Colonic H-K-ATPase β-subunit: identification in apical membranes and regulation by dietary K depletion.

Colonic H-K-ATPase β-subunit: identification in apical membranes and regulation by dietary K depletion.
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结肠 H-K-ATP 酶 β 亚基:顶膜中的识别和饮食 K 消耗的调节。

DOI:
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发表时间:
1999
期刊:
American Journal of Physiology - Cell Physiology
影响因子:
--
通讯作者:
H. Binder
H. Binder
中科院分区:
--
文献类型:
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作者:
P. Sangan;S. Kolla;V. Rajendran;M. Kashgarian;H. Binder

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P 型 ATP 酶需要 α 和 β 亚基才能发挥功能。虽然结肠顶膜 H-K-ATP 酶 (HKcα) 的 α 亚基已被鉴定和研究,但其 β 亚基尚未鉴定。我们克隆了假定的 β 亚基大鼠结肠 H-K-ATP 酶 (HKcβ) cDNA,该 cDNA 编码具有单个跨膜结构域的 279 个氨基酸蛋白质,并且与其他大鼠 β 亚基具有序列同源性。 Northern印迹分析表明,该HKcβ在多种大鼠组织中表达,包括远端和近端结肠,并且在睾丸和肺中高表达。在 K-耗尽大鼠的远端结肠中,HKcβ mRNA 丰度比正常值上调三倍,但在近端结肠、睾丸或肺中则没有。相反,K-耗尽大鼠远端结肠中 Na-K-ATPase β1 mRNA 丰度没有改变。 Na 消耗也会刺激远端结肠的活性 K 吸收,但不会增加 HKcβ mRNA 丰度。使用针对谷胱甘肽 S-转移酶-HKcβ 融合蛋白的多克隆抗体进行蛋白质印迹分析,确定了大鼠远端结肠顶膜和基底外侧膜中 45 kDa HKcβ 蛋白的表达,但 K 耗竭仅增加顶膜中 HKcβ 蛋白的表达。 HKcβ 和 HKcα 蛋白之间的物理关联通过使用 HKcβ 抗体对大鼠远端结肠顶膜和 HKcα 抗体进行免疫沉淀进行的蛋白质印迹分析来证明。该 β 亚基 mRNA 响应 K 耗竭的组织特异性上调、其蛋白质的定位、远端结肠顶膜 K 耗竭导致的上调以及其与 HKcα 蛋白的物理关联,提供了令人信服的证据,表明 HKcβ 是结肠 H-K-ATP 酶的推定 β 亚基。
P-type ATPases require both α- and β-subunits for functional activity. Although an α-subunit for colonic apical membrane H-K-ATPase (HKcα) has been identified and studied, its β-subunit has not been identified. We cloned putative β-subunit rat colonic H-K-ATPase (HKcβ) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat β-subunits. Northern blot analysis demonstrates that this HKcβ is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKcβ mRNA abundance is upregulated threefold compared with normal in distal colon but not proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase β1 mRNA abundance is unaltered in distal colon of K-depleted rats. Na depletion, which also stimulates active K absorption in distal colon, does not increase HKcβ mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKcβ fusion protein established expression of a 45-kDa HKcβ protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKcβ protein expression only in apical membranes. Physical association between HKcβ and HKcα proteins was demonstrated by Western blot analysis performed with HKcβ antibody on immunoprecipitate of apical membranes of rat distal colon and HKcα antibody. Tissue-specific upregulation of this β-subunit mRNA in response to K depletion, localization of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKcα protein provide compelling evidence that HKcβ is the putative β-subunit of colonic H-K-ATPase.
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