The amino acid transporter asc-1 is not involved in cystinuria.
The amino acid transporter asc-1 is not involved in cystinuria.
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DOI：
10.1111/j.1523-1755.2004.00908.x
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发表时间：
2004-10
影响因子：
19.6
通讯作者：
M. Pineda;M. Font;M. Bassi;M. Manzoni;G. Borsani;V. Marigo;Esperanza Fernández;R. D. Río;J. Purroy;A. Zorzano;V. Nunes;M. Palacín
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文献类型：
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作者：
M. Pineda;M. Font;M. Bassi;M. Manzoni;G. Borsani;V. Marigo;Esperanza Fernández;R. D. Río;J. Purroy;A. Zorzano;V. Nunes;M. Palacín
人氨基酸转运蛋白asc-1（SLC 7A 10）对小的中性氨基酸（包括半胱氨酸）表现出底物选择性，在肾脏中表达，位于胱氨酸尿症B基因附近并呈现序列变体（例如，E112D）。我们克隆了人asc-1，评估其转运特性，定位其在肾脏中的表达，寻找胱氨酸尿症患者中的突变，并测试变体E112 D的转运功能。方法：我们采用了基于EST的同源克隆策略。通过在非洲爪蟾卵母细胞和HeLa细胞中与4F 2 hc共表达来评估asc-1的转运特性。原位杂交检测肾组织中asc-1 mRNA的定位。从血细胞DNA中扩增外显子和内含子-外显子边界，并通过单链构象多态性（SSCP）进行突变筛查。结果与卵母细胞不同，Asc-1在HeLa细胞中最可能通过与内源性4F 2 hc相互作用到达质膜，并呈现出与共表达asc-1/4F 2 hc的卵母细胞相似的转运特征。Asc-1介导丙氨酸以易化扩散运输模式大量流出。asc-1 mRNA的表达定位于Henle袢、远端小管和集合管。最后，在胱氨酸尿症先证者中鉴定了SLC 7A 10多态性，SLC 7A 10序列变异体E112 D显示出完全的转运活性。结论asc-1在近曲小管中表达缺失，提示asc-1在氨基酸重吸收过程中不起作用。在SLC 7A 10中未发现引起胱氨酸尿症的突变。易化扩散转运模式和远端肾单位表达提示asc-1在渗透适应中的作用。
BACKGROUND The human amino acid transporter asc-1 (SLC7A10) exhibits substrate selectivity for small neutral amino acids, including cysteine, is expressed in kidney, is located close to the cystinuria B gene and presents sequence variants (e.g., E112D) in some cystinuria patients. We have cloned human asc-1, assessed its transport characteristics, localized its expression in kidney, searched for mutations in cystinuria patients, and tested the transport function of variant E112D. METHODS We used an EST-based homology cloning strategy. Transport characteristics of asc-1 were assessed by coexpression with 4F2hc in Xenopus oocytes and HeLa cells. Localization of asc-1 mRNA in kidney was assessed by in situ hybridization. Exons and intron-exon boundaries were polymerase chain reaction (PCR)-amplified from blood cell DNA and mutational screening was performed by single-stranded conformational polymorphism (SSCP). RESULTS Asc-1 reaches the plasma membrane in HeLa cells, unlike in oocytes, most probably by interaction with endogenous 4F2hc and presents similar transport characteristics to those in oocytes coexpressing asc-1/4F2hc. Asc-1 mediates a substantial efflux of alanine in a facilitated diffusion mode of transport. Expression of asc-1 mRNA localized to Henle's loop, distal tubules, and collecting ducts. Finally, SLC7A10 polymorphisms were identified in cystinuria probands and the SLC7A10 sequence variant E112D showed full transport activity. CONCLUSION The lack of expression of asc-1 in the proximal tubule indicates that it plays no role in the bulk of renal reabsorption of amino acids. No mutations causing cystinuria have been found in SLC7A10. The facilitated diffusion mode of transport and the expression in distal nephron suggest a role for asc-1 in osmotic adaptation.