Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter
Function of essential chloride and arginine residue in nucleotide binding to vesicular nucleotide transporter
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DOI：
10.1093/jb/mvz002
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发表时间：
2019-06-01
影响因子：
2.7
通讯作者：
Omote, Hiroshi
中科院分区：
文献类型：
--
作者：
Iwai, Yuma;Kamatani, Setsuko;Omote, Hiroshi
囊泡核苷酸转运蛋白（VNUT）通过转运核苷酸的能力在嘌呤能信号传导中起关键作用。VNUT属于SLC17家族，该家族包括泡状谷氨酸转运蛋白（VGLUTs）和I型Na+/磷酸盐共转运蛋白。所有这些转运体都表现出膜电位和Cl依赖的有机阴离子转运活性，并在跨膜区域具有必需的精氨酸。以前，我们报道过酮酸通过调节Cl-活化来抑制这些转运体。尽管这种调控对控制信号传递很重要，但Cl依赖性调控的机制尚不清楚。在这里，我们使用荧光ATP类似物三硝基苯-ATP （TNP-ATP）检测了Cl-和必需精氨酸残基在ATP与VNUT结合中的功能作用。VNUT可增强TNP-ATP的荧光，而VGLUT无增强作用。浓度依赖性曲线显示，tnf -ATP是VNUT的高亲和荧光探针，其K-d为4.8 μ m。tnf -ATP结合与ATP具有竞争性，对转运活性具有相似的特异性。Cl-和酮酸的添加不影响对TNP-ATP的表观亲和力。Arg119对Ala突变体保留了tnf - atp结合能力，但亲和力略有降低。总的来说，这些结果表明Cl-和必需精氨酸对ATP结合并不重要。
Vesicular nucleotide transporter (VNUT) plays a key role in purinergic signalling through its ability to transport nucleotides. VNUT belongs to the SLC17 family, which includes vesicular glutamate transporters (VGLUTs) and Type I Na+/phosphate cotransporters. All of these transporters exhibit membrane potential and Cl--dependent organic anion transport activity and have essential arginine in the transmembrane region. Previously, we reported that ketoacids inhibit these transporters through modulation of Cl- activation. Although this regulation is important to control signal transmission, the mechanisms underlying Cl--dependent regulation are unclear. Here, we examined the functional roles of Cl- and essential arginine residue on ATP binding to VNUT using the fluorescent ATP analogue trinitrophenyl-ATP (TNP-ATP). The fluorescence of TNP-ATP was enhanced by VNUT, whereas no enhancement was observed by VGLUT. Concentration-dependence curves showed that TNP-ATP was a high-affinity fluorescent probe for VNUT, with a K-d of 4.8 mu M. TNP-ATP binding was competitive to ATP and showed similar specificity to transport activity. Addition of Cl- and ketoacids did not affect the apparent affinity for TNP-ATP. The Arg119 to Ala mutant retained TNP-ATP binding ability with slightly reduced affinity. Overall, these results indicated that Cl- and essential arginine were not important for ATP binding.