Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA
Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA
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DOI：
10.1152/ajpcell.00382.2003
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发表时间：
2004-06-01
影响因子：
5.5
通讯作者：
Casey, JR
中科院分区：
文献类型：
--
作者：
Loiselle, FB;Morgan, PE;Casey, JR
人类 NBC3 是一种电中性 Na+/HCO3- 协同转运蛋白，在心脏、骨骼肌和肾脏中表达，在 HCO3- 代谢中发挥重要作用。胞浆酶碳酸酐酶 II (CAII) 在许多组织中催化 CO2 + H2O 可逆箭头 HCO3- + H+ 反应。我们研究了 NBC3 是否像一些 Cl-HCO3- 交换蛋白一样可以结合 CAII，以及 PKA 是否可以通过调节 CAII 结合来调节 NBC3 活性。 CAII 结合 NBC3 (NBC3Ct) 的 COOH 末端结构域，K-d = 101 nM；在酸性pH下相互作用更强。 HEK-293 细胞与 NBC3 和 CAII 的共转染将 CAII 招募到质膜上。 CAII 共有结合位点的诱变表明，NBC3 的 D1135-D1136 区域对于 CAII/NBC3 相互作用和最佳功能至关重要，因为 NBC3 D1135N/D1136N 仅保留了野生型活性的 29 +/- 22%。相对于未处理的 NBC3 转染细胞，功能显性失活 CAII 突变体 V143Y 与 NBC3 的共表达或向 NBC3 转染细胞中添加 100 μM 8-溴腺苷分别使细胞内 pH (pH(i)) 恢复率降低 31 +/- 3 或 38 +/- 7%。这些效应是相加的，将 pHi 恢复率降低了 69 +/- 12%，表明 PKA 通过独立于 CAII 的机制降低了转运活性。通过质谱法和 [gamma-P-32] ATP 标记对 PKA 依赖性磷酸化的测量表明 NBC3Ct 不是 PKA 底物。这些结果表明 NBC3 和 CAII 相互作用以使 HCO3- 传输速率最大化。尽管 PKA 降低了 NBC3 转运活性，但其作用独立于 NBC3/CAII 相互作用，并且不涉及 NBC3Ct 的磷酸化。
Human NBC3 is an electroneutral Na+/ HCO3- cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO3- metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO2 + H2O reversible arrow HCO3- + H+ in many tissues. We investigated whether NBC3, like some Cl- HCO3- exchange proteins, could bind CAII and whether PKA could regulate NBC3 activity through modulation of CAII binding. CAII bound the COOH-terminal domain of NBC3 (NBC3Ct) with K-d = 101 nM; the interaction was stronger at acid pH. Cotransfection of HEK-293 cells with NBC3 and CAII recruited CAII to the plasma membrane. Mutagenesis of consensus CAII binding sites revealed that the D1135-D1136 region of NBC3 is essential for CAII/NBC3 interaction and for optimal function, because the NBC3 D1135N/D1136N retained only 29 +/- 22% of wild-type activity. Coexpression of the functionally dominant-negative CAII mutant V143Y with NBC3 or addition of 100 muM 8-bromoadenosine to NBC3 transfected cells reduced intracellular pH (pH(i)) recovery rate by 31 +/- 3, or 38 +/- 7%, respectively, relative to untreated NBC3 transfected cells. The effects were additive, together decreasing the pHi recovery rate by 69 +/- 12%, suggesting that PKA reduces transport activity by a mechanism independently of CAII. Measurements of PKA-dependent phosphorylation by mass spectroscopy and labeling with [gamma-P-32] ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO3- transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.