豚鼠远端结肠推定的H⁺-K⁺-ATP酶的功能表达

Functional expression of putative H+-K+-ATPase from guinea pig distal colon.

作者信息

Asano S, Hoshina S, Nakaie Y, Watanabe T, Sato M, Suzuki Y, Takeguchi N

机构信息

Molecular Genetics Research Center, Toyama Medical and Pharmaceutical University, Toyama 930-01, Japan.

出版信息

Am J Physiol. 1998 Sep;275(3):C669-74. doi: 10.1152/ajpcell.1998.275.3.C669.

DOI:10.1152/ajpcell.1998.275.3.C669

PMID:9730950

Abstract

A guinea pig cDNA encoding the putative colonic H+-K+-ATPase alpha-subunit (T. Watanabe, M. Sato, K. Kaneko, T. Suzuki, T. Yoshida, and Y. Suzuki; GenBank accession no. D21854) was functionally expressed in HEK-293, a human kidney cell line. The cDNA for the putative colonic H+-K+-ATPase was cotransfected with cDNA for either rabbit gastric H+-K+-ATPase or Torpedo Na+-K+-ATPase beta-subunit. In both expressions, Na+-independent, K+-dependent ATPase (K+-ATPase) activity was detected in the membrane fraction of the cells, with a Michaelis-Menten constant for K+ of 0.68 mM. The expressed K+-ATPase activity was inhibited by ouabain, with its IC50 value being 52 microM. However, the activity was resistant to Sch-28080, an inhibitor specific for gastric H+-K+-ATPase. The ATPase was not functionally expressed in the absence of the beta-subunits. Therefore, it is concluded that the cDNA encodes the catalytic subunit (alpha-subunit) of the colonic H+-K+-ATPase. Although the beta-subunit of the colonic H+-K+-ATPase has not been identified yet, both gastric H+-K+-ATPase and Na+-K+-ATPase beta-subunits were found to act as a surrogate for the colonic beta-subunit for the functional expression of the ATPase. The present colonic H+-K+-ATPase first expressed in mammalian cells showed the highest ouabain sensitivity in expressed colonic H+-K+-ATPases so far reported (rat colonic in Xenopus oocytes had an IC50 = 0.4-1 mM; rat colonic in Sf9 cells had no ouabain sensitivity).

摘要

编码假定的结肠H⁺-K⁺-ATP酶α亚基的豚鼠cDNA（T. 渡边、M. 佐藤、K. 金子、T. 铃木、T. 吉田和Y. 铃木；GenBank登录号D21854）在人肾细胞系HEK-293中实现了功能表达。将假定的结肠H⁺-K⁺-ATP酶的cDNA与兔胃H⁺-K⁺-ATP酶或电鳐Na⁺-K⁺-ATP酶β亚基的cDNA共转染。在这两种表达中，在细胞的膜部分检测到了不依赖Na⁺、依赖K⁺的ATP酶（K⁺-ATP酶）活性，K⁺的米氏常数为0.68 mM。所表达的K⁺-ATP酶活性被哇巴因抑制，其IC50值为52 μM。然而，该活性对胃H⁺-K⁺-ATP酶特异性抑制剂Sch-28080具有抗性。在没有β亚基的情况下，该ATP酶未实现功能表达。因此，可以得出结论，该cDNA编码结肠H⁺-K⁺-ATP酶的催化亚基（α亚基）。虽然结肠H⁺-K⁺-ATP酶的β亚基尚未被鉴定，但发现胃H⁺-K⁺-ATP酶和Na⁺-K⁺-ATP酶β亚基都可替代结肠β亚基用于该ATP酶的功能表达。目前首次在哺乳动物细胞中表达的结肠H⁺-K⁺-ATP酶在迄今报道的已表达结肠H⁺-K⁺-ATP酶中显示出最高的哇巴因敏感性（非洲爪蟾卵母细胞中的大鼠结肠IC50 = 0.4 - 1 mM；Sf9细胞中的大鼠结肠对哇巴因不敏感）。