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首页> 外文期刊>American Journal of Physiology >Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA.
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Regulation of the human NBC3 Na+/HCO3- cotransporter by carbonic anhydrase II and PKA.

机译：碳酸酐酶II和PKA对人NBC3 Na + / HCO3-共转运蛋白的调节。

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摘要

Human NBC3 is an electroneutral Na(+)/HCO(3)(-) cotransporter expressed in heart, skeletal muscle, and kidney in which it plays an important role in HCO(3)(-) metabolism. Cytosolic enzyme carbonic anhydrase II (CAII) catalyzes the reaction CO(2) + H(2)O left arrow over right arrow HCO(3)(-) + H(+) in many tissues. We investigated whether NBC3, like some Cl(-)/HCO(3)(-) 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 microM 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 pH(i) 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-(32)P]ATP showed that NBC3Ct was not a PKA substrate. These results demonstrate that NBC3 and CAII interact to maximize the HCO(3)(-) transport rate. Although PKA decreased NBC3 transport activity, it did so independently of the NBC3/CAII interaction and did not involve phosphorylation of NBC3Ct.
机译：人NBC3是在心脏，骨骼肌和肾脏中表达的电中性Na（+）/ HCO（3）（-）协同转运蛋白，在其中它在HCO（3）（-）代谢中起重要作用。胞质酶碳酸酐酶II（CAII）在许多组织中催化反应CO（2）+ H（2）O左箭头超过右箭头HCO（3）（-）+ H（+）。我们调查了NBC3是否像某些Cl（-）/ HCO（3）（-）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以K（d）= 101 nM结合NBC3（NBC3Ct）的COOH末端结构域;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募集到质膜上。共有CAII结合位点的诱变表明，NBC3的D1135-D1136区对于CAII / NBC3相互作用和最佳功能至关重要，因为NBC3 D1135N / D1136N仅保留了29 +/- 22％的野生型活性。与NBC3共表达功能优势阴性CAII突变体V143Y或向NBC3转染的细胞中添加100 microM 8-溴腺苷使细胞内pH（pH（i））回收率降低31 +/- 3或38 +/- 7％，相对于未处理的NBC3转染细胞。这些作用是累加的，共同使pH（i）的回收率降低了69 +/- 12％，这表明PKA通过一种独立于CAII的机制降低了转运活性。质谱法测定PKA依赖性磷酸化并用[γ-（32）P] ATP标记表明NBC3Ct不是PKA底物。这些结果表明NBC3和CAII相互作用以最大化HCO（3）（-）的运输速度。尽管PKA降低了NBC3转运活性，但它独立于NBC3 / CAII相互作用而这样做，并且不涉及NBC3Ct的磷酸化。

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