White K E, Gesek F A, Reilly R F, Friedman P A
Department of Pharmacology and Toxicology, Dartmouth Medical School, Hanover, New Hampshire, USA.
Kidney Int. 1998 Sep;54(3):897-906. doi: 10.1046/j.1523-1755.1998.00056.x.
Plasma membrane NCX1 Na+/Ca2+ exchangers mediate cellular Ca2+ efflux. Renal distal convoluted tubule (DCT) cells express transcripts encoding three alternatively spliced NCX1 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.
Sense 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 (delta[Ca2+]i), in single cells, when the electrochemical gradient for Na+ was reversed.
The change of [Ca2+]i in cells treated with antisense ODNs to a downstream or upstream region common to all NCX1 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 delta[Ca2+]i by 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 delta[Ca2+]i by 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 recognizes NCX1 confirmed that protein expression was inhibited after treatment with the exon B antisense ODNs.
These findings show that Na+-dependent cellular Ca2+ efflux in DCT cells is primarily mediated by NACA2 and NACA3.
质膜钠钙交换体NCX1介导细胞内钙离子外流。肾远曲小管（DCT）细胞表达编码三种可变剪接的NCX1亚型的转录本：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细胞中钠钙交换体活性和表达的作用。
将靶向交换体转录本的正义和反义ODN导入经链球菌溶血素O通透处理的DCT细胞。当钠离子的电化学梯度反转时，通过测量单细胞中游离细胞内钙离子浓度（Δ[Ca2+]i）的钠离子依赖性变化来评估钠钙交换活性。
用针对所有NCX1亚型共有的下游或上游区域的反义ODN处理的细胞中，[Ca2+]i的变化在位于假定的第九跨膜结构域的下游区域为173 nM（-66%），用针对位于细胞内环可变部分5'端上游区域的ODN处理时为226 nM（-39%）。针对NACA2和NACA3中均存在的外显子B的反义ODN使Δ[Ca2+]i降低了209 nM（-44%），而针对NACA6（外显子A）的特异性反义ODN则无作用。针对NACA2和NACA6中存在的外显子C的特异性反义ODN使Δ[Ca2+]i降低了226 nM（-39%）。对远曲小管细胞原代培养物制备的mRNA进行的Northern分析显示含有外显子B而非外显子A的转录本。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。
这些发现表明，DCT细胞中钠离子依赖性细胞内钙离子外流主要由NACA2和NACA3介导。
