The Journal of General Physiology, Vol 98, 131-161, Copyright © 1991 by The Rockefeller University Press
Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin
JF Garcia-Diaz
Department of Physiology, Boston University School of Medicine, Massachusetts 02118.
Whole-cell and single channel currents were studied in cells from frog (R.
pipiens and R. catesbiana) skin epithelium, isolated by collagenase and
trypsin treatment, and kept in primary cultures up to three days.
Whole-cell currents did not exhibit any significant time-dependent kinetics
under any ionic conditions used. With an external K gluconate Ringer
solution the currents showed slight inward rectification with a reversal
potential near zero and an average conductance of 5 nS at reversal. Ionic
substitution of the external medium showed that most of the cell
conductance was due to K and that very little, if any, Na conductance was
present. This confirmed that most cells originate from inner epithelial
layers and contain membranes with basolateral properties. At voltages more
positive than 20 mV outward currents were larger with K in the medium than
with Na or N-methyl-D-glucamine. Such behavior is indicative of a multi-ion
transport mechanism. Whole-cell K current was inhibited by external Ba and
quinidine. Blockade by Ba was strongly voltage dependent, while that by
quinidine was not. In the presence of high external Cl, a component of
outward current that was inhibited by the anion channel blocker
diphenylamine-2-carboxylate (DPC) appeared in 70% of the cells. This
component was strongly outwardly rectifying and reversed at a potential
expected for a Cl current. At the single channel level the event most
frequently observed in the cell-attached configuration was a K channel with
the following characteristics: inward-rectifying I-V relation with a
conductance (with 112.5 mM K in the pipette) of 44 pS at the reversal
potential, one open and at least two closed states, and open probability
that increased with depolarization. Quinidine blocked by binding in the
open state and decreasing mean open time. Several observations suggest that
this channel is responsible for most of the whole-cell current observed in
high external K, and for the K conductance of the basolateral membrane of
the intact epithelium. On a few occasions a Cl channel was observed that
activated upon excision and brief strong depolarization. The I-V relation
exhibited strong outward rectification with a single channel conductance of
48 pS at 0 mV in symmetrical 112 mM Cl solutions. Kinetic analysis showed
the presence of two open and at least two closed states. Open time
constants and open probability increased markedly with
depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)
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