Cabado A G, Yu F H, Kapus A, Lukacs G, Grinstein S, Orlowski J
Division of Cell Biology, Research Institute, Hospital for Sick Children, M5G 1X8 Toronto, Ontario, Canada.
J Biol Chem. 1996 Feb 16;271(7):3590-9. doi: 10.1074/jbc.271.7.3590.
Agents known to increase cAMP levels in renal and intestinal epithelia decrease sodium absorption by inhibiting NHE3, an isoform of the Na+/H+ exchanger expressed at high levels in apical membranes of these cells. In contrast, the ubiquitous, housekeeping isoform of the exchanger (NHE1) is stimulated by cAMP in some cell types. Optimal activity of NHE3 as well as NHE1 requires the presence of ATP. To gain insight into the molecular mechanisms of ATP dependence and cAMP regulation of NHE3, a series of mutations were constructed by progressively truncating segments of the C-terminal cytoplasmic domain of the transporter at amino acid positions 684, 638, and 579 (named NHE3delta684, NHE3delta638, and NHE3delta579). In addition, chimeric antiporters were constructed with the N-terminal transmembrane domain of NHE3 linked to the entire cytoplasmic region of NHE1 (chimera NHE3/1) or vice versa (chimera NHE1/3). These constructs were heterologously expressed in antiport-deficient Chinese hamster ovary cells, and their activities were assessed by fluorimetric measurements of intracellular pH and by radioisotope determinations of Na+ influx. Forskolin, which directly stimulates adenylate cyclase, inhibited NHE3 as well as NHE1/3, but not NHE3/1, suggesting that the cytoplasmic domain of NHE3 was sufficient to confer sensitivity to inhibition by cAMP. Forskolin also inhibited the truncated mutant NHE3delta684 to an extent similar to that for wild type NHE3. However, the inhibitory effect was greatly reduced in NHE3delta638 and more profound truncations (NHE3delta579 obliterated the effect of forskolin. These findings suggest that a region found between amino acids 579 and 684 is essential for the cAMP response of NHE3. In contrast, comparable ATP dependence was observed in all exchanger constructs examined. These observations indicate that ATP dependence is conferred by a region of the molecule in or adjacent to the transmembrane domain, which is most conserved between isoforms. It is concluded that different sites, and therefore different mechanisms, underlie inhibition of NHE3 by cAMP and by depletion of ATP.
已知可提高肾和肠上皮细胞中环磷酸腺苷（cAMP）水平的物质，通过抑制NHE3来减少钠的吸收。NHE3是钠/氢交换体的一种亚型，在这些细胞的顶端膜中高水平表达。相比之下，该交换体普遍存在的管家亚型（NHE1）在某些细胞类型中受到cAMP的刺激。NHE3以及NHE1的最佳活性都需要ATP的存在。为了深入了解NHE3对ATP的依赖性和cAMP调节的分子机制，通过在氨基酸位置684、638和579逐步截短转运蛋白C末端胞质结构域的片段，构建了一系列突变体（命名为NHE3delta684、NHE3delta638和NHE3delta579）。此外，构建了嵌合反向转运体，将NHE3的N末端跨膜结构域与NHE1的整个胞质区域相连（嵌合体NHE3/1），反之亦然（嵌合体NHE1/3）。这些构建体在缺乏反向转运体的中国仓鼠卵巢细胞中进行异源表达，并通过荧光法测量细胞内pH值和放射性同位素测定钠内流来评估它们的活性。直接刺激腺苷酸环化酶的福斯可林抑制了NHE3以及NHE1/3，但不抑制NHE3/1，这表明NHE3的胞质结构域足以赋予对cAMP抑制的敏感性。福斯可林对截短突变体NHE3delta684的抑制程度与野生型NHE3相似。然而，在NHE3delta638中抑制作用大大降低，而更深的截短（NHE3delta579）则消除了福斯可林的作用。这些发现表明，氨基酸579至684之间的区域对于NHE3的cAMP反应至关重要。相比之下，在所检测的所有交换体构建体中观察到了相当的ATP依赖性。这些观察结果表明，ATP依赖性是由跨膜结构域内或其附近的分子区域赋予的，该区域在各亚型之间最为保守。得出的结论是，cAMP和ATP耗竭对NHE3的抑制作用基于不同的位点，因此机制也不同。
