Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes.
Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes.
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非洲爪蟾卵母细胞 K-Cl 协同转运蛋白的功能和分子特征。
DOI：
10.1152/ajpcell.2001.281.2.c670
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Gamba,G
中科院分区：
文献类型：
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作者：
Mercado,A;delosHeros,P;Vázquez,N;Meade,P;Mount,DB;Gamba,G
钾-氯共转运体(KCC)在许多细胞和物种中具有广泛的生理作用。我们在这里报道了非洲爪哇的非洲爪哇细胞表达一种与哺乳动物KCC在功能和分子上具有显著相似性的K-Cl共转运蛋白。在等渗条件下，去卵泡卵母细胞在半胱氨酸反应性化合物N-乙基马来酰亚胺(NEM)和氯化汞(HgCl2)激活后，表现出依赖于Cl-−的86Rb+摄取机制。细胞肿胀抑制蛋白磷酸酶-1可以阻止这种K-Cl共转运体的激活，而NEM对转运体的激活不能被磷酸酶抑制所阻断。动力学表征表明，Rb+和Cl−的表观米氏常数分别为2 7.7±3.0和15.4±4.7 mm，对K+迁移的阴离子选择性为：Cl−=PO=BR−>；i−>；SCN−>；卵母细胞钾氯转运体对包括环状利尿剂在内的几种抑制剂很敏感，对速尿和布美他尼的表观半最大抑制值分别为2 0 0和5 0 0μM。从非洲爪鼠卵母细胞RNA中克隆了编码非洲爪鼠K-Cl共转运蛋白的部分基因，其转录本在非洲爪鼠组织中广泛表达。预测的COOH端蛋白片段与哺乳动物KCCs的KCC1/KCC3亚群具有特定的同源性，其功能特征与KCC1最相似(Mercado A，Song L，Vazquez N，mount DB，and Gamba G.J Biol Chem275：30326-30334,2000)。
The K-Cl cotransporters (KCCs) have a broad range of physiological roles, in a number of cells and species. We report here thatXenopus laevisoocytes express a K-Cl cotransporter with significant functional and molecular similarity to mammalian KCCs. Under isotonic conditions, defolliculated oocytes exhibit a Cl−-dependent86Rb+uptake mechanism after activation by the cysteine-reactive compoundsN-ethylmaleimide (NEM) and mercuric chloride (HgCl2). The activation of this K-Cl cotransporter by cell swelling is prevented by inhibition of protein phosphatase-1 with calyculin A; NEM activation of the transporter was not blocked by phosphatase inhibition. Kinetic characterization reveals apparent values for the Michaelis-Menten constant of 27.7 ± 3.0 and 15.4 ± 4.7 mM for Rb+and Cl−, respectively, with an anion selectivity for K+transport of Cl−= PO= Br−> I−> SCN−> gluconate. The oocyte K-Cl cotransporter was sensitive to several inhibitors, including loop diuretics, with apparent half-maximal inhibition values of 200 and 500 μM for furosemide and bumetanide, respectively. A partial cDNA encoding theXenopusK-Cl cotransporter was cloned from oocyte RNA; the corresponding transcript is widely expressed inXenopustissues. The predicted COOH-terminal protein fragment exhibited particular homology to the KCC1/KCC3 subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount DB, and Gamba G.J Biol Chem275: 30326–30334, 2000).