AJP - Cell Physiology, Vol 265, Issue 2 C548-C555, Copyright © 1993 by American Physiological Society
Immunoisolation of a K+ channel from basolateral membranes of Necturus enterocytes
W. P. Dubinsky, O. Mayorga-Wark, L. T. Garretson and S. G. Schultz
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77225.
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.
