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 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.
Dubinsky
W P
WP
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77225.
Mayorga-Wark
O
O
Garretson
L T
LT
Schultz
S G
SG
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002352:Carrier Proteins / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods*; D007421:Intestine, Small / Q000378:metabolism* / Q000648:ultrastructure; D008081:Liposomes / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D009338:Necturus maculosus / Q000378:metabolism*; D010446:Peptide Fragments / Q000276:immunology; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism*; D011233:Precipitin Tests
D000906:Antibodies; D002352:Carrier Proteins; D008081:Liposomes; D010446:Peptide Fragments; D015221:Potassium Channels; D011188:Potassium