Morita Shin-ya, Kobayashi Aya, Takanezawa Yasukazu, Kioka Noriyuki, Handa Tetsurou, Arai Hiroyuki, Matsuo Michinori, Ueda Kazumitsu
Laboratory of Cellular Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Hepatology. 2007 Jul;46(1):188-99. doi: 10.1002/hep.21591.
Human ABCB4 (multidrug resistance [MDR]3 P-glycoprotein) is expressed in the canalicular membrane of the hepatocyte. ABCB4 has been shown to be required for phosphatidylcholine (PC) secretion into the bile and to translocate PC across the plasma membrane. To further investigate the function of ABCB4, we established a cell line stably expressing ABCB4 (human embryonic kidney [HEK]/ABCB4). The efflux of phospholipids from HEK/ABCB4 cells was remarkably increased by the addition of taurocholate. In addition, the cholesterol efflux from HEK/ABCB4 cells was also enhanced in the presence of taurocholate. Light scattering measurements suggested that the taurocholate monomer plays an important role in ABCB4-mediated lipid secretion. On the other hand, the efflux of phospholipids and cholesterol was not mediated by ABCB1 (MDR1) even in the presence of taurocholate. Taurocholate promoted the efflux of phospholipids and cholesterol from HEK/ABCB4 cells more efficiently than glycocholate and cholate. ABCB4-K435M and ABCB4-K1075M, Walker A lysine mutants, did not mediate the phospholipid and cholesterol efflux in the presence of taurocholate, suggesting that ATP hydrolysis is essential for the efflux. Verapamil completely inhibited the taurocholate-dependent efflux of phospholipids and cholesterol from HEK/ABCB4 cells. Mass spectrometry revealed that, in the presence of taurocholate, HEK/ABCB4 cells preferentially secreted PC compared to sphingomyelin. PC vesicles induced cholesterol diffusion from cell membrane, but did not accept cholesterol from ABCB4.
ABCB4 mediates the efflux of phospholipids into the canalicular lumen in the presence of bile salts, and plays a crucial role in bile formation and lipid homeostasis.
人ABCB4（多药耐药[MDR]3 P-糖蛋白）在肝细胞的胆小管膜中表达。ABCB4已被证明是磷脂酰胆碱（PC）分泌到胆汁中以及将PC转运穿过质膜所必需的。为了进一步研究ABCB4的功能，我们建立了稳定表达ABCB4的细胞系（人胚肾[HEK]/ABCB4）。添加牛磺胆酸盐后，HEK/ABCB4细胞中磷脂的流出显著增加。此外，在牛磺胆酸盐存在下，HEK/ABCB4细胞的胆固醇流出也增强。光散射测量表明，牛磺胆酸盐单体在ABCB4介导的脂质分泌中起重要作用。另一方面，即使在牛磺胆酸盐存在下，磷脂和胆固醇的流出也不是由ABCB1（MDR1）介导的。牛磺胆酸盐比甘氨胆酸盐和胆酸盐更有效地促进了HEK/ABCB4细胞中磷脂和胆固醇的流出。沃克A赖氨酸突变体ABCB4-K435M和ABCB4-K1075M在牛磺胆酸盐存在下不介导磷脂和胆固醇的流出，表明ATP水解对于流出至关重要。维拉帕米完全抑制了牛磺胆酸盐依赖性的HEK/ABCB4细胞中磷脂和胆固醇的流出。质谱分析表明，在牛磺胆酸盐存在下，与鞘磷脂相比，HEK/ABCB4细胞优先分泌PC。PC囊泡诱导胆固醇从细胞膜扩散，但不接受来自ABCB4的胆固醇。
ABCB4在胆盐存在下介导磷脂向胆小管腔的流出，并在胆汁形成和脂质稳态中起关键作用。
