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

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 ␣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.

Molecular evidence for a role for K+-Cl− cotransporters in the kidney

“…The expression of these transporters has been documented in several epithelial cells either at the apical or basolateral membranes (17). Recent work has suggested an important role for KCC3 and KCC4 in acid secretion in the stomach (13)(14)(15). KCC3 knockout mice develop arterial hypertension (44), and KCC4 knockout mice exhibit renal tubular acidosis (6).…”

Functional Association between K+-Cl- Cotransporter-4 and H+,K+-ATPase in the Apical Canalicular Membrane of Gastric Parietal Cells

“…KCC3a and KCC4 are mainly expressed in epithelial-type cells (13,14). Recently we found that KCC3a is expressed in the basolateral membrane of gastric parietal cells located at the luminal region of gastric glands and coimmunoprecipitated with Na ϩ ,K ϩ -ATPase (15). Exogenous expression of KCC3a in LLC-PK1 cells up-regulates Na…”

Function of K+-Cl- Cotransporters in the Acid Secretory Mechanism of Gastric Parietal Cells

“…The exogenous expression of KCC3a recruited a1NaK into lipid rafts, accompanying an increase of the ATP-hydrolyzing activity and the phosphorylation level of Na ϩ , K ϩ -ATPase. 18) In contrast, exogenously expressed KCC3b did not co-immunoprecipitate with a1NaK, accompanying no change in the Na (Fig. 2), and the NH 2 -terminus of KCC3a may be a key region for association with Na (Fig.…”

“…Recently we found that KCC3a and KCC4 were expressed in the basolateral and apical canalicular membranes of gastric parietal cells, respectively 18,19) (Fig. 1).…”

“…Therefore, KCC3a may be coupled with a1NaK and up-regulates the pump activity in lipid rafts of the cells. 18) In the tetracycline-inducing expression system of KCC3a in …”