Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.
Phosphorylation and transport in the Na-K-2Cl cotransporters, NKCC1 and NKCC2A, compared in HEK-293 cells.
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DOI：
10.1371/journal.pone.0017992
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发表时间：
2011-03-25
期刊：
影响因子：
3.7
通讯作者：
Flatman PW
中科院分区：
文献类型：
--
作者：
Hannemann A;Flatman PW
Na-K-2Cl协同转运蛋白有助于确定细胞组成和体积。NKCC 1广泛分布，而NKCC 2仅存在于肾脏中，在肾脏中起着重吸收20%过滤的NaCl的重要作用。NKCC 2的调控是知之甚少，因为它的分布有限，并与它的表达在哺乳动物细胞培养的困难。在这里，我们比较磷酸化的N-末端的协同转运蛋白，测量磷酸化特异性抗体，与布美他尼敏感的K+（86 Rb+）（活性）的运输在HEK-293细胞中稳定表达fNKCC 1或fNKCC 2A，从雪貂肾克隆。通过在37°C下工作，将转染的转运蛋白的活性与内源性转运蛋白的活性区分开。在低渗低[Cl−]培养基中预孵育细胞以减少通量测量期间的细胞[Cl−]和体积后，fNKCC 1和fNKCC 2A活性最高。两种转运蛋白的磷酸化增加了一倍以上。与哇巴因预孵育也强烈刺激fNKCC 1和fNKCC 2A，并大大增加磷酸化，而在无Na+培养基中预孵育最大限度地刺激fNKCC 1，并使其磷酸化加倍，但抑制fNKCC 2A，其磷酸化略有增加。激酶抑制剂减半的磷酸化和活性的两种转运蛋白，而抑制磷酸酶与calyculin A强烈增加磷酸化的两种转运蛋白，但只有轻微刺激fNKCC 1和抑制fNCCC 2A。因此，激酶抑制减少磷酸化和运输，和运输刺激只看到磷酸化增加时，但运输并不总是增加磷酸化。这表明N-末端的磷酸化决定了转运蛋白移动离子的潜在能力，但最终活性也取决于其他因素。仅使用磷酸特异性抗体对全细胞裂解物不能可靠地推断转运。
Na-K-2Cl cotransporters help determine cell composition and volume. NKCC1 is widely distributed whilst NKCC2 is only found in the kidney where it plays a vital role reabsorbing 20% of filtered NaCl. NKCC2 regulation is poorly understood because of its restricted distribution and difficulties with its expression in mammalian cell cultures. Here we compare phosphorylation of the N-termini of the cotransporters, measured with phospho-specific antibodies, with bumetanide-sensitive transport of K+ (86Rb+) (activity) in HEK-293 cells stably expressing fNKCC1 or fNKCC2A which were cloned from ferret kidney. Activities of transfected transporters were distinguished from those of endogenous ones by working at 37°C. fNKCC1 and fNKCC2A activities were highest after pre-incubation of cells in hypotonic low-[Cl−] media to reduce cell [Cl−] and volume during flux measurement. Phosphorylation of both transporters more than doubled. Pre-incubation with ouabain also strongly stimulated fNKCC1 and fNKCC2A and substantially increased phosphorylation, whereas pre-incubation in Na+-free media maximally stimulated fNKCC1 and doubled its phosphorylation, but inhibited fNKCC2A, with a small increase in its phosphorylation. Kinase inhibitors halved phosphorylation and activity of both transporters whereas inhibition of phosphatases with calyculin A strongly increased phosphorylation of both transporters but only slightly stimulated fNKCC1 and inhibited fNCCC2A. Thus kinase inhibition reduced phosphorylation and transport, and transport stimulation was only seen when phosphorylation increased, but transport did not always increase with phosphorylation. This suggests phosphorylation of the N-termini determines the transporters' potential capacity to move ions, but final activity also depends on other factors. Transport cannot be reliably inferred solely using phospho-specific antibodies on whole-cell lysates.
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DOI：
10.1152/ajpcell.1998.274.2.c299
发表时间：
1998-02-01
影响因子：
5.5
作者：
Lytle, C;McManus, TJ;Haas, M
通讯作者：
Haas, M
影响因子：
5.5
作者：
ELLIS, D
通讯作者：
ELLIS, D
DOI：
10.1074/jbc.m109.060004
发表时间：
2009-12-18
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Hannemann A;Christie JK;Flatman PW
通讯作者：
Flatman PW
DOI：
10.1152/ajprenal.00107.2002
发表时间：
2002-11-01
影响因子：
4.2
作者：
Gagnon, É;Forbush, B;Isenring, P
通讯作者：
Isenring, P
DOI：
10.1016/0005-2736(82)90564-8
发表时间：
1982-01-01
期刊：
BIOCHIMICA ET BIOPHYSICA ACTA
影响因子：
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作者：
BAKKERGRUNWALD, T;OGDEN, P;LAMB, JF
通讯作者：
LAMB, JF