Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase.
Regulation of Cl- transport in T84 cell clones expressing a mutant regulatory subunit of cAMP-dependent protein kinase.
复制标题
表达 cAMP 依赖性蛋白激酶突变调节亚基的 T84 细胞克隆中 Cl- 转运的调节。
DOI：
10.1073/pnas.87.22.8975
复制
发表时间：
1990
影响因子：
11.1
通讯作者：
McKnight,GS
中科院分区：
文献类型：
--
作者：
Rogers,KV;Goldman,PS;Frizzell,RA;McKnight,GS
分泌盐的上皮细胞的顶端膜中的Cl-通道被cAMP和Ca 2+第二信使系统激活，并且在囊性纤维化患者中已经证明了它们的激素调节功能障碍。我们用含有cAMP依赖性蛋白激酶调节亚基的突变形式的表达载体转染了上皮细胞系T84。表达该构建体的稳定转化体具有降低的基础cAMP依赖性蛋白激酶活性，并且响应于cAMP而不增加激酶活性超过对照细胞的基础水平。毛喉素、血管活性肠肽和前列腺素E2均刺激突变体和对照克隆中的细胞内cAMP积累;然而，响应于升高的cAMP的Cl-通道的激活在突变体克隆中被阻断，表明cAMP依赖性蛋白激酶的直接参与。相反，Ca 2+离子载体保留其激活表达突变调控亚基的T84细胞中的Cl-通道的能力，表明通过Ca 2+激活通道不需要cAMP依赖性蛋白激酶活性的参与。这些克隆将有助于进一步研究盐分泌上皮细胞中cAMP-和Ca 2（+）-依赖性调节途径之间的相互作用。它们也可用于识别与cAMP第二信使途径相互作用的Ca 2（+）依赖性Cl-通道激活的介质。
Cl- channels in the apical membranes of salt-secreting epithelia are activated by both cAMP and Ca2+ second-messenger systems, and dysfunctions in their hormonal regulation have been demonstrated in patients with cystic fibrosis. We have transfected the epithelial cell line T84 with an expression vector containing a mutant form of the regulatory subunit of the cAMP-dependent protein kinase. Stable transformants that express this construct have reduced basal cAMP-dependent protein kinase activity and do not increase kinase activity beyond the basal level of control cells in response to cAMP. Forskolin, vasoactive intestinal peptide, and prostaglandin E2 each stimulate intracellular cAMP accumulation in both mutant and control clones; however, the activation of Cl- channels in response to elevated cAMP is blocked in mutant clones, indicating direct involvement of the cAMP-dependent protein kinase. In contrast, Ca2+ ionophores retain their ability to activate the Cl- channel in T84 cells expressing the mutant regulatory subunit, suggesting that activation of the channel by means of Ca2+ does not require the participation of cAMP-dependent protein kinase activity. These clones will be useful for further studies of the interactions between the cAMP- and Ca2(+)-dependent regulatory pathways in salt-secreting epithelial cells. They can also be used to identify the mediators of Ca2(+)-dependent Cl- channel activation in isolation from interactions with the cAMP second-messenger pathway.