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J. Biol. Chem., Vol. 283, Issue 11, 6869-6877, March 14, 2008

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K⁺-Cl^- Cotransporter-3a Up-regulates Na⁺,K⁺-ATPase in Lipid Rafts of Gastric Luminal Parietal Cells^*

Takuto Fujii, Yuji Takahashi, Yasuo Itomi, Kyosuke Fujita, Magotoshi Morii, Yoshiaki Tabuchi, Shinji Asano^¶, Kazuhiro Tsukada^||, Noriaki Takeguchi, and Hideki Sakai¹

From the Departments of Pharmaceutical Physiology and ^||Surgery II, Graduate School of Medicine and Pharmaceutical Sciences, and the Life Science Research Center, University of Toyama, Toyama 930-0194, Japan and the ^¶Department of Molecular Physiology, College of Information Science and Engineering, Ritsumeikan University, Shiga 525-8577, Japan

Gastric parietal cells migrate from the luminal to the basal region of the gland, and they gradually lose acid secretory activity. So far, distribution and function of K⁺-Cl^- cotransporters (KCCs) in gastric parietal cells have not been reported. We found that KCC3a but not KCC3b mRNA was highly expressed, and KCC3a protein was predominantly expressed in the basolateral membrane of rat gastric parietal cells located in the luminal region of the glands. KCC3a and the Na⁺,K⁺-ATPase 1-subunit (1NaK) were coimmunoprecipitated, and both of them were highly localized in a lipid raft fraction. The ouabain-sensitive K⁺-dependent ATP-hydrolyzing activity (Na⁺,K⁺-ATPase activity) was significantly inhibited by a KCC inhibitor (R-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid (DIOA)). The stable exogenous expression of KCC3a in LLC-PK1 cells resulted in association of KCC3a with endogenous 1NaK, and it recruited 1NaK in lipid rafts, accompanying increases of Na⁺,K⁺-ATPase activity and ouabain-sensitive Na⁺ transport activity that were suppressed by DIOA, whereas the total expression level of 1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with 1NaK. In conclusion, KCC3a forms a functional complex with 1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates 1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells.

Received for publication, October 10, 2007 , and in revised form, December 20, 2007.

^* This work was supported in part by Grants-in-Aid for Scientific Research 15390062 and 18390064 from the Japan Society for the Promotion of Science and Grants-in-Aid for Scientific Research 16657042 and 18059012 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ To whom correspondence should be addressed: Dept. of Pharmaceutical Physiology, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. E-mail: sakaih{at}pha.u-toyama.ac.jp.

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