NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells.
NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells.
复制标题
反义寡核苷酸对小鼠远曲小管细胞中 NCX1 Na/Ca 交换蛋白的抑制作用。
DOI：
10.1046/j.1523-1755.1998.00056.x
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发表时间：
1998
期刊：
影响因子：
--
通讯作者：
Friedman,PA
中科院分区：
文献类型：
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作者：
White,KE;Gesek,FA;Reilly,RF;Friedman,PA
小鼠远曲小管细胞中反义寡核苷酸对 NCX1 Na/Ca 交换器的抑制。背景质膜 NCX1 Na+/Ca2+ 交换器介导细胞 Ca2+ 流出。肾远曲小管 (DCT) 细胞表达编码三种选择性剪接的 NCX1 同工型的转录物：NACA2（外显子 B、C、D）、NACA3（外显子 B 和 D）和 NACA6（外显子 A、C、D）。我们使用反义寡脱氧核苷酸 (ODN) 来确定这些 NACA 亚型对 DCT 细胞中 Na+/Ca2+ 交换器活性和表达的功能。方法将靶向交换器转录物的正义和反义 ODN 引入用链球菌溶血素 O 透化的 DCT 细胞中。通过测量细胞内游离 Ca2+ 浓度的 Na+ 依赖性变化来评估 Na+/Ca2+ 交换活性(Δ[Ca2+]i)，在单细胞中，当 Na+ 的电化学梯度反转时。结果用反义 ODN 处理的细胞中，[Ca2+]i 的变化对于所有 NCX1 亚型共有的下游或上游区域为 173 nm(-66%)，位于假定的第九跨膜结构域的下游区域，以及 226 nm(-39%)，ODN 位于假定的第九跨膜结构域5'至细胞内环的可变部分。 NACA2 和 NACA3 中都存在的外显子 B 反义 ODN 将 Δ[Ca2+]i 降低了 209 nm (-44%)，而针对 NACA6（外显子 A）特异的反义 ODN 则没有影响。 NACA2 和 NACA6 中存在的外显子 C 特异性反义 ODN 将 Δ[Ca2+]i 降低了 226 nm（-39%）。对从远端小管细胞的原代培养物中制备的 mRNA 进行 Northern 分析，发现含有外显子 B 的转录物，但未发现含有外显子 A 的转录物。使用识别NCX1的多克隆抗体进行的免疫荧光分析证实，用外显子B反义ODN处理后蛋白质表达受到抑制。结论这些发现表明DCT细胞中Na+依赖性细胞Ca2+流出主要由NACA2和NACA3介导。
NCX1 Na/Ca exchanger inhibition by antisense oligonucleotides in mouse distal convoluted tubule cells.BackgroundPlasma membraneNCX1 Na+/Ca2+exchangers mediate cellular Ca2+efflux. Renal distal convoluted tubule (DCT) cells express transcripts encoding three alternatively splicedNCX1 isoforms: NACA2 (exons B, C, D), NACA3 (exons B and D), and NACA6 (exons A, C, D). We used antisense oligodeoxynucleotides (ODNs) to determine the function of these NACA isoforms on Na+/Ca2+exchanger activity and expression in DCT cells.MethodsSense and antisense ODNs targeting exchanger transcripts were introduced into DCT cells permeabilized with streptolysin O. Na+/Ca2+exchange activity was assessed by measuring Na+-dependent changes of free intracellular Ca2+concentration (Δ[Ca2+]i), in single cells, when the electrochemical gradient for Na+was reversed.ResultsThe change of [Ca2+]iin cells treated with antisense ODNs to a downstream or upstream region common to allNCX1 isoforms was 173 nm(-66%) to the downstream region located in the putative ninth transmembrane domain, and 226 nm(-39%) with ODNs to an upstream region located 5′ to the variable portion of the intracellular loop. Antisense ODNs to exon B, present in both NACA2 and NACA3, decreased Δ[Ca2+]iby 209 nm(-44%), while antisense ODNs specific for NACA6 (exon A) were without effect. Antisense ODNs specific for exon C, present in NACA2 and NACA6, decreased Δ[Ca2+]iby 226 nm(-39%). Northern analysis of mRNA prepared from primary cultures of distal tubule cells revealed exon B- but not exon A-containing transcripts. Immunofluorescence analysis using a polyclonal antibody that recognizesNCX1 confirmed that protein expression was inhibited after treatment with the exon B antisense ODNs.ConclusionThese findings show that Na+-dependent cellular Ca2+efflux in DCT cells is primarily mediated by NACA2 and NACA3.