Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease
Retinal stimulates ATP hydrolysis by purified and reconstituted ABCR, the photoreceptor-specific ATP-binding cassette transporter responsible for Stargardt disease
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DOI：
10.1074/jbc.274.12.8269
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发表时间：
1999-03-19
影响因子：
4.8
通讯作者：
Nathans, J
中科院分区：
文献类型：
--
作者：
Sun, H;Molday, RS;Nathans, J
P-糖蛋白是一种ABC转运蛋白，在哺乳动物细胞中介导多药耐药，其许多底物在体外可刺激其ATP酶活性。在本研究中，我们使用此属性作为标准，以寻找天然和人工基板和/或变构调节ABCR，杆光感受器特异性ABC转运负责Stargardt病，早发性黄斑变性。从牛视杆外节中免疫亲和纯化ABCR至表观均一性，并将其重组到脂质体中。候选配体全反式视黄醇刺激ABCR的ATP酶活性3-4倍，在10-15 μ M时具有半数最大效应。11-顺式-和13-顺式-视黄醛显示相似的活性。全反式视黄醇刺激ABCR的ATP酶活性，具有Michaelis-Menten行为，表明与ATP水解途径中的限速酶-底物中间体相关的简单非合作结合。在37种结构不同的非类维生素A化合物中，包括9种以前表征的P-糖蛋白底物或敏化剂，当在20 μ M下测试时，只有4种显示出显著的ATP酶刺激。这四种化合物的剂量-反应曲线表明与ABCR的多个结合位点和/或相互作用模式，其中两种化合物，胺碘酮和毛地黄皂苷，可以与全反式视黄醛协同作用，这意味着它们与ABCR上的一个或多个位点相互作用不同于全反式视黄醛相互作用的位点，与视黄醛不同，胺碘酮似乎与游离的和ATP结合的ABCR相互作用，结合Stargardt病的临床观察和ABCR定位于视杆外段椎间盘膜，这些数据表明，类维生素A，最有可能是视网膜，是ABCR在视杆外段转运的天然底物。这些观察结果对于理解Stargardt病的视觉周期和发病机制以及鉴定可以改变Stargardt病或与ABCR功能受损相关的其他视网膜病的自然史的化合物具有重要意义。
Many substrates for P-glycoprotein, an ABC transporter that mediates multidrug resistance in mammalian cells, have been shown to stimulate its ATPase activity in vitro. In the present study, we used this property as a criterion to search for natural and artificial substrates and/or allosteric regulators of ABCR, the rod photoreceptor-specific ABC transporter responsible for Stargardt disease, an early onset macular degeneration. ABCR was immunoaffinity purified to apparent homogeneity from bovine rod outer segments and reconstituted into liposomes, All-trans-retinal, a candidate ligand, stimulates the ATPase activity of ABCR 3-4-fold, with a half-maximal effect at 10-15 mu M. 11-cis- and 13-cis-retinal show similar activity. All-trans-retinal stimulates the ATPase activity of ABCR with Michaelis-Menten behavior indicative of simple noncooperative binding that is associated with a rate-limiting enzyme-substrate intermediate in the pathway of ATP hydrolysis. Among 37 structurally diverse non-retinoid compounds, including nine previously characterized substrates or sensitizers of P-glycoprotein, only four show significant ATPase stimulation when tested at 20 mu M. The dose-response curves of these four compounds are indicative of multiple binding sites and/or modes of interaction with ABCR, Two of these compounds, amiodarone and digitonin, can act synergistically with all-trans retinal, implying that they interact with a site or sites on ABCR different from the one with which all-trans-retinal interacts, Unlike retinal, amiodarone appears to interact with both free and ATP-bound ABCR, Together with clinical observations on Stargardt disease and the localization of ABCR to rod outer segment disc membranes, these data suggest that retinoids, and most likely retinal, are the natural substrates for transport by ABCR in rod outer segments. These observations have significant implications for understanding the visual cycle and the pathogenesis of Stargardt disease and for the identification of compounds that could modify the natural history of Stargardt disease or other retinopathies associated with impaired ABCR function.