Lysine 539 of human band 3 is not essential for ion transport or inhibition by stilbene disulfonates.
The anion transporter from human red blood cells, band 3, has been expressed in Xenopus laevis frog oocytes microinjected with mRNA prepared from the cDNA clone. About 10% of the protein is present at the plasma membrane as determined by immunoprecipitation of covalently bound 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS) with anti-DIDS antibody. The expressed band 3 transport chloride at a rate comparable to that in erythrocytes. Transport of chloride is inhibited by stilbene disulfonates, niflumic acid, and dipyridamole at concentrations similar to those that inhibit transport in red blood cells: DIDS and 4,4'-dinitro-2,2'-stilbene disulfonate inhibit chloride uptake with Kiapp of 34 nM and 2.5 microM, respectively. Lysine 539 has been tentatively identified as the site of stilbene disulfonate binding. Site-directed mutagenesis of this lysine to five different amino acids has no effect on transport. Inhibition by stilbene disulfonates or their covalent binding was not affected when Lys-539 was substituted by Gln, Pro, Leu, or His. However, substitution by Ala resulted in weaker inhibition and covalent binding. These results indicate that lysine 539 is not part of the anion transport site and that it is not essential for stilbene disulfonate binding and inhibition.
Garcia
A M
AM
Whitehead Institute for Biomedical Research, Cambridge, Massachusetts.
Lodish
H F
HF
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D001457:Anion Exchange Protein 1, Erythrocyte / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002712:Chlorides / Q000378:metabolism; D004176:Dipyridamole / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism; D005260:Female; D006801:Humans; D007700:Kinetics; D008239:Lysine; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D009865:Oocytes / Q000378:metabolism; D011485:Protein Binding; D013267:Stilbenes / Q000378:metabolism* / Q000494:pharmacology; D014982:Xenopus laevis
D001457:Anion Exchange Protein 1, Erythrocyte; D002712:Chlorides; D015345:Oligonucleotide Probes; D013267:Stilbenes; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; D004176:Dipyridamole; C022306:4,4'-dinitro-2,2'-stilbenedisulfonic acid; D008239:Lysine; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid