A novel I551F variant of the Na/HCO3 cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity
A novel I551F variant of the Na/HCO3 cotransporter NBCe1-A shows reduced cell surface expression, resulting in diminished transport activity
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Na/HCO3 协同转运蛋白 NBCe1-A 的新型 I551F 变体显示细胞表面表达减少，导致转运活性减弱
DOI：
10.1152/ajprenal.00584.2020
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发表时间：
2021
影响因子：
4.2
通讯作者：
Shibata Shigeru
中科院分区：
文献类型：
--
作者：
Yamazaki Osamu;Yamashita Maho;Li Jinping;Ochiai-Homma Fumika;Yoshida Tadashi;Hirahashi Junichi;Furukawa Taiji;Kozuma Ken;Fujigaki Yoshihide;Seki George;Hayashi Matsuhiko;Shibata Shigeru
编码Na+/协同转运蛋白(NBCe1)的SLC4A4基因纯合突变可引起近端肾小管酸中毒，并伴有肾外症状。尽管目前已在近端肾小管性酸中毒患者中发现SLC4A4基因17‘端突变位点，但其他非同义单核苷酸变异体(SNV)的生理意义仍未确定。在这里，我们研究了NBCe1中SNV的功能性质。从国家生物技术信息中心的数据库中，我们鉴定了13个SNV，这些SNV以前没有在NBCe1-A的高度保守的跨膜结构域中被鉴定过。免疫细胞化学分析表明，I551F变异体主要存在于人胚胎肾(HEK)-293细胞的胞浆中，而所有其他SNV的亚细胞分布没有显示出明显的变化。在HEK-293细胞中的Western印迹分析表明，I551F变异体表现出糖基化受损和细胞表面水平降低69%。为了更详细地确定I551的作用，我们研究了各种人工突变体在非极化的HEK-293细胞和极化的Madin-Darby犬肾细胞中的意义，这表明只有I551F替代导致细胞质滞留。此外，对异种卵母细胞的功能分析表明，I551F突变体的活性显著降低，相当于野生型的39%，而任何其他SNV和人工I551突变体的活性没有明显变化。最后，在HEK-293细胞中的免疫荧光实验表明，I551F变异体在细胞质中保留了野生型NBCe1-A。这些数据表明，NBCe1-A的I551F变体主要通过细胞质滞留表现出转运活性受损，并表明该变体可能通过与野生型NBCe1-A形成复合体而产生显性的负面影响。近端小管中的生电Na+/共转运体1-A(NBCe1-A)调节酸碱平衡和液体容量稳态。从国家生物技术信息中心的数据库中，我们鉴定了NBCe1-A的I551F变异体，该变异体表现出糖基化、细胞表面表达和转运活性降低。我们还发现，I551F变异体对野生型NBCe1-A具有显性负效应，提示其具有生理意义。
Homozygous mutations inSLC4A4, which encodes the electrogenic Na+/cotransporter (NBCe1), cause proximal renal tubular acidosis associated with extrarenal symptoms. Although 17` mutated sites inSLC4A4have thus far been identified among patients with proximal renal tubular acidosis, the physiological significance of other nonsynonymous single-nucleotide variants (SNVs) remains largely undetermined. Here, we investigated the functional properties of SNVs in NBCe1. From the National Center for Biotechnology Information dbSNP database, we identified 13 SNVs that have not previously been characterized in the highly conserved, transmembrane domains of NBCe1-A. Immunocytochemical analysis revealed that the I551F variant was present predominantly in the cytoplasm in human embryonic kidney (HEK)-293 cells, whereas all other SNVs did not show as dramatic a change in subcellular distribution. Western blot analysis in HEK-293 cells demonstrated that the I551F variant showed impaired glycosylation and a 69% reduction in cell surface levels. To determine the role of I551 in more detail, we examined the significance of various artificial mutants in both nonpolarized HEK-293 cells and polarized Madin-Darby canine kidney cells, which indicated that only I551F substitution resulted in cytoplasmic retention. Moreover, functional analysis usingXenopusoocytes demonstrated that the I551F variant had a significantly reduced activity corresponding to 39% of that of the wild-type, whereas any other SNVs and artificial I551 mutants did not show significant changes in activity. Finally, immunofluorescence experiments in HEK-293 cells indicated that the I551F variant retained wild-type NBCe1-A in the cytoplasm. These data demonstrate that the I551F variant of NBCe1-A shows impaired transport activity predominantly through cytoplasmic retention and suggest that the variant can have a dominant negative effect by forming complexes with wild-type NBCe1-A.NEW & NOTEWORTHYElectrogenic Na+/cotransporter 1-A (NBCe1-A) in the proximal tubule regulates the acid/base balance and fluid volume homeostasis. From the National Center for Biotechnology Information dbSNP database, we identified the I551F variant of NBCe1-A, which showed reduced glycosylation, cell surface expression, and transport activity. We also found that the I551F variant can exert a dominant negative effect on wild-type NBCe1-A, suggesting its physiological significance.