AJP - Cell Physiology, Vol 261, Issue 4 C713-C717, Copyright © 1991 by American Physiological Society
Reconstitution of isolated Ca(2+)-activated K+ channel proteins from basolateral membranes of rabbit colonocytes
S. Liu, W. P. Dubinsky, M. K. Haddox and S. G. Schultz
Department of Physiology and Cell Biology, University of Texas Medical School, Houston 77225.
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.
