Immunoisolation of a K+ channel from basolateral membranes of Necturus enterocytes.
Immunoisolation of a K+ channel from basolateral membranes of Necturus enterocytes.
复制标题
Necturus 肠细胞基底外侧膜的 K 通道的免疫分离。
DOI：
10.1152/ajpcell.1993.265.2.c548
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Schultz,SG
中科院分区：
文献类型：
--
作者：
Dubinsky,WP;Mayorga-Wark,O;Garretson,LT;Schultz,SG
我们已经报道了一种由果蝇Shaker B K+通道蛋白的NH 2-末端22个氨基酸组成的肽，该肽负责该A型通道的失活，其阻断了存在于斑花蜜桔小肠肠上皮细胞基底外侧膜中的电压门控K+通道的内部开口。我们现在证明，这种“失活”肽的抗体相互作用的蛋白质溶解和完整的基底外侧膜从Necturus肠细胞。用溶解的基底外侧膜蛋白的完整补体重构的Asolectin囊泡显示86 Rb+摄取，其被四乙基铵离子抑制并通过与这些抗体的免疫沉淀被废除。此外，含有从抗体亲和柱洗脱的蛋白质的asolectin囊泡显示86 Rb+摄取，其通过煮沸而被废除。最后，将免疫分离的蛋白质重建到平面磷脂双层中，揭示了一个K+通道，其单通道特性与天然基底外侧膜中的电压门控通道相同。我们的数据是一致的概念，即150 kDa的蛋白质存在于Necturus肠上皮细胞的基底外侧膜具有内向整流K+通道活性，这种蛋白质是抗原性相似的A型K+通道存在于果蝇的飞行肌肉和编码的振动筛B基因座。
We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsible for the inactivation of this A-type channel, blocks the inner, open mouth of a voltage-gated K+ channel present in the basolateral membrane of Necturus maculosa small intestinal enterocytes. We now demonstrate that antibodies to this "inactivating" peptide interact with proteins in solubilized and intact basolateral membranes from Necturus enterocytes. Asolectin vesicles reconstituted with the full complement of solubilized basolateral membrane proteins display 86Rb+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipitation with these antibodies. Furthermore, asolectin vesicles containing protein eluted from an antibody-affinity column display 86Rb+ uptake that is abolished by boiling. Finally, reconstitution of the immunoisolated protein into planar phospholipid bilayers disclosed a K+ channel whose single-channel properties are identical to those of the voltage-gated channel in the native basolateral membranes. Our data are consistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ channel activity and that this protein is antigenically similar to the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.