EXPRESSION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN A MODEL EPITHELIUM
EXPRESSION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR IN A MODEL EPITHELIUM
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DOI：
10.1152/ajplung.1994.266.4.l405
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发表时间：
1994-04-01
影响因子：
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通讯作者：
WELSH, MJ
中科院分区：
文献类型：
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作者：
SHEPPARD, DN;CARSON, MR;WELSH, MJ
囊性纤维化跨膜传导调节因子（CFTR）是一种由腺苷3 '，5'-环磷酸（cAMP）依赖性磷酸化和细胞内核苷酸调节的Cl-通道。与其他重组离子通道一样，CFTR的功能通常使用电压钳技术在单细胞中进行研究。然而，由于CFTR通常位于上皮细胞的顶膜中，我们希望开发一种系统来研究在上皮细胞中表达的重组CFTR的功能。我们选择Fischer大鼠甲状腺（FRT）上皮细胞有两个原因。首先，当在可渗透的过滤器支持物上生长时，FRT细胞形成具有高跨上皮电阻的极化上皮。第二，它们的顶膜中没有内源性cAMP调节的Cl-通道。我们表达CFTR在FRT上皮细胞瞬时，使用重组牛痘病毒，或稳定，使用逆转录病毒。为了测量顶端膜Cl-电流，我们用制霉菌素将基底外侧膜渗透到单价离子，并施加大的跨上皮Cl-浓度梯度。cAMP激动剂刺激感染野生型CFTR（vTF-CFTR）的FRT上皮细胞的顶端膜Cl-电流，但不刺激感染对照病毒（vTF 7 -3）或含有Δ F508突变的CFTR（vTF-Δ F508）的FRT上皮细胞的顶端膜Cl-电流。这些Cl-电流具有与表达内源性或重组CFTR的细胞中cAMP激活的Cl-电流相似的性质。然而，CFTR也在基底外侧膜中表达：当我们在跨上皮Cl-浓度梯度存在下透化顶膜时，我们在基底外侧膜中观察到cAMP激活的Cl-电流。免疫细胞化学证实，CFTR是目前在感染FRT上皮细胞的顶侧和基底侧膜。这不是过度表达的结果，因为即使在CFTR的低表达水平下也观察到基底外侧cAMP激活的Cl-电流。因此，在FRT上皮细胞中表达重组CFTR为研究CFTR的功能提供了一个强大而方便的系统。这种方法在检查其他离子通道的功能时也可能证明是有用的。
Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl- channel regulated by adenosine 3',5'-cyclic monophosphate (cAMP)dependent phosphorylation and by intracellular nucleotides. The function of CFTR, like other recombinant ion channels, has generally been studied in single cells using voltage-clamp techniques. However, because CFTR is normally located in the apical membrane of epithelia we wanted to develop a system to study the function of recombinant CFTR expressed in an epithelium. We chose Fischer rat thyroid (FRT) epithelia for two reasons. First, when grown on permeable filter supports, FRT cells form polarized epithelia with a high transepithelial resistance. Second, they have no endogenous cAMP-regulated Cl- channels in their apical membrane. We expressed CFTR in FRT epithelia either transiently, using recombinant vaccinia virus, or stably, using a retrovirus. To measure apical membrane Cl- currents, we permeabilized the basolateral membrane to monovalent ions with nystatin and imposed a large transepithelial Cl- concentration gradient. cAMP agonists stimulated apical membrane Cl- currents in FRT epithelia infected with wild-type CFTR (vTF-CFTR) but not in FRT epithelia infected with either control virus (vTF7-3) or CFTR containing the Delta F508 mutation (vTF-Delta F508). These Cl- currents had properties similar to those of cAMP-activated Cl- currents in cells expressing endogenous or recombinant CFTR. However, CFTR was also expressed in the basolateral membrane: when we permeabilized the apical membrane in the presence of a transepithelial Cl- concentration gradient, we observed cAMP-activated Cl- currents in the basolateral membrane. Immunocytochemistry confirmed that CFTR was present in both the apical and basolateral membranes of infected FRT epithelia. This was not a consequence of overexpression, since basolateral cAMP-activated Cl- currents were observed even at low levels of expression of CFTR. Thus the expression of recombinant CFTR in FRT epithelia provides a powerful and convenient system to study the function of CFTR. This method may also prove useful when examining the function of other ion channels.