Reconstitution of isolated Ca(2+)-activated K+ channel proteins from basolateral membranes of rabbit colonocytes.
Using calmodulin-affinity chromatography, we have isolated a fraction of proteins from solubilized basolateral membranes of rabbit colonocytes which when reconstituted into planar phospholipid bilayers disclosed Ca(2+)-activated single K+ channel activities. The properties of the reconstituted channels are identical to those of native membrane vesicles incorporated into these bilayers with respect to their high selectivity for K+ over C-, high ("maxi") conductance, voltage gating, and inhibition by trifluoperazine. Two-dimensional sodium dodecyl sulfate gel electrophoresis of these proteins revealed three major protein species with molecular masses of 120, 60, and 35 kDa, which constituted 70, 10, and 20%, respectively, of the total protein. The results of other studies strongly suggest that the 35-kDa protein may be the Ca(2+)-activated K+ channel protein in these membranes.
Liu
S
S
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77225.
Dubinsky
W P
WP
Haddox
M K
MK
Schultz
S G
SG
D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin; D002846:Chromatography, Affinity; D003106:Colon / Q000737:chemistry* / Q000166:cytology; D004594:Electrophysiology; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000378:metabolism*; D010743:Phospholipids; D015221:Potassium Channels / Q000378:metabolism* / Q000502:physiology; D011817:Rabbits
D002147:Calmodulin; D008051:Lipid Bilayers; D008565:Membrane Proteins; D010743:Phospholipids; D015221:Potassium Channels; D002118:Calcium