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Renal basolateral membrane anion transporter characterized by a fluorescent disulfonic stilbene

The Journal of Membrane Biology volume 100, pages 1–12 (1987)Cite this article

Summary

The fluorescence enhancement of 4,4′-dibenzamido-2,2′-disulfonic stilbene (DBDS) upon binding to membranes was used to examine proximal tubule stilbene binding sites. Equilibrium binding studies of DBDS to renal brush border (BBMV) and basolateral membrane vesicles (BLMV) were performed using a fluorescence enhancement technique developed for red blood cells (A.S. Verkman, J.A. Dix and A.K. Solomon,J. Gen. Physiol. 81:421–449, 1983). In the absence of transportable anions, DBDS bound reversibly to a single class of sites on BLMV isolated from rabbit (K d =3.8 μm) and rat (3.2 μm); 100 μm dihydro-4,4′-diisothiocyano-2,2′-disulfonic stilbene (H2DIDS) blocked >95% of binding. H2DIDS inhibitable DBDS binding was not detected using rat or rabbit BBMV. In rabbit BLMV, DBDSK d doubled with 10mm SO4, 50mm HCO3 and 100mm Cl, but was not altered by Na or pH (6–8). In stopped-flow experiments the exponential time constant for DBDS binding slowed with SO4, HCO3 and Cl, but was unaffected by Na. These results are consistent with competitive binding of DBDS and anions at an anion transport site. To relate DBDS binding data to anion transport inhibition we used35SO4 uptake to characterize several modes of rabbit BLM anion transport: H/SO4 and Na/SO4 cotransport, and Cl/SO4 countertransport. Each transport process was electroneutral and was inhibited by H2DIDS, furosemide, probenecid, chlorothiazide and DBDS. The apparentK t 's for DBDS (3–20 μm) were similar toK d for DBDS binding. These studies define a class of anion transport sites on the proximal tubule basolateral membrane measureable optically by a fluorescent stilbene.

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Abbreviations

BBMV:

brush border membrane vesicle

Bicin:

N,N′-bis(2-hydroxyethyl)glycine

Bis-Tris:

bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane

BLMV:

basolateral membrane vesicle

CCCP:

carbonylcyanide-m-chlorophenylhydrazone

DBDS:

4,4′-dibenzamido-2,2′-disulfonic stilbene

DIDS:

4,4′-diisothiocyano-2,2′-disulfonic stilbene

H2DIDS:

dihydro-4,4′-diisothiocyano-2,2′-disulfonic stilbene

HEPES:

4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid

NMG:

N-methylglucamine

SITS:

4-acetamido-4′-isothiocyano-2,2′-disulfonic stilbene

Tris:

tris(hydroxymethyl)aminomethane

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Author notes

  1. Pei-Yuan Chen

    Present address: Department of Internal Medicine, Taipei Medical College, 252 Wu-Hsing St., Taipei, Taiwan

Affiliations

  1. Division of Nephrology, Cardiovascular Research Institute, University of California, 94143, San Francisco, California

    Pei-Yuan Chen & A. S. Verkman

Authors
  1. Pei-Yuan Chen
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  2. A. S. Verkman
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Chen, PY., Verkman, A.S. Renal basolateral membrane anion transporter characterized by a fluorescent disulfonic stilbene. J. Membrain Biol. 100, 1–12 (1987). https://doi.org/10.1007/BF02209135

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  • DOI: https://doi.org/10.1007/BF02209135

Key Words

  • proximal tubule
  • basolateral membrane
  • anion transport
  • disulfonic stilbene
  • fluorescence