712:Chlorides / Q000378:metabolism; D005576:Colforsin / Q000494:pharmacology; D003110:Colonic Neoplasms; D000242:Cyclic AMP / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D007455:Iodine / Q000493:pharmacokinetics; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000378:metabolism*; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000648:ultrastructure
D000906:Antibodies; D002712:Chlorides; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D010455:Peptides; D005576:Colforsin; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D007455:Iodine; D000242:Cyclic AMP; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid