IRBIT reduces the apparent affinity for intracellular Mg2+ in inhibition of the electrogenic Na+-HCO3- cotransporter NBCe1-B
IRBIT reduces the apparent affinity for intracellular Mg2+ in inhibition of the electrogenic Na+-HCO3- cotransporter NBCe1-B
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DOI：
10.1016/j.bbrc.2012.06.127
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发表时间：
2012-08-03
影响因子：
3.1
通讯作者：
Ishikawa, Toru
中科院分区：
文献类型：
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作者：
Yamaguchi, Soichiro;Ishikawa, Toru
Na+-HCO3-共转运体NBCe1-B可受细胞内镁离子(Mg-I(2+))的调节。我们先前报道，在全细胞电压钳条件下，哺乳动物细胞异源表达的牛NBCe1-B(bNBCe1-B)电流被Mg-I(2+)强烈抑制，这种抑制可能是通过静电相互作用介导的，并通过截断胞内NBCe1-B特异的N-末端区域而解除。有趣的是，天然表达于牛腮腺腺泡(BPA)细胞的NBCe1-B类电流对镁离子抑制的敏感性远低于bNBCe1-B电流。在这里，我们假设这种明显的差异可能与IRBIT有关，IRBIT是一种先前发现的NBCe1-B相互作用蛋白。RT-PCR、Western印迹和免疫荧光共聚焦显微镜显示，Irbit不仅在胞浆中表达，而且与NBCe1-B共定位于BPA细胞的质膜区域。用抗NBCe1抗体在牛腮腺细胞裂解液中将IRBIT与NBCe1-B共沉淀。全细胞膜片钳实验表明，共表达Irbit降低了稳定表达于HEK293细胞的bNBCe1-B电流对镁离子的敏感性。综上所述，这些结果表明，Irbit可能降低了哺乳动物细胞中抑制NBCe1-B活性的对镁离子的表观亲和力。(C)2012 Elsevier Inc.保留所有权利。
The electrogenic Na+-HCO3- cotransporter NBCe1-B can be regulated by intracellular Mg2+ (Mg-i(2+)). We previously reported that under whole-cell voltage-clamp conditions, bovine NBCe1-B (bNBCe1-B) currents heterologously expressed in mammalian cells are strongly inhibited by Mg-i(2+), and the inhibition is likely mediated by electrostatic interaction and relieved by truncation of the cytosolic NBCe1-B specific N-terminal region. Intriguingly, NBCe1-B-like currents natively expressed in bovine parotid acinar (BPA) cells are much less sensitive to Mg-i(2+) inhibition than bNBCe1-B currents. Here, we hypothesized that this apparent discrepancy may involve IRBIT, a previously identified NBCe1-B-interacting protein. RT-PCR, Western blot and immunofluorescence confocal microscopy revealed that IRBIT was not only expressed in the cytosol, but also colocalized with NBCe1-B in the region of plasma membranes of BPA cells. IRBIT was coimmunoprecipitated with NBCe1-B by an anti-NBCe1 antibody in bovine parotid cell lysate. Whole-cell patch-clamp experiments showed that coexpression of IRBIT lowered the Mg-i(2+) sensitivity of bNBCe1-B currents stably expressed in HEK293 cells. Collectively, these results suggest that IRBIT may reduce the apparent affinity for Mg-i(2+) in inhibition of NBCe1-B activity in mammalian cells. (C) 2012 Elsevier Inc. All rights reserved.