We studied whether K ؉ -Cl ؊ cotransporters (KCCs) are involved in gastric HCl secretion. We found that KCC4 is expressed in the gastric parietal cells more abundantly at the luminal region of the gland than at the basal region. KCC4 was found in the stimulation-associated vesicles (SAV) derived from the apical canalicular membrane but not in the intracellular tubulovesicles, whereas H ؉ ,K ؉ -ATPase was expressed in both of them. In contrast, KCC1, KCC2, and KCC3 were not found in either SAV or tubulovesicles.

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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 ouabainsensitive 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 associ-ation 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.