McConnell K R, Aronson P S
Department of Medicine, Yale University School of Medicine, New Haven, Connecticut 06510.
J Biol Chem. 1994 Aug 26;269(34):21489-94.
Cl-formate, Cl-oxalate, and SO4-CO3 exchange participate in Cl and organic anion transport across the brush border membrane of the rabbit proximal tubule. To determine the functional similarity of these transporters to each other and to band 3, we characterized, in isolated membrane vesicles, the inhibition of these transporters by compounds known to inhibit erythrocyte band 3. 4,4'-Dinitro-2,2'-disulfonic stilbene (DNDS), diphenylamine-2-carboxylate (DPC), flufenamate, and 4-aceto-4'-isothiocyano-2,2'-disulfonic stilbene (SITS) were effective inhibitors of Cl-oxalate and SO4-CO3 exchange, suggesting at least some common structural motifs between these exchangers and band 3. Cl-formate exchange was relatively insensitive to DNDS and DPC but sensitive to flufenamate (IC50 = 43 microM). Sensitivity to DNDS but not 4-amino-4'-amino-2,2'-disulfonic stilbene, a feature of band 3, was seen only for the SO4-CO3 exchanger. None of the exchangers had significant affinity for dipyridamole, furosemide, or probenecid. Finally, the presence of DPC or flufenamate increased the IC50 for reversible inhibition by DNDS, consistent with at least a partial overlap between the disulfonic stilbene and diphenylamine carboxylate binding sites of the Cl-oxalate exchanger. We next examined the effect of irreversible SITS binding. The Cl-oxalate exchanger was inhibited 90%, consistent with its high affinity for reversible inhibition by stilbenes. SITS pretreatment caused 50% inhibition of the Cl-formate exchanger, consistent with the reduced affinity of this exchanger for reversible binding of stilbenes. Despite the high sensitivity of the SO4-CO3 exchanger to reversible inhibition by stilbenes, SITS pretreatment caused < 20% irreversible inhibition of this exchanger. Finally, we characterized the stilbene inhibition of the Cl-oxalate exchanger in more detail. The presence of oxalate increased the IC50 for reversible inhibition by DNDS or SITS, implying that oxalate can directly compete at the reversible stilbene binding site of the Cl-oxalate exchanger. However, oxalate could not protect against covalent inactivation of the Cl-oxalate exchanger by SITS, indicating the presence of a separate site for irreversible binding of disulfonic stilbenes. These results suggest a dissociation between the sensitivities of proximal tubule anion exchangers to reversible and irreversible inhibition by disulfonic stilbenes. In contrast to band 3, the Cl-oxalate exchanger must possess separate sites for reversible and irreversible interaction with stilbenes, with only the former overlapping the substrate binding site.
氯离子 - 甲酸根、氯离子 - 草酸根和硫酸根 - 碳酸根交换参与了氯离子和有机阴离子跨兔近端小管刷状缘膜的转运。为了确定这些转运体彼此之间以及与带3蛋白的功能相似性，我们在分离的膜囊泡中，研究了已知可抑制红细胞带3蛋白的化合物对这些转运体的抑制作用。4,4'-二硝基 - 2,2'-二磺酸芪（DNDS）、二苯胺 - 2 - 羧酸盐（DPC）、氟灭酸和4 - 乙酰基 - 4'-异硫氰酸基 - 2,2'-二磺酸芪（SITS）是氯离子 - 草酸根和硫酸根 - 碳酸根交换的有效抑制剂，这表明这些交换体与带3蛋白之间至少存在一些共同的结构基序。氯离子 - 甲酸根交换对DNDS和DPC相对不敏感，但对氟灭酸敏感（IC50 = 43 microM）。仅硫酸根 - 碳酸根交换体表现出对DNDS敏感而对4 - 氨基 - 4'-氨基 - 2,2'-二磺酸芪不敏感，这是带3蛋白的一个特征。这些交换体对双嘧达莫、呋塞米或丙磺舒均无显著亲和力。最后，DPC或氟灭酸的存在增加了DNDS可逆抑制的IC50，这与氯离子 - 草酸根交换体中二磺酸芪和二苯胺羧酸盐结合位点至少部分重叠一致。接下来我们研究了不可逆的SITS结合的影响。氯离子 - 草酸根交换体被抑制了90%，这与其对芪类化合物可逆抑制的高亲和力一致。SITS预处理导致氯离子 - 甲酸根交换体被抑制50%，这与该交换体对芪类化合物可逆结合的亲和力降低一致。尽管硫酸根 - 碳酸根交换体对芪类化合物的可逆抑制高度敏感，但SITS预处理导致该交换体的不可逆抑制小于20%。最后，我们更详细地研究了芪类化合物对氯离子 - 草酸根交换体的抑制作用。草酸根的存在增加了DNDS或SITS可逆抑制的IC50，这意味着草酸根可以在氯离子 - 草酸根交换体的可逆芪类结合位点直接竞争。然而，草酸根不能保护氯离子 - 草酸根交换体免受SITS的共价失活作用，这表明存在一个二磺酸芪不可逆结合的单独位点。这些结果表明近端小管阴离子交换体对二磺酸芪可逆和不可逆抑制的敏感性之间存在差异。与带3蛋白不同，氯离子 - 草酸根交换体必须具有与芪类化合物可逆和不可逆相互作用的单独位点，只有前者与底物结合位点重叠。
