Human pancreatic duct cells secrete HCO3- ions mediated by a Cl-/HCO3- exchanger and a HCO3- channel that may be a carbonic anhydrase IV (CA IV) in a channel-like conformation. This secretion is regulated by CFTR (Cystic Fibrosis Transmembrane conductance Regulator). In CF cells homozygous for the deltaF508 mutation, the defect in targeting of CFTR to plasma membranes leads to a disruption in the secretion of Cl- and HCO3 ions along with a defective targeting of other proteins. In this study, we analyzed the targeting of membrane CA IV in the human pancreatic duct cell line CFPAC-1, which expresses a deltaF508 CFTR, and in the same cells transfected with the wild-type CFTR (CFPAC-PLJ-CFTR6) or with the vector alone (CFPAC-PLJ6). The experiments were conducted on cells in the stationary phase the polarized state of which was checked by the distribution of occludin and actin. We show that both cell lines express a 35-kDa CA IV at comparable levels. Analysis of fractions of plasma membranes purified on a Percoll gradient evidenced lower levels of CA IV (8-fold) in the CFPAC-1 than in the CFPAC-PLJ-CFTR6 cells. Quantitative analyses showed that 6- to 10-fold fewer cells in the CFPAC-1 cell line exhibited membrane CA IV-immunoreactivity than in the CFPAC-PLJ-CFTR6 cell line. Taken together, these results suggest that the targeting of CA IV to apical plasma membranes is impaired in CFPAC-1 cells. CA IV/gamma-adaptin double labeling demonstrated the presence of CA IV in the trans-Golgi network (TGN) of numerous CFPAC-1 cells, indicating that trafficking was disrupted on the exit face of the TGN. The retargeting of CA IV observed in CFPAC-PLJ-CFTR6 cells points to a relationship between the traffic of CFTR and CA IV. On the basis of these observations, we propose that the absence of CA IV in apical plasma membranes due to the impairment in targeting in cells expressing a deltaAF508 CFTR largely contributes to the disruption in HCO3- secretion in CF epithelia.

中文翻译：

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在表达突变的（deltaF508）CFTR的培养的人胰管细胞中，碳酸酐酶IV对质膜的靶向作用发生了改变。

人胰管细胞分泌由Cl- / HCO3-交换子和HCO3-通道介导的HCO3-离子，该通道可能是呈通道状构象的碳酸酐酶IV（CA IV）。该分泌受CFTR（囊性纤维化跨膜电导调节剂）调节。在针对deltaF508突变纯合的CF细胞中，将CFTR靶向质膜的缺陷导致Cl-和HCO3离子分泌的中断，以及其他蛋白质的靶向缺陷。在这项研究中，我们分析了在表达deltaF508 CFTR的人胰管细胞系CFPAC-1中以及在野生型CFTR（CFPAC-PLJ-CFTR6）或同单独的载体（CFPAC-PLJ6）。实验是在固定相的细胞上进行的，该细胞的极化状态通过occludin和actin的分布进行检查。我们显示这两个细胞系以可比较的水平表达35 kDa CA IV。在Percoll梯度上纯化的质膜级分的分析表明，CFPAC-1中的CA IV水平（8倍）低于CFPAC-PLJ-CFTR6细胞。定量分析显示，CFPAC-1细胞系中的膜CA IV免疫反应性比CFPAC-PLJ-CFTR6细胞系少6至10倍。综上所述，这些结果表明，在CFPAC-1细胞中，CA IV对顶质膜的靶向性受损。CA IV /γ-adaptin双重标记证明了CA IV在众多CFPAC-1细胞的反高尔基网络（TGN）中的存在，表示TGN出口面上的贩运活动受到干扰。在CFPAC-PLJ-CFTR6细胞中观察到的CA IV的重新定向指向CFTR流量与CA IV之间的关系。基于这些观察，我们提出由于表达deltaAF508 CFTR的细胞的靶向作用受损而导致的根尖质膜中缺乏CA IV很大程度上有助于CF上皮细胞HCO3-分泌的破坏。