AJP - Cell Physiology, Vol 254, Issue 4 C512-C518, Copyright © 1988 by American Physiological Society
Sodium channels in membrane vesicles from cultured toad bladder cells
C. Asher, A. Moran, B. C. Rossier and H. Garty
Department of Membrane Research, Weizmann Institute of Science, Rehovot, Israel.
Electrical potential-driven 22Na+ fluxes were measured in membrane vesicles
prepared from TBM-18(c123) cells (a clone of the established cell line
TB-M). Fifty to seventy percent of the tracer uptake in vesicles derived
from cells that were cultivated on a porous support were blocked by the
diuretic amiloride. The amiloride inhibition constant was less than 0.1
microM, indicating that this flux is mediated by the apical Na+-specific
channels. Vesicles prepared from cells that were not grown on a porous
support exhibited much smaller amiloride-sensitive fluxes. Two
Ca2+-dependent processes that down-regulate the channel conductance and
were previously identified in native epithelia were found in the cultured
cells as well. Vesicles isolated from cells that were preincubated with 5 X
10(-7) M aldosterone for 16-20 h exhibited higher amiloride-sensitive
conductance than vesicles derived from control, steroid-depleted cells.
Thus membrane derived from TBM-18(c123) cells can be used to characterize
the epithelial Na+ channel and its hormonal regulation.
