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,Hideki Sakai

doi:10.1074/jbc.m708429200

Abstract

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 alpha1-subunit (alpha1NaK) 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 alpha1NaK, and it recruited alpha1NaK 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 alpha1NaK in the cells was not significantly altered. On the other hand, the expression of KCC4 induced no association with alpha1NaK. In conclusion, KCC3a forms a functional complex with alpha1NaK in the basolateral membrane of luminal parietal cells, and it up-regulates alpha1NaK in lipid rafts, whereas KCC3a is absent in basal parietal cells.

Highlights

Among the SLC12 family, Naϩ-Kϩ-2ClϪ cotransporter-1 (NKCC1) is present in the basolateral membrane of gastric parietal cells [6]
The band size of the gastric mucosa (180 kDa) was apparently greater than that in the kidney (150 kDa). These results suggest that KCC3a mRNA and protein are predominantly expressed in gastric mucosa
2) KCC3a is associated with ␣1NaK, and both of them are highly localized in lipid rafts

Summary

Introduction

Among the SLC12 family, Naϩ-Kϩ-2ClϪ cotransporter-1 (NKCC1) is present in the basolateral membrane of gastric parietal cells [6]. ATPase activity of LLC-PK1 (30 ␮g of protein) and membrane fractions of the rabbit gastric glands (30 ␮g of protein) and rabbit kidney (3 ␮g of protein) was measured in a 1 ml of solution containing 120 mM NaCl, 15 mM KCl, 3 mM MgSO4, 1 or 3 mM ATP, 50 ␮M SCH 28080, and 40 mM Tris-HCl, pH 7.4, in the presence or absence of 100 ␮M ouabain. KCC3a was colocalized with Naϩ,Kϩ-ATPase ␣1-subunit (␣1NaK) (Fig. 2, J–L), suggesting that it is present in the basolateral membrane of gastric parietal cells.

Results

Conclusion