Voltage dependence of cellular current and conductances in frog skin

Abstract

Knowledge of the voltage dependencies of apical and basolateral conductances is important in determining the factors that regulate transcellular transport. To gain this knowledge it is necessary to distinguish between cellular and paracellular currents and conductances. This is generally done by sequentially measuring transepithelial current/voltage (It/Vt) and conductance/voltage (gt/Vt) relationships before and after the abolition of cellular sodium transport with amiloride. Often, however, there are variable time-dependent and voltage-dependent responses to voltage perturbation both in the absence and presence of amiloride, pointing to effects on the paracellular pathway. We have here investigated these phenomena systematically and found that the difficulties were significantly lessened by the use of an intermittent technique, measuring It and gt before and after brief (less than 10 sec) exposure to amiloride at each setting of Vt. I/V relationships were characterized by these means in frog skins (Rana pipiens, Northern variety, and Rana temporaria). Cellular current, Ic, decreased with hyperpolarization (larger serosa positive clamps) of Vt. Derived Ic/Vt relationships between Vt = 0 and 175 mV (serosa positive) were slightly concave upwards. Because values of cell conductance, gc, remained finite, it was possible to demonstrate reversal of Ic. Values of the reversal potential Vr averaged 156 +/- 14 (SD, n = 18) mV. Simultaneous microelectrode measurements permitted also the calculation of apical and basolateral conductances, ga and gb. The apical conductance decreased monotonically with increasing positivity of Vt (and Va). In contrast, in the range in which the basolateral conductance could be evaluated adequately (Vt less than 125 mV), gb increased with more positive values of Vt (and Vb). That is, there was an inverse relation between gb and cellular current at the quasi-steady state, 10-30 sec after the transepithelial voltage step.

Whole-cell and single channel K+ and Cl- currents in epithelial cells of frog skin.

“…With this hypothesis it is possible to estimate the number of channels in a cell from the relation: (Schoen and Erlij, 1985;Nagel et al, 1988). Such behavior is comparable to that of the whole-cell currents in Na medium reported here in the voltage range between reversal and 20 mV.…”

“…Noise analysis of the basolateral membrane has been hampered by the attenuating effect of the high resistance apical membrane (Van Driessche and Hillyard, 1985). Different approaches have been used to elucidate the I-V relation and conductance of the basolateral membrane, with results that do not generally agree (Nagel, 1985;Schoen and Erlij, 1985;Nagel, Garcia-Diaz, and Essig, 1988). Such problems complicate the study of regulation of transport at this membrane and its possible relation with alterations in Na absorption.…”

Effects of cyclic AMP and theophylline on chloride conductance across toad skin.

“…Baseline conductance (at short circuit) was 0 3 mS cm-2 and decreased slightly after clamping Vt to -80 mV. Such a response, which is presumably due to decrease in apical membrane Na+ conductance (Nagel, Garcia-Diaz & Essig, 1988a) associated with minimal increase in voltageactivated gcl, was occasionally observed although it is uncharacteristic for skins from Bufo. (Frog skin is often lacking responsiveness of gcl to voltage activation under control conditions and its sensitization by agents such as theophylline or procaine is also unpredictable; Nagel, 1989;Nagel & Dorge, 1990.)…”

Effects of Aminoperimidine on Electrolyte Transport across Amphibian Skin

“…fR a ) below 0.96 was considered as the limiting value for valid cellular impalement. Since frog skin used for microelectrode analysis had negligible G Cl , correction for paracellular current recirculation [18] was not required.…”

“…Techniques for the determination of intracellular potentials from isolated amphibian skins have been reported previously [18]. In brief, microelectrodes were pulled from borosilicate glass on a Brown-Flaming P80C puller.…”