Physiological function and localization of a novel rCLCA molecule related to Cl-flux in salivary tissues

已知上皮氯离子转运对唾液的产生很重要，尽管依赖于钙离子的氯离子转运的分子实体还没有得到很好的描述。我们发现了CLCA家族的一个新成员，rCLCA，它在颌下腺(SMG)中强烈表达。预测的ORF编码903-A.A.一种含有N-末端区域的蛋白质，位于细胞外。瞬时稳定地将rCLCA基因导入HEK293细胞，可在细胞膜部分表达120 kDa和86 kDa的蛋白质。后者似乎是前者的蛋白质分解产物。链霉亲和素用抗rCLCA抗体识别从表面生物素标记的细胞中免疫沉淀的86 kDa蛋白，表明蛋白水解物位于质膜。转染rCLCA后，尼氟米酸敏感的钙离子电导在I-V关系中呈外向整流。这个分子也被发现在早期和循环中的内体中表达，这些内体由其特定的标记标记。RT-PCR检测到rCLCA在胃、肺、肝脏和SMG中均有表达。在SMG的纹状管中有较强的免疫染色，而在腺泡细胞中未见表达。免疫印迹的膜部分产生N-糖链连接的120-130和90 kDa的条带。为了探讨rCLCA是否参与大鼠SMG在毒鼠碱刺激过程中对Cl~+的重吸收，我们测定了rCLCA siRNA处理的腺体唾液中的电解质含量。颌下管逆行注射siRNA后，唾液中的氯离子浓度显著增加。这些结果表明，rCLCA在大鼠SMG导管系统依赖的跨上皮钙离子转运中具有生理意义。

Epithelial Cl^- transport is known to be important for saliva production although a molecular entity for Ca^<2+>-dependent C1^- transport has not been well characterized. We identified a new member of the CLCA family, rCLCA, which is strongly expressed in the submandibular gland (SMG). The predicted ORF encodes a 903-a.a. protein containing an N-terminal region located extracellularly. Transient and stable transfection of HEK293 cells with rCLCA cDNA resulted in expression of 120^- and 86-kDa proteins in the membrane fraction. The latter appeared to be a proteolytic product of the former. Streptavidin recognized the 86-kDa protein immunoprecipitated from surface-biotinylated cells using the anti-rCLCA antibody, suggesting that the proteolytic product locates on the plasma membrane. The transfection with rCLCA revealed a marked niflumic acid-sensitive Ca^<2+>-dependent Cl^- conductance evoked by ionomycin with an outward rectification in the I-V relationship. This molecule was also found to be expressed in early and recycling endosomes labeled by their specific markers. RT-PCR revealed expression of rCLCA in the stomach, lung and liver as well as the SMG. Intense immunostaining was detected in the striated ducts of SMG, but not in the acinar cells. Immunoblot of the membrane fraction yielded N-glycan-linked 120-130- and 90-kDa bands. To address the question as to whether rCLCA is involved in Cl^- reabsorption in rat SMG during muscarinic stimulation, we evaluated the electrolyte content of saliva from the glands pretreated with rCLCA siRNA. Retrograde injection into a given submandibular duct with the siRNA significantly increased Cl^- concentration in saliva collected after administration of pilocarpine. These results represent a physiological significance of rCLCA in transepithelial Ca^<2+>-dependent Cl^- transport in the ductal system of rat SMG.