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J. Biol. Chem., Vol. 281, Issue 26, 17845-17855, June 30, 2006

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Na⁺/H⁺ Exchanger NHE3 Activity and Trafficking Are Lipid Raft-dependent^*

From the Departments of Physiology and Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

A previous study showed that 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. 50% of surface (biotinylated) NHE3 in OK cells distributed in LR by density gradient centrifugation. Disruption of LR with methyl--cyclodextrin (MCD) decreased NHE3 activity and increased K'(H⁺)_i, but K_m(Na+) was not affected. The MCD effect was completely reversed by repletion of cholesterol, but not by an inactive analog of cholesterol (cholestane-3,5,6-triol). The MCD effect was specific for NHE3 activity because it did not alter Na⁺-dependent L-Ala uptake. MCD 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 MCD on NHE3 activity were not additive, indicating a common inhibitory mechanism. In contrast, 8-bromo-cAMP and MCD inhibition of NHE3 was additive, indicating different mechanisms for inhibition of NHE3 activity. Approximately 50% of BB NHE3 and only 11% of intracellular NHE3 in polarized OK cells were in LR. In summary, the BB pool of NHE3 in LR is functionally active because MCD 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 MCD resemble those with second messenger regulation of NHE3, these results suggest that the NHE3 C terminus may be involved in the MCD sensitivity of NHE3.

Received for publication, February 23, 2006 , and in revised form, April 11, 2006.

^* This work was supported in part by NIDDK Grants R01-DK26523, R01-DK61765, P01-DK44484, K01-DK62264, and R24-DK64388 (to The Hopkins Basic Research Digestive Diseases Development Core Center) from the National Institutes of Health and by The Hopkins Center for Epithelial Disorders. Part of this work was presented at Digestive Disease Week, New Orleans, LA, May 15–20, 2004, and has been published in abstract form (Murtazina, R., Kovbasnjuk, O., Donowitz, M., and Li, X. (2004) Gastroenterology 126, A295). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² To whom correspondence may be addressed: Dept. of Medicine, Div. of Gastroenterology, The Johns Hopkins University School of Medicine, 925 Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205. Tel.: 410-955-9675; Fax: 410-955-9677; E-mail: mdonowit{at}jhmi.edu. ³ To whom correspondence may be addressed: Dept. of Medicine, Div. of Gastroenterology, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. Tel.: 443-287-4804; Fax: 410-955-9677; E-mail: xuhang{at}jhmi.edu.

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