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Cell K activity, acK, was measured in the short-circuited frog skin by simultaneous cell punctures from the apical surface with open-tip and K-selective microelectrodes. Strict criteria for acceptance of impalements included constancy of the open-tip microelectrode resistance, agreement within 3% of the fractional apical voltage measured with open-tip and K-selective microelectrodes, and constancy of the differential voltage recorded between the open-tip and the K microelectrodes 30-60 sec after application of amiloride or substitution of apical Na. Skins were bathed on the serosal surface with NaCl Ringer and, to reduce paracellular Cl conductance and effects of amiloride on paracellular conductance, with NaNO3 Ringer on the apical surface. Under control conditions acK was nearly constant among skins (mean +/- SD = 92 +/- 8 mM, 14 skins) in spite of a wide range of cellular currents (5 to 70 microA/cm2). Cell current (and transcellular Na transport) was inhibited by either apical addition of amiloride or substitution of Na by other cations. Although in some experiments the expected small increase in acK after inhibition of cell current was observed, on the average the change was not significant (98 +/- 11 mM after amiloride, 101 +/- 12 mM after Na substitution), even 30 min after the inhibition of cell current. The membrane potential, which in the control state ranged from -42 to -77 mV, hyperpolarized after inhibition of cell current, initially to -109 +/- 5 mV, then depolarizing to a stable value (-88 +/- 5 mV) after 15-25 min. At this time K was above equilibrium (EK = 98 +/- 2 mV), indicating that the active pump mechanism is still operating after inhibition of transcellular Na transport. The measurement of acK permitted the calculation of the passive K current and pump current under control conditions, assuming a "constant current source" with almost all of the basolateral conductance attributable to K. We found a significant correlation between pump current and cell current with a slope of 0.31, indicating that about one-third of the cell current is carried by the pump, i.e., a pump stoichiometry of 3Na/2K.

In studies of apical membrane current-voltage relationships, in order to avoid laborious intracellular microelectrode techniques, tight epithelia are commonly exposed to high serosal K concentrations. This approach depends on the assumptions that high serosal K reduces the basolateral membrane resistance and potential to insignificantly low levels, so that transepithelial values can be attributed to the apical membrane. We have here examined the validity of these assumptions in frog skins (Rana pipiens pipiens). The skins were equilibrated in NaCl Ringer's solutions, with transepithelial voltage Vt clamped (except for brief perturbations delta Vt) at zero. The skins were impaled from the outer surface with 1.5 M KCl-filled microelectrodes (Rel greater than 30 M omega). The transepithelial (short-circuit) current It and conductance gt = -delta It/delta Vt, the outer membrane voltage Vo (apical reference) and voltage-divider ratio (Fo = delta Vo/delta Vt), and the microelectrode resistance Rel were recorded continuously. Intermittent brief apical exposure to 20 microM amiloride permitted estimation of cellular (c) and paracellular (p) currents and conductances. The basolateral (inner) membrane conductance was estimated by two independent means: either from values of gt and Fo before and after amiloride or as the ratio of changes (-delta Ic/delta Vi) induced by amiloride. On serosal substitution of Na by K, within about 10 min, Ic declined and gt increased markedly, mainly as a consequence of increase in gp. The basolateral membrane voltage Vi (= -Vo) was depolarized from 75 +/- 4 to 2 +/- 1 mV [mean +/- SEM (n = 6)], and was partially repolarized following amiloride to 5 +/- 2 mV.(ABSTRACT TRUNCATED AT 250 WORDS)