Lysine 539 of human band 3 is not essential for ion transport or inhibition by stilbene disulfonates

A M Garcia,H F Lodish

doi:10.1016/s0021-9258(19)47157-3

A M Garcia, H F Lodish

Open Access

https://doi.org/10.1016/s0021-9258(19)47157-3

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Abstract

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.

Highlights

From thet Whitehead Institute for Biomedical Research and $Departmentof Biology, Massachussetts Institute of Technology, Cambridge, Massachussetts 02122
About 10%of theprotein is present at the DIDS,’ H2-DIDS, and SITS, allows covalent reaction with plasma membrane as determined by immunoprecipi- the amino group of a lysine side chain which is accessible only tation of covalently bound 4,4’-diisothiocyano-2,2’- from the outside of the red blood cell membrane and only disulfonic acid stilbene (DIDS) with anti-DIDS anti- when the transport site is facing outward
Inhibition bystilbene disulfonates or DIDS and otherstilbene disulfonates is modified in the three their covalent binding was not affected whenLys-539 cases mentioned above, theseresults strongly suggest that was substituted by Gln, Pro, Leu, or His

Summary

RESULTS

Resulting complex had a molar ratio of DIDS per KLH. Rabbits Expression of Human Band 3-Total membranes prepared were immunized with 0.4mgof KLH-DIDS in Freund's complete adjuvant, followedbytwo boosts in Freund's incomplete adjuvant. In compared to those on HB3 in erythrocytes. Ues determined for erythrocytes (1).At the concentrations Expression and Properties of Lys-539 Mutants-Fig. 4 used, none of the inhibitors had a significant effect on the shows that oocytes injected with mRNA coding for HB3 with basal anion transport by the oocytes, indicating thatthe five different substitutions of Lys-539 ( K / A ,K / H , KIL, KIP, endogenous frog oocyte anion transport systems have prop- and KIQ lanes), make comparable amounts of a polypeptide erties different from those of band 3.

Niflumic acid

DISCUSSION

Findings

Labeling conditions