IMMUNOISOLATION OF A K+ CHANNEL FROM BASOLATERAL MEMBRANES OF NECTURUS ENTEROCYTES

We have reported that a peptide composed of the NH2-terminal 22 amino acids of the Drosophila Shaker B K+ channel protein, which is responsi ble for the inactivation of this A-type channel, blocks the inner, ope n mouth of a voltage-gated K+ channel present in the basolateral membr ane of Necturus maculosa small intestinal enterocytes. We now demonstr ate that antibodies to this ''inactivating'' peptide interact with pro teins in solubilized and intact basolateral membranes from Necturus en terocytes. Asolectin vesicles reconstituted with the full complement o f solubilized basolateral membrane proteins display Rb86+ uptake that is inhibited by tetraethylammonium ion and abolished by immunoprecipit ation with these antibodies. Furthermore, asolectin vesicles containin g protein eluted from an antibody-affinity column display Rb-86+ uptak e that is abolished by boiling. Finally, reconstitution of the immunoi solated protein into planar phospholipid bilayers discloses a K+ chann el whose single-channel properties are identical to those of the volta ge-gated channel in the native basolateral membranes. Our data are con sistent with the notion that a 150-kDa protein present in basolateral membranes of Necturus enterocytes possesses inwardly rectifying K+ cha nnel activity and that this protein is antigenically similar the type A K+ channel present in the flight muscles of Drosophila melanogaster and encoded by the Shaker B locus.