Functional association between K+-Cl- cotransporter-4 and H+,K+-ATPase in the apical canalicular membrane of gastric parietal cells.
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. KCC4 coimmunoprecipitated with H+,K+-ATPase in the lysate of SAV. Interestingly the MgATP-dependent uptake of (36)Cl(-) into the SAV was suppressed by either the H+,K+-ATPase inhibitor (SCH28080) or the KCC inhibitor ((R)-(+)-[(2-n-butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]acetic acid). The KCC inhibitor suppressed the H+ uptake into SAV and the H+,K+-ATPase activity of SAV, but the inhibitor had no effects on these activities in the freeze-dried leaky SAV. These results indicate that the K+-Cl(-) cotransport by KCC4 is tightly coupled with H+/K+ antiport by H+,K+-ATPase, resulting in HCl accumulation in SAV. In the tetracycline-regulated expression system of KCC4 in the HEK293 cells stably expressing gastric H+,K+-ATPase, KCC4 was coimmunoprecipitated with H+,K+-ATPase. The rate of recovery of intracellular pH in the KCC4-expressing cells after acid loading through an ammonium pulse was significantly faster than that in the KCC4-non-expressing cells. Our results suggest that KCC4 and H+,K+-ATPase are the main machineries for basal HCl secretion in the apical canalicular membrane of the resting parietal cell. They also may contribute in part to massive acid secretion in the stimulated state.
Fujii
Takuto
T
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan.
Takahashi
Yuji
Y
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan.
Ikari
Akira
A
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan.
Morii
Magotoshi
M
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan.
Tabuchi
Yoshiaki
Y
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan.
Tsukada
Kazuhiro
K
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan.
Takeguchi
Noriaki
N
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan.
Sakai
Hideki
H
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, the Department of Pharmaco-Biochemistry, University of Shizuoka, Shizuoka 422-8526, Japan, and the Laboratory of Physical Science, Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, Mie 513-8670, Japan. Electronic address: sakaih@pha.u-toyama.ac.jp.
D000818:Animals; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D006851:Hydrochloric Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007093:Imidazoles / Q000494:pharmacology; D051379:Mice; D010295:Parietal Cells, Gastric / Q000166:cytology / Q000378:metabolism*; D054328:Proton Pump Inhibitors; D011817:Rabbits; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism; D013552:Swine; D027981:Symporters / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000096922:K Cl- Cotransporters
D004791:Enzyme Inhibitors; D017506:H(+)-K(+)-Exchanging ATPase; D006851:Hydrochloric Acid; D007093:Imidazoles; D054328:Proton Pump Inhibitors; D027981:Symporters; C119651:SLC12A7 protein, human; D000096922:K Cl- Cotransporters; C492282:Slc12a7 protein, mouse; C492283:Slc12a7 protein, rat; C035235:Sch 28080