MOLECULAR-CLONING AND CHARACTERIZATION OF THE HUMAN CARDIAC NA+/CA2+ EXCHANGER CDNA
MOLECULAR-CLONING AND CHARACTERIZATION OF THE HUMAN CARDIAC NA+/CA2+ EXCHANGER CDNA
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DOI：
10.1073/pnas.89.10.4769
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发表时间：
1992-05-15
影响因子：
11.1
通讯作者：
IZUMO, S
中科院分区：
文献类型：
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作者：
KOMURO, I;WENNINGER, KE;IZUMO, S
Na+/Ca ~（2+）交换器在许多可兴奋细胞的Ca ~（2+）处理中起重要作用。特别地，Na+/Ca 2+交换器在心脏肌膜中以高水平表达，并且是Ca 2+从细胞中挤出的主要机制。此外，交换器已被建议发挥关键作用洋地黄行动和缺血后再灌注损伤的心肌细胞。我们在这里报告的分离和鉴定的cDNA编码的人心脏Na+/Ca ~（2+）交换。从筛选的5 × 105噬菌体噬斑中分离出12个重叠克隆，对应于交换器cDNA序列的5.6个酶。该序列预测了一个973个氨基酸的多肽与一个推定的前导肽，11个潜在的跨膜区域，和一个大的推定的细胞质环之间的第五和第六跨膜螺旋。当从克隆的cDNA在体外合成RNA并注射到非洲爪蟾卵母细胞中时，其诱导高水平的Na+/Ca 2+交换活性的表达，证实该克隆编码功能性Na+/Ca 2+交换器。Southern印迹分析表明，心脏交换基因在人类基因组中以单拷贝形式存在，但不能排除其他相关基因的存在。北方杂交和S1定位分析表明，心脏型交换蛋白mRNA在心脏中表达最丰富，其次是在脑中。心脏型交换器mRNA也在视网膜和骨骼肌和平滑肌中以非常低的水平表达。编码交换器的mRNA水平在胎儿心脏中显著低于成人心脏，但在终末期心力衰竭患者的心肌中没有变化。
The Na+/Ca2+ exchanger plays important roles in Ca2+ handling in many excitable cells. In particular, the Na+/Ca2+ exchanger is expressed at high levels in the cardiac sarcolemma and is the dominant mechanism of Ca2+ extrusion from the cells. In addition, the exchanger has been suggested to play key roles in digitalis action and in postischemic reperfusion injury of cardiac myocytes. We report here the isolation and characterization of the cDNA encoding the human cardiac Na+/Ca2+ exchanger. Twelve overlapping clones corresponding to 5.6 kilobases of the exchanger cDNA sequence were isolated from 5 x 10(5) phage plaques screened. The sequence predicted a 973-amino acid polypeptide with a putative leader peptide, 11 potential membrane-spanning regions, and one large putative cytoplasmic loop between the fifth and sixth transmembrane helices. When RNA was synthesized in vitro from the cloned cDNA and injected into Xenopus oocytes, it induced expression of Na+/Ca2+ exchange activity at high levels, confirming that this clone encodes the functional Na+/Ca2+ exchanger. Southern blot analysis indicated that the cardiac exchanger gene exists as a single copy in the human genome, although existence of other related genes cannot be ruled out. Northern blot and S1 mapping analyses revealed that the cardiac type exchanger mRNA is expressed most abundantly in the heart and next in the brain. The cardiac-type exchanger mRNA was also expressed in the retina and in skeletal and smooth muscles at very low levels. The levels of mRNA encoding the exchanger were significantly lower in fetal hearts than in adult hearts but were unchanged in the myocardium from patients with end-stage heart failure.