The functional role of arginine 901 at the C-terminus of the human anion transporter band 3 protein.
To determine which arginine residues are responsible for band 3-mediated anion transport, we analyzed hydroxyphenylglyoxal (HPG)-modified band 3 protein in native erythrocyte membranes. HPG-modification leads to inhibition of the transport of phosphoenolpyruvate, a substrate for band 3-mediated transport. We analyzed the HPG-modified membranes by reverse phase-HPLC, and determined that arginine 901 was modified by HPG. To determine the role of Arg 901 in the conformational change induced by anion exchange, we analyzed HPG-modification of the membranes when 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethypyrocarbonate (DEPC) was present. DNDS and DEPC fix band 3 in the outward and inward conformations, respectively. HPG-modification was unaffected in the presence of DEPC but decreased in the presence of DNDS. In addition to that, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), which specifically reacts with the outward conformation of band 3, did not react with HPG-modified membranes. Furthermore, we expressed a band 3 mutant in which Arg 901 was replaced by alanine (R901A) on yeast membranes. The kinetic parameters indicated that the R901A mutation affected the rate of conformational change of the band 3 protein. From these results, we conclude that the most C-terminal arginine, Arg 901, has a functional role in the conformational change that is necessary for anion transport.
Takazaki
Shinya
S
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582.
Abe
Yoshito
Y
Kang
Donchon
D
Li
Chunyan
C
Jin
Xiuri
X
Ueda
Tadashi
T
Hamasaki
Naotaka
N
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D001457:Anion Exchange Protein 1, Erythrocyte / Q000737:chemistry* / Q000378:metabolism*; D027321:Anion Transport Proteins / Q000737:chemistry* / Q000378:metabolism*; D001120:Arginine / Q000737:chemistry* / Q000378:metabolism*; D006801:Humans; D017136:Ion Transport / Q000187:drug effects / Q000502:physiology; D010658:Phenylglyoxal / Q000737:chemistry / Q000494:pharmacology; D013329:Structure-Activity Relationship
D001457:Anion Exchange Protein 1, Erythrocyte; D027321:Anion Transport Proteins; D001120:Arginine; D010658:Phenylglyoxal; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid