Finn A L, Tsai L M, Falk R J
Department of Medicine, University of North Carolina, Chapel Hill 27599.
Proc Natl Acad Sci U S A. 1989 Oct;86(19):7649-52. doi: 10.1073/pnas.86.19.7649.
Monoclonal antibodies raised by injecting Necturus gallbladder cells into mice were tested for their ability to inhibit the apical chloride conductance induced by elevation of cellular cAMP. Five of these monoclonal antibodies bound to the apical cells, as shown by indirect immunofluorescence microscopy, and inhibited the chloride conductance; one antibody that bound only to subepithelial smooth muscle, by indirect immunofluorescence microscopy, showed no inhibition of chloride transport. The channel or a closely related molecule is present in the membrane whether or not the pathway is open, since, in addition to inhibiting the conductance of the open channel, the antibody also bound to the membrane in the resting state and prevented subsequent opening of the channel. The antibody was shown to recognize, by ELISA, epitopes from the Necturus gallbladder and small intestine. Finally, by Western blot analysis of Necturus gallbladder homogenates, the antibody was shown to recognize two protein bands of Mr 219,000 and Mr 69,000. This antibody should permit isolation and characterization of this important ion channel.
将美西螈胆囊细胞注射到小鼠体内产生的单克隆抗体，对其抑制细胞内环磷酸腺苷（cAMP）升高所诱导的顶端氯离子传导的能力进行了测试。通过间接免疫荧光显微镜观察，这些单克隆抗体中有五种与顶端细胞结合，并抑制了氯离子传导；通过间接免疫荧光显微镜观察，有一种仅与上皮下平滑肌结合的抗体，未显示出对氯离子转运的抑制作用。无论该通道是否开放，通道或与之密切相关的分子都存在于膜中，因为除了抑制开放通道的传导外，该抗体还与静息状态下的膜结合，并阻止通道随后的开放。通过酶联免疫吸附测定（ELISA）表明，该抗体可识别来自美西螈胆囊和小肠的表位。最后，通过对美西螈胆囊匀浆的蛋白质印迹分析，该抗体显示可识别分子量为219,000和69,000的两条蛋白带。这种抗体应有助于分离和鉴定这种重要的离子通道。
