Phosphorylation-induced modulation of pNBC1 function: distinct roles for the amino- and carboxy-termini
Phosphorylation-induced modulation of pNBC1 function: distinct roles for the amino- and carboxy-termini
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DOI：
10.1113/jphysiol.2003.042226
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发表时间：
2003-06-15
影响因子：
5.5
通讯作者：
Kurtz, I
中科院分区：
文献类型：
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作者：
Gross, E;Fedotoff, O;Kurtz, I
人类 NBC1 (SLC4A4) 基因编码电动碳酸氢钠协同转运蛋白 kNBC1 和 pNBC1，分别在肾脏和胰腺中高度表达。这些协同转运蛋白的 HCO3-:Na+ 化学计量是离子流方向的重要决定因素。最近，我们在表达 kNBC1 的小鼠近曲小管 (mPCT) 细胞系中发现，8-Br-cAMP 通过蛋白激酶 A (PKA) 依赖性 Ser(982) 磷酸化将协同转运蛋白的化学计量从 3:1 转变为 2:1。 pNBC1 具有相同的羧基末端共有磷酸化 PKA 位点 (KKGS(1026))，并且在其氨基末端有一个附加位点 (KRKT49)。在这项研究中，我们确定了这些位点在调节 pNBC1 功能中的潜在作用。结果表明，在表达 pNBC1 的 mPCT 细胞中，8-Br-cAMP 处理后，PKA 依赖性 Ser(1026) 磷酸化将化学计量从 3:1 转变为 2:1。该效应本质上是静电效应，因为用 Asp 替换 Ser(1026) 会导致类似的化学计量变化。除了改变化学计量外，8-Br-cAMP 还导致表达 pNBC1 的细胞的 4,4'-二硝基芪-2,2'-二磺酸 (DNDS) 敏感的基底外侧膜电导 (G(DS)) 显着增加，但不显着增加表达 kNBC1 的细胞。虽然该效应不涉及 Thr(49) 的磷酸化，而 Thr(49) 是内源性磷酸化，但用 Asp 或 Ala 替换该残基消除了 8-Br-cAMP 诱导的 G(DS) 增加。在 mPEC 胰管细胞系中，内源性 pNBC1 以 2:1 的 HCO3-:Na+ 化学计量发挥作用，8-Br-cAMP 将 G(DS) 增加约 90%，而不改变化学计量或诱导协同转运蛋白的磷酸化。结果表明，Ser(1026) 的磷酸化介导 pNBC1 化学计量中 cAMP 依赖性的变化，而 Thr(49) 在 cAMP 诱导的 G(DS) 增加中起着重要作用。
The human NBC1 (SLC4A4) gene encodes the electrogenic sodium bicarbonate cotransporters kNBC1 and pNBC1, which are highly expressed in the kidney and pancreas, respectively. The HCO3-:Na+ stoichiometry of these cotransporters is an important determinant of the direction of ion flux. Recently we showed in a mouse proximal tubule (mPCT) cell line expressing kNBC1, that 8-Br-cAMP shifts the stoichiometry of the cotransporter from 3:1 to 2:1 via protein kinase A (PKA)-dependent phosphorylation of Ser(982). pNBC1 has the identical carboxy-terminal consensus phosphorylation PKA site (KKGS(1026)), and an additional site in its amino-terminus (KRKT49). In this study we determined the potential role of these sites in regulating the function of pNBC1. The results demonstrated that in mPCT cells expressing pNBC1, PKA-dependent phosphorylation of Ser(1026) following 8-Br-cAMP treatment shifted the stoichiometry from 3:1 to 2:1. The effect was electrostatic in nature as replacing Ser(1026) With Asp resulted in a similar stoichiometry shift. In addition to shifting the stoichiometry, 8-Br-cAMP caused a significant increase in the 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)-sensitive basolateral membrane conductance (G(DS)) of cells expressing pNBC1, but not kNBC1. Although, the effect did not involve phosphorylation of Thr(49), which was endogenously phosphorylated, replacing this residue with Asp or Ala abolished the 8-Br-cAMP-induced increase in G(DS). In the mPEC pancreatic duct cell line, where endogenous pNBC1 functions with a HCO3-:Na+ stoichiometry of 2:1, 8-Br-cAMP increased G(DS) by similar to90 % without altering the stoichiometry or inducing phosphorylation of the cotransporter. The results demonstrate that phosphorylation of Ser(1026) mediates the cAMP-dependent shift in the stoichiometry of pNBC1, whereas Thr(49) plays an essential role in the cAMP-induced increase in G(DS).