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

唾液组织中与 Cl 通量相关的新型 rCLCA 分子的生理功能和定位

• 批准号: 17591958
• 负责人: YAMAZAKI Jun
• 金额: $ 2.34万
• 依托单位: Fukuoka Dental College
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17591958/
• 关键词: C1^- channels; salivary tissues; duct; epithelium; endosome; transporting vesicle; saliva; post-translational modification; CLCA; Rab; Ca^<2+>活性化Cl^-チャネル; 細胞内小器官; エンドソ-ム; Cl^-輸送; 顎下腺; 唾液分泌; N型糖鎖; 口腔乾燥症

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.

上皮细胞Cl^-转运对唾液的产生很重要，尽管Ca^2+依赖性Cl ^-转运的分子实体还没有得到很好的表征。我们确定了CLCA家族的新成员，rCLCA，这是强烈表达的下颌下腺（SMG）。预测的ORF编码903-a.a.含有位于细胞外的N-末端区域的蛋白质。用rCLCA cDNA瞬时和稳定转染HEK 293细胞，导致120^-和86-kDa蛋白质在膜组分中表达。后者似乎是前者的蛋白水解产物。链霉亲和素识别的86 kDa的蛋白免疫沉淀的表面生物素化的细胞使用抗rCLCA抗体，这表明蛋白水解产物位于质膜上。用rCLCA转染显示，离子霉素引起了显著的尼氟灭酸敏感性Ca^2+依赖性Cl^-电导，并在I-V关系中具有外向整流。还发现该分子在由其特异性标记物标记的早期和再循环内体中表达。RT-PCR显示rCLCA在胃、肺、肝和SMG中均有表达。SMG的纹状管中检测到强烈的免疫染色，但在腺泡细胞中未检测到。膜部分的免疫印迹产生N-聚糖连接的120-130-和90-kDa条带。为了解决rCLCA是否参与毒蕈碱刺激期间大鼠SMG中Cl^-重吸收的问题，我们评估了用rCLCA siRNA预处理的腺体的唾液的电解质含量。将siRNA逆行注射到给定的下颌下腺导管中显著增加了毛果芸香碱给药后收集的唾液中的Cl^-浓度。这些结果代表了rCLCA在大鼠SMG导管系统跨上皮Ca^<2+>依赖性Cl^-转运中的生理意义。

项目成果

CLCAスプライシング変異型の細胞特異的な発現と細胞接着に対する抑制作用

CLCA剪接变体的细胞特异性表达及其对细胞粘附的抑制作用

• 发表时间: 2008
• 作者: 山崎 純;岡村 和彦;他
• 通讯作者: 他

翻訳後修飾を受けたCa^<2+>活性化Cl^-チャネル関連分子の細胞内局在の差異

翻译后修饰的Ca^2+-激活Cl^-通道相关分子亚细胞定位的差异

• 发表时间: 2007
• 作者: 山崎 純;岡村 和彦;北村 憲司
• 通讯作者: 北村 憲司

ラット顎下腺における CLCAアイソフォームの選択的発現と機能

大鼠颌下腺中 CLCA 亚型的选择性表达和功能

• 发表时间: 2007
• 作者: 山崎 純;岡村 和彦
• 通讯作者: 岡村 和彦

Cloning and Characterization of CLCA Protein Expressed in Ductal Cells of Rat Salivary Glands

大鼠唾液腺导管细胞表达的 CLCA 蛋白的克隆和表征

• 发表时间: 2006
• 作者: Jun Yamazaki;Kazuhiko Okamura;Kazunari Ishibashi;et. al.
• 通讯作者: et. al.

Involvement of apical P2Y2 receptor-regulated CFTR activity in muscarinic stimulation of Cl- reabsorption in rat submandibular gland

• DOI: 10.1152/ajpregu.00758.2007
• 发表时间: 2008-05-01
• 期刊: AMERICAN JOURNAL OF PHYSIOLOGY-REGULATORY INTEGRATIVE AND COMPARATIVE PHYSIOLOGY
• 影响因子: 2.8
• 作者: Ishibashi, Kazunari;Okamura, Kazuhiko;Yamazaki, Jun
• 通讯作者: Yamazaki, Jun