Racette K J, Gabriel S E, Gaspar K J, Forsyth G W
University of Saskatchewan, Saskatoon, Canada.
Am J Physiol. 1996 Aug;271(2 Pt 1):C478-85. doi: 10.1152/ajpcell.1996.271.2.C478.
Conductive chloride transport in the small intestine is an important factor controlling fluid movement from the blood to the lumen of the gut. Several proteins with potential conductive chloride ion channel activity are expressed in the enterocyte cell population. However, it is not clear whether one or more than one protein species is normally responsible for mediating conductive chloride transport. We have raised monoclonal antibodies that inhibit conductive chloride transport in apical membrane vesicles prepared from porcine ileal enterocytes. These monoclonal antibodies have been used to identify a unique protein involved with this conductive chloride transport. Here, we report that anti-chloride conductance monoclonal antibodies did not detect any antigen in Western blots of enterocyte apical membrane protein. Dot blotting and immunoprecipitation experiments indicated that the antigen recognized by these monoclonal antibodies was not the cystic fibrosis transmembrane conductance regulator. The antigen was localized to both villus and crypt regions of ileum on immunohistochemistry. A 90-kDa protein species was immunoprecipitated from a primary enterocyte cell line by these monoclonal antibodies. This 90-kDa protein may be a chloride ion channel or may play some regulatory role in conductive chloride transport in enterocyte apical membrane vesicles.
小肠中的氯化物传导性运输是控制液体从血液向肠腔流动的一个重要因素。几种具有潜在氯化物离子通道传导活性的蛋白质在肠上皮细胞群体中表达。然而，尚不清楚是一种还是多种蛋白质通常负责介导氯化物传导性运输。我们制备了能抑制从猪回肠肠上皮细胞制备的顶端膜囊泡中氯化物传导性运输的单克隆抗体。这些单克隆抗体已被用于鉴定一种与这种氯化物传导性运输有关的独特蛋白质。在此，我们报告抗氯化物电导单克隆抗体在肠上皮细胞顶端膜蛋白的蛋白质印迹中未检测到任何抗原。斑点印迹和免疫沉淀实验表明，这些单克隆抗体识别的抗原不是囊性纤维化跨膜电导调节因子。通过免疫组织化学方法，该抗原定位于回肠的绒毛和隐窝区域。这些单克隆抗体从原代肠上皮细胞系中免疫沉淀出一种90 kDa的蛋白质。这种90 kDa的蛋白质可能是一种氯离子通道，或者可能在肠上皮细胞顶端膜囊泡的氯化物传导性运输中发挥某种调节作用。
