title: Interactions between anion exchange and other membrane proteins in rabbit kidney medullary collecting duct cells.
abstract: In separated outer medullary collecting duct (MCD) cells, the time course of binding of the fluorescent stilbene anion exchange inhibitor, DBDS (4,4'-dibenzamido-2,2'-stilbene disulfonate), to the MCD cell analog of band 3, the red blood cell (rbc) anion exchange protein, can be measured by the stopped-flow method and the reaction time constant, tau TDBDS, can be used to report on the conformational state of the band 3 analog. In order to validate the method we have now shown that the ID50D,DBDS,MCD (0.5 +/- 0.1 microM) for the H2-DIDS (4,4'-diisothiocyano-2,2'-dihydrostilbene disulfonate) inhibition of tau DBDS is in agreement with the ID50,Cl-MCD (0.94 +/- 0.07 microM) for H2-DIDS inhibition of MCD cell Cl- flux, thus relating tau DBDS directly to anion exchange. The specific cardiac glycoside cation transport inhibitor, ouabain, not only modulates DBDS binding kinetics, but also increases the time constant for Cl- exchange by a factor of two, from tau Cl- = 0.30 +/- 0.02 sec to 0.56 +/- 0.06 sec (30 mM NaHCO3). The ID50,DBDS,MCD for the ouabain effect on DBDS binding kinetics is 0.003 +/- 0.001 microM, so that binding is about an order of magnitude tighter than that for inhibition of rbc K+ flux (KI,K+,rbc = 0.017 microM). These experiments indicate that the Na+,K+-ATPase, required to maintain cation gradients across the MCD cell membrane, is close enough to the band 3 analog that conformational information can be exchanged. Cytochalasin E (CE), which binds to the spectrin/actin complex in rbc and other cells. modulates DBDS binding kinetics with a physiological ID50,DBDS,MCD (0.076 +/- 0.005 microM); 2 microM CE also more than doubles the Cl- exchange time constant from 0.20 +/- 0.04 sec to 0.50 +/- 0.08 sec (30 mM NaHCO3). These experiments indicate that conformational information can also be exchanged between the MCD cell band 3 analog and the MCD cell cytoskeleton.
authors.affiliation: Biophysical Laboratory, Harvard Medical School, Boston, Massachusetts 02115.
authors.forename: A
authors.initials: A
authors.lastname: Janoshazi
authors.forename: J L
authors.initials: JL
authors.lastname: Seifter
authors.forename: A K
authors.initials: AK
authors.lastname: Solomon
mesh_terms: D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000031:analogs & derivatives*; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D027321:Anion Transport Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D002712:Chlorides / Q000378:metabolism; D003572:Cytochalasins / Q000494:pharmacology; D066298:In Vitro Techniques; D007684:Kidney Tubules / Q000378:metabolism*; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism*; D008954:Models, Biological; D010042:Ouabain / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D013050:Spectrometry, Fluorescence
chemical_list: D027321:Anion Transport Proteins; D002352:Carrier Proteins; D002712:Chlorides; D003572:Cytochalasins; D008565:Membrane Proteins; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C007215:cytochalasin E; D010042:Ouabain; C018564:dihydro-DIDS; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid