Murtazina Rakhilya, Kovbasnjuk Olga, Donowitz Mark, Li Xuhang
Departments of Physiology and Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, 720 Rutland Avenue, Baltimore, MD 21205, USA.
J Biol Chem. 2006 Jun 30;281(26):17845-55. doi: 10.1074/jbc.M601740200. Epub 2006 Apr 27.
A previous study showed that approximately 25-50% of rabbit ileal brush border (BB) Na(+)/H(+) exchanger NHE3 is in lipid rafts (LR) (Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537-552). Here, we examined the role of LR in NHE3 transport activity using a simpler system: opossum kidney (OK) cells (a renal proximal tubule epithelial cell line) containing NHE3. approximately 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl-beta-cyclodextrin (MbetaCD) decreased NHE3 activity and increased K'(H+)(i), but K(m)((Na+)) was not affected. The MbetaCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3beta,5alpha,6beta-triol). The MbetaCD effect was specific for NHE3 activity because it did not alter Na(+)-dependent l-Ala uptake. MbetaCD did not alter OK cell BB topology and did not change the surface amount of NHE3, but greatly reduced the rate of NHE3 endocytosis. The effects of inhibiting phosphatidylinositol 3-kinase and of MbetaCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MbetaCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only approximately 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MbetaCD treatment decreased NHE3 basal activity. The LR pool is necessary for multiple kinetic aspects of normal NHE3 activity, including V(max) and K'(H+)(i), and also for multiple aspects of NHE3 trafficking, including at least basal endocytosis and phosphatidylinositol 3-kinase-dependent basal exocytosis. Because the C-terminal domain of NHE3 is necessary for its regulation and because the changes in NHE3 kinetics with MbetaCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MbetaCD sensitivity of NHE3.
先前的一项研究表明，兔回肠刷状缘（BB）Na⁺/H⁺交换体NHE3约25 - 50%存在于脂筏（LR）中（Li, X., Galli, T., Leu, S., Wade, J. B., Weinman E. J., Leung, G., Cheong, A., Louvard, D., and Donowitz, M. (2001) J. Physiol. (Lond.) 537, 537 - 552）。在此，我们使用一个更简单的系统：含有NHE3的负鼠肾（OK）细胞（一种肾近端小管上皮细胞系），来研究脂筏在NHE3转运活性中的作用。通过密度梯度离心，OK细胞中约50%的表面（生物素化）NHE3分布在脂筏中。用甲基 - β - 环糊精（MβCD）破坏脂筏会降低NHE3活性并增加K'(H⁺)(i)，但K(m)((Na⁺))不受影响。补充胆固醇可完全逆转MβCD的作用，但胆固醇的无活性类似物（胆甾烷 - 3β,5α,6β - 三醇）则不能。MβCD对NHE3活性的影响具有特异性，因为它不会改变Na⁺依赖性的L - Ala摄取。MβCD不会改变OK细胞刷状缘的拓扑结构，也不会改变NHE3的表面量，但会大大降低NHE3的内吞速率。抑制磷脂酰肌醇3 - 激酶和MβCD对NHE3活性的影响不是相加的，表明存在共同的抑制机制。相反，8 - 溴 - cAMP和MβCD对NHE3的抑制是相加的，表明抑制NHE3活性的机制不同。在极化的OK细胞中，约50%的刷状缘NHE3和仅约11%的细胞内NHE3存在于脂筏中。总之，脂筏中的NHE3刷状缘池具有功能活性，因为MβCD处理会降低NHE3的基础活性。脂筏池对于正常NHE3活性的多个动力学方面是必需的，包括V(max)和K'(H⁺)(i)，并且对于NHE3转运的多个方面也是必需的，包括至少基础内吞作用和磷脂酰肌醇3 - 激酶依赖性基础胞吐作用。由于NHE3的C末端结构域对其调节是必需的，并且MβCD引起的NHE3动力学变化类似于NHE3的第二信使调节引起的变化，这些结果表明NHE3的C末端可能参与了NHE3对MβCD的敏感性。
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