Hallén S, Björquist A, Ostlund-Lindqvist A M, Sachs G
UCLA and the Wadsworth Veterans Administration Hospital, Los Angeles, California 90073, USA.
Biochemistry. 2002 Dec 17;41(50):14916-24. doi: 10.1021/bi0205404.
Drug intervention that prevents reabsorption of circulating bile acids by the apical (ileal) sodium/bile acid cotransporter (ASBT) may be a promising new therapy for lowering of plasma cholesterol. 2164U90 is a benzothiazepine-based competitive inhibitor of bile acid transport with K(i) values of approximately 10 and 0.068 microM for the homologous human and mouse apical transporters, respectively. Hybrid human-mouse and mouse-human transporters were engineered to identify regions involved in this 150-fold difference in the inhibition constant for 2164U90. A mouse-human chimera with only the most C-terminal hydrophobic domain and the C-terminus of the transporter originating from the human variant was found to have a sensitivity to 2164U90 inhibition similar to that of the human transporter. Conversely, a human-mouse hybrid transporter encompassing the same C-terminal region from the mouse sequence but now inserted into the human sequence demonstrated the greater inhibition seen with the mouse wild type ASBT. Amino acid substitutions, individually or in combinations, of six candidate nonconserved residues between mouse and human transporters in this C-terminal domain showed replacements of Thr294 by Ser and Val295 by Ile to be responsible for the difference in the sensitivity toward 2164U90 seen between the species. The hamster apical SBAT encompassing Ser/Ile in these positions shared the lower sensitivity to 2164U90, as seen with the human ASBT, even though it is identical to the mouse SBAT in the remaining four positions of this region. In addition, the rat ASBT which is identical to the mouse ASBT in this domain also had the high sensitivity to 2164U90 inhibition found for the mouse ASBT. Methanethiosulfonates (MTS) are known to inactivate the sodium/bile acid transporters through alkylation of a cysteine in the most C-terminal hydrophobic domain (1). Inactivation of the human ASBT due to MTS modification of cysteine 270 was shown to be largely abolished when the transporter was preincubated with 2164U90, suggesting that the binding of this benzothiazepine is in the vicinity of position 270. Thus, the domain containing the two most C-terminal putative transmembrane regions of the SBATs, H8-H9, previously shown to constitute part of the binding pocket for bile acids, interacts also with the bile acid transport competitive inhibitor, 2164U90.
通过顶端（回肠）钠/胆汁酸共转运体（ASBT）阻止循环胆汁酸重吸收的药物干预可能是一种有前景的降低血浆胆固醇的新疗法。2164U90是一种基于苯并噻氮䓬的胆汁酸转运竞争性抑制剂，对同源的人类和小鼠顶端转运体的K(i)值分别约为10和0.068微摩尔。构建了人-鼠杂交和鼠-人转运体，以确定参与2164U90抑制常数中150倍差异的区域。发现一种仅具有最末端疏水结构域且转运体C末端源自人类变体的鼠-人嵌合体对2164U90抑制的敏感性与人类转运体相似。相反，一个包含来自小鼠序列相同C末端区域但现在插入人类序列的人-鼠杂交转运体表现出与小鼠野生型ASBT相同的更大抑制作用。在这个C末端结构域中，对小鼠和人类转运体之间六个候选非保守残基进行单个或组合的氨基酸替换，结果显示Thr294被Ser替换以及Val295被Ile替换是造成物种间对2164U90敏感性差异的原因。在这些位置包含Ser/Ile的仓鼠顶端SBAT与人类ASBT一样，对2164U90的敏感性较低，尽管它在该区域的其余四个位置与小鼠SBAT相同。此外，在这个结构域中与小鼠ASBT相同的大鼠ASBT对2164U90抑制也具有高敏感性。已知甲硫基磺酸盐（MTS）通过使最末端疏水结构域中的半胱氨酸烷基化来使钠/胆汁酸转运体失活（1）。当转运体与2164U90预孵育时，由于半胱氨酸270的MTS修饰导致的人类ASBT失活在很大程度上被消除，这表明这种苯并噻氮䓬的结合在位置270附近。因此，先前显示构成胆汁酸结合口袋一部分的SBATs的包含两个最末端假定跨膜区域的结构域H8-H9，也与胆汁酸转运竞争性抑制剂2164U90相互作用。
