Molecular cloning of a sixth member of the K+-dependent Na+/Ca2+ exchanger gene family, NCKX6
Molecular cloning of a sixth member of the K+-dependent Na+/Ca2+ exchanger gene family, NCKX6
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DOI：
10.1074/jbc.m310908200
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发表时间：
2004-02-13
影响因子：
4.8
通讯作者：
Lytton, J
中科院分区：
文献类型：
--
作者：
Cai, XJ;Lytton, J
利用生物信息学和分子克隆工具从小鼠和人体组织中鉴定和分离编码K+依赖性Na+/Ca2+交换器家族第6个成员NCKX6的cDNA克隆。小鼠NCKX6蛋白全长585个氨基酸，与先前鉴定的交换子在α -重复区域具有62%的序列相似性，但在这些区域之外几乎没有一级序列相似性。4 kb的NCKX6转录本在所有被检查的组织中都大量表达，因此比之前描述的NC(K)X家族成员分布更广泛。该新基因的两个选择性剪接产物被鉴定为编码不同长度的蛋白质。短异构体与全长异构体在c端疏水结构域不同，这是由于两个外显子的缺失导致了阅读框的移位。两种NCKX6亚型均在HEK-293细胞中表达。通过数字成像对fura-2转染的HEK-293细胞的功能分析表明，短异构体具有K+依赖性的Na+/Ca2+交换活性，而全长异构体则没有。后者保留在内质网内，而短异构体则存在于转染细胞的质膜上。使用NCKX6特异性抗体检测天然组织中NCKX6表达的免疫荧光研究显示，NCKX6强烈标记心脏肌层膜。因此，NCKX6的发现揭示了Na+/Ca2+交换超家族的新成员，其在所有组织中的普遍表达表明，K+依赖性Na+/Ca2+交换在维持不同组织和细胞类型的细胞Ca2+稳态中起重要作用。
Bioinformatic and molecular cloning tools were used to identify and isolate cDNA clones from mouse and human tissues that encode the sixth member of the K+-dependent Na+/Ca2+ exchanger family, NCKX6. The mouse NCKX6 protein is 585 amino acids long and shares about 62% sequence similarity with previously identified exchangers in the alpha-repeat regions but has little primary sequence similarity outside these regions. NCKX6 transcripts of 4 kb are abundantly expressed in all tissues examined and are thus more broadly distributed than previously described NC(K)X family members. Two alternatively spliced products of this novel gene were identified that encode proteins of different length. The short isoform differs from the full-length isoform at the C-terminal hydrophobic domain as a result of a shift in the reading frame caused by the deletion of two exons. Both NCKX6 isoforms were expressed in HEK-293 cells. Functional analysis by digital imaging of fura-2 loaded transfected HEK-293 cells demonstrated that the short isoform exhibited K+-dependent Na+/Ca2+ exchange activity whereas the full-length isoform did not. The latter was retained within the endoplasmic reticulum, whereas the short isoform was present at the plasma membrane in transfected cells. Immunofluorescence studies examining NCKX6 expression in native tissue using an NCKX6-specific antibody showed intense labeling of the cardiac sarcolemmal membrane. The discovery of NCKX6 therefore reveals a novel member of the Na+/Ca2+ exchanger superfamily whose ubiquitous expression in all tissues suggests an important role for K+-dependent Na+/Ca2+ exchange in maintaining cellular Ca2+ homeostasis in diverse tissues and cell types.