Band 3 (AE1, SLC4A1)-mediated transport of stilbenedisulfonates. I: Functional identification of the proton-activated stilbenedisulfonate influx site.
Stilbenedisulfonates (SD) bind to a "primary" SD (PSD) site on the outer membrane surface of band 3, and inhibit anion exchange (AE) allosterically. Yet, evidence [Membr. Biochem. 2 (1979) 297] suggests that SD can be transported by band 3, thus raising questions about the relative locations of the transport and SD binding sites. A "second" class of DBDS (4,4'-dibenzamido-2,2'-stilbenedisulfonate) binding sites has been discovered, which is activated by protons (pK approximately 5.0), and is located on the membrane domain of band 3 [Biochem. J. 388 (2005) 343]. Here we show that the "second" class of DBDS binding sites, not the PSD site, lies on the SD transport pathway. We compare the pH dependence of DBDS influx to DBDS binding using: (a) control cells, (b) cells selectively crosslinked at the PSD site by treatment with 300 microM BS3 (bis(sulfosuccinimidyl)suberate), and (c) cells with DIDS (4,4'-diisothiocyanato-2,2'-stilbenedisulfonate) bound covalently to the PSD site. DBDS binds to the "second" class of sites on band 3 in all three types of cells. DBDS does not bind to the PSD sites of BS3- or DIDS-modified cells. Proton-activated DBDS influx was observed using control and BS3-modified cells, but not when using DIDS-modified cells. The results with DIDS suggest that the PSD site and the transport site overlap. However, this interpretation is disproved by experiments with BS3-modified cells, where the PSD site is blocked, yet DBDS transport and binding to the "second" class of sites both take place.
Salhany
James M
JM
The Veterans Administration Medical Center, University of Nebraska Medical Center, Omaha, NE 68198-4510, USA. jsalhan@attglobal.net
Cordes
Karen S
KS
Sloan
Renee L
RL
D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000031:analogs & derivatives* / Q000378:metabolism / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport / Q000187:drug effects; D004910:Erythrocyte Membrane / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D011522:Protons
D001457:Anion Exchange Protein 1, Erythrocyte; D011522:Protons; C454201:SLC4A1 protein, human; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C036010:4,4'-dibenzamido-2,2'-stilbenedisulfonic acid