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首页> 外文期刊>American Journal of Physiology >Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes.
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Ca2+-dependent inhibition of NHE3 requires PKC alpha which binds to E3KARP to decrease surface NHE3 containing plasma membrane complexes.

机译：依赖Ca2 +的NHE3抑制作用需要与E3KARP结合的PKCα才能降低含有表面NHE3的质膜复合物。

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摘要

The intestinal brush border (BB) Na+/H+ exchanger isoform 3 (NHE3) is acutely inhibited by elevation in the concentration of free intracellular Ca2+ ([Ca2+]i) by the cholinergic agonist carbachol and Ca2+ ionophores in a protein kinase C (PKC)-dependent manner. We previously showed that elevating [Ca2+]i with ionomycin rapidly inhibited NHE3 activity and decreased the amount of NHE3 on the plasma membrane in a manner that depended on the presence of the PDZ domain-containing protein E3KARP (NHE3 kinase A regulatory protein, also called NHERF2). The current studies were performed in PS120 fibroblasts (NHE-null cell line) stably transfected with NHE3 and E3KARP to probe the mechanism of PKC involvement in Ca2+ regulation of NHE3. Pretreatment with the general PKC inhibitor, GF109203X prevented ionomycin inhibition of NHE3 without altering basal NHE3 activity. Similarly, the Ca2+-mediated inhibition of NHE3 activity was blocked after pretreatment with the conventional PKC inhibitor Go-6976 and a specific PKCalpha pseudosubstrate-derived inhibitor peptide. [Ca2+]i elevation caused translocation of PKCalpha from cytosol to membrane. PKCalpha bound to the PDZ1 domain of GST-E3KARP in vitro in a Ca2+-dependent manner. PKCalpha and E3KARP coimmunoprecipitated from cell lysates; this occurred to a lesser extent at basal [Ca2+]i and was increased with ionomycin exposure. Biotinylation studies demonstrated that [Ca2+]i elevation induced oligomerization of NHE3 in total lysates and decreased the amount of plasma membrane NHE3. Treatment with PKC inhibitors did not affect the oligomerization of NHE3 but did prevent the decrease in surface amount of NHE3. These results suggest that PKCalpha is not necessary for the Ca2+-dependent formation of the NHE3 plasma membrane complex, although it is necessary for decreasing the membrane amounts of NHE3, probably by stimulating NHE3 endocytosis.
机译：肠内刷缘（BB）Na + / H +交换异构体3（NHE3）被蛋白激酶C（PKC）中的胆碱能激动剂卡巴胆碱和Ca2 +离子载体提高了游离细胞内Ca2 +（[Ca2 +] i）的浓度，从而被急性抑制。依赖的方式。我们先前发现，用离子霉素升高[Ca2 +] i可以迅速抑制NHE3活性，并以一种依赖于含PDZ域的蛋白质E3KARP（NHE3激酶A调节蛋白，也称为NHERF2）。目前的研究是在用NHE3和E3KARP稳定转染的PS120成纤维细胞（NHE无效细胞系）中进行的，以探讨PKC参与NHE3的Ca2 +调节的机制。用一般的PKC抑制剂GF109203X进行预处理可防止离子霉素抑制NHE3，而不会改变基础NHE3的活性。同样，在用常规PKC抑制剂Go-6976和特定的PKCalpha假底物衍生的抑制剂肽进行预处理后，阻断了Ca2 +介导的NHE3活性抑制。 [Ca2 +] i升高引起PKCalpha从胞质溶胶转移到膜。 PKCalpha在体外以Ca2 +依赖的方式与GST-E3KARP的PDZ1域结合。从细胞裂解物中共免疫沉淀PKCalpha和E3KARP;这种情况发生在基底[Ca2 +] i的程度较小，并且随着离子霉素的暴露而增加。生物素化研究表明，[Ca2 +] i升高会引起总裂解物中NHE3的寡聚，并减少质膜NHE3的量。用PKC抑制剂处理不会影响NHE3的低聚，但可以防止NHE3表面量的减少。这些结果表明，PKCalpha对于NHE3质膜复合物的Ca2 +依赖性形成不是必需的，尽管它可能是通过刺激NHE3内吞作用来减少NHE3的膜量所必需的。

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