A Substrate Binding Hinge Domain Is Critical for Transport-related Structural Changes of Organic Cation Transporter 1
A Substrate Binding Hinge Domain Is Critical for Transport-related Structural Changes of Organic Cation Transporter 1
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DOI：
10.1074/jbc.m112.388793
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发表时间：
2012-09-07
影响因子：
4.8
通讯作者：
Koepsell, Hermann
中科院分区：
文献类型：
--
作者：
Egenberger, Brigitte;Gorboulev, Valentin;Koepsell, Hermann
有机阳离子转运体是膜电位依赖的促进扩散系统。功能研究、广泛的诱变和同源性建模表明了以下机制。具有大的向外开放裂缝的转运蛋白构象结合细胞外底物，通过底物被封闭的状态，转变为具有向内开放裂缝的构象，释放底物，随后转变回向外开放状态。在大鼠有机阳离子转运蛋白（rOct1），电压和配体依赖性的运动的荧光标记的半胱氨酸的电压钳荧光测定。对于荧光检测，半胱氨酸残基被引入裂缝形成跨膜α-螺旋（TMH）5，8和11的细胞外部分。在非洲爪蟾卵母细胞中表达突变体后，用四甲基罗丹明-6-马来酰亚胺标记半胱氨酸，并通过电压钳荧光法监测电压依赖性构象变化。在TMH 11的中心结构域引入一个半胱氨酸取代甘氨酸478。该结构域包含两个参与底物结合的氨基酸和两个允许螺旋弯曲的甘氨酸残基（Gly-477和Gly-478）。Cys-478可以用转运底物类似物[2-（三甲基铵）-乙基]甲硫基磺酸酯修饰，但不能被四甲基罗丹明-6-马来酰亚胺修饰。电压依赖性运动的指示剂位置TMH 5，8，和11被改变基板的应用程序表明大的构象变化在运输过程中。G478C交换降低了转运体的周转率，并阻断了TMH 5和11的电压依赖性运动。Cys-478的[2-（三甲基铵）-乙基]甲硫基磺酸盐修饰阻断了TMH8的底物结合、转运活性和运动。这些数据表明，Gly-478位于TMH 11的一个重要的铰链结构域，其中底物结合诱导运输相关的结构变化。
Organic cation transporters are membrane potential-dependent facilitative diffusion systems. Functional studies, extensive mutagenesis, and homology modeling indicate the following mechanism. A transporter conformation with a large outward-open cleft binds extracellular substrate, passes a state in which the substrate is occluded, turns to a conformation with an inward-open cleft, releases substrate, and subsequently turns back to the outward-open state. In the rat organic cation transporter (rOct1), voltage- and ligand-dependent movements of fluorescence-labeled cysteines were measured by voltage clamp fluorometry. For fluorescence detection, cysteine residues were introduced in extracellular parts of cleft-forming transmembrane alpha-helices (TMHs) 5, 8, and 11. Following expression of the mutants in Xenopus laevis oocytes, cysteines were labeled with tetramethylrhodamine-6-maleimide, and voltage-dependent conformational changes were monitored by voltage clamp fluorometry. One cysteine was introduced in the central domain of TMH 11 replacing glycine 478. This domain contains two amino acids that are involved in substrate binding and two glycine residues (Gly-477 and Gly-478) allowing for helix bending. Cys-478 could be modified with the transported substrate analog [2-(trimethylammonium)-ethyl]methanethiosulfonate but was inaccessible to tetramethylrhodamine-6-maleimide. Voltage-dependent movements at the indicator positions of TMHs 5, 8, and 11 were altered by substrate applications indicating large conformational changes during transport. The G478C exchange decreased transporter turnover and blocked voltage-dependent movements of TMHs 5 and 11. [2-(Trimethylammonium)-ethyl] methanethiosulfonate modification of Cys-478 blocked substrate binding, transport activity, and movement of TMH8. The data suggest that Gly-478 is located within a mechanistically important hinge domain of TMH 11 in which substrate binding induces transport-related structural changes.