Centre for Integrative Physiology, College of Medicine and Veterinary Medicine, The University of Edinburgh, Edinburgh, United Kingdom.
PLoS One. 2011 Mar 25;6(3):e17992. doi: 10.1371/journal.pone.0017992.
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(+) ((86)Rb(+)) (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.
钠钾 2 氯协同转运蛋白有助于确定细胞组成和体积。NKCC1 广泛分布，而 NKCC2 仅存在于肾脏中，在肾脏中它发挥着重要作用，可重吸收过滤的 20%的 NaCl。由于 NKCC2 的分布有限且在哺乳动物细胞培养物中表达困难，因此其调节机制仍不清楚。在这里，我们比较了用磷酸特异性抗体测量的协同转运蛋白 N 端的磷酸化与稳定表达 fNKCC1 或 fNKCC2A 的 HEK-293 细胞中钾（86Rb+）（活性）的 bumetanide 敏感转运，这些细胞从雪貂肾脏中克隆。通过在 37°C 下工作，可以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来。fNKCC1 和 fNKCC2A 的活性在细胞在低渗低-[Cl-]培养基中孵育后达到最高，以在通量测量期间降低细胞 [Cl-]和体积。两种转运蛋白的磷酸化都增加了一倍以上。哇巴因预孵育也强烈刺激 fNKCC1 和 fNKCC2A，并大大增加磷酸化，而无钠培养基预孵育则最大限度地刺激 fNKCC1 并使其磷酸化增加一倍，但抑制 fNKCC2A，其磷酸化略有增加。激酶抑制剂将两种转运蛋白的磷酸化和活性减半，而 calyculin A 抑制磷酸酶强烈增加两种转运蛋白的磷酸化，但仅轻微刺激 fNKCC1 并抑制 fNCCC2A。因此，激酶抑制降低了磷酸化和转运，只有在磷酸化增加时才会看到转运刺激，但转运不一定随磷酸化增加而增加。这表明 N 端的磷酸化决定了转运蛋白移动离子的潜在能力，但最终活性还取决于其他因素。不能仅使用全细胞裂解物上的磷酸特异性抗体可靠地推断出转运。
