Functional Association between K+-Cl- Cotransporter-4 and H+,K+-ATPase in the Apical Canalicular Membrane of Gastric Parietal Cells
Functional Association between K+-Cl- Cotransporter-4 and H+,K+-ATPase in the Apical Canalicular Membrane of Gastric Parietal Cells
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DOI：
10.1074/jbc.m806562200
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发表时间：
2009-01-02
影响因子：
4.8
通讯作者：
Sakai, Hideki
中科院分区：
文献类型：
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作者：
Fujii, Takuto;Takahashi, Yuji;Sakai, Hideki
我们研究了 K+-Cl- 协同转运蛋白 (KCC) 是否参与胃 HCl 分泌。我们发现 KCC4 在胃壁细胞中的表达量在腺体的管腔区域比在基底区域更丰富。 KCC4 在源自心尖小管膜的刺激相关小泡 (SAV) 中发现，但在细胞内小管小泡中没有发现，而 H+,K+-ATPase 在两者中均表达。相比之下，KCC1、KCC2 和 KCC3 在 SAV 或管状小泡中均未发现。 KCC4 与 SAV 裂解液中的 H+,K+-ATP 酶共免疫沉淀。有趣的是，SAV 中 Cl-36(-) 的 MgATP 依赖性摄取可被 H+、K+-ATP 酶抑制剂 (SCH28080) 或 KCC 抑制剂（(R)-(+)-[(2-n-丁基-6,7-二氯-2-环戊基-2,3-二氢-1-氧代-1H-茚-5-基)氧基]乙酸）抑制。 KCC抑制剂抑制了SAV的H+摄取和SAV的H+,K+-ATP酶活性，但该抑制剂对冻干渗漏SAV中的这些活性没有影响。这些结果表明，KCC4 的 K+-Cl- 共转运与 H+,K (+)-ATPase 的 H+/K+ 反向转运紧密耦合，导致 HCl 在 SAV 中积累。在稳定表达胃H+,K+-ATPase的HEK293细胞中四环素调节的KCC4表达系统中，KCC4与H+,K+-ATPase进行免疫共沉淀。通过铵脉冲加酸后，表达 KCC4 的细胞中细胞内 pH 的恢复速度明显快于不表达 KCC4 的细胞。我们的结果表明，KCC4 和 H+,K+-ATP 酶是静息壁细胞顶小管膜中基础 HCl 分泌的主要机制。它们还可能在一定程度上导致受刺激状态下的大量酸分泌。
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 Cl-36(-) 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-cyclo-pentyl-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-nonexpressing 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.