Roles of external and cellular Cl− ions on the activation of an apical electrodiffusional Cl− pathway in toad skin

Abstract

This study is concerned with the short-circuit current, Isc, responses of the Cl(-)-transporting cells of toad skin submitted to sudden changes of the external Cl- concentration, [Cl]o. Sudden changes of [Cl]o, carried out under apical membrane depolarization, allowed comparison of the roles of [Cl]o and [Cl]cell on the activation of the apical Cl- pathways. Equilibration of short-circuited skins symmetrically in K-Ringer's solutions of different Cl- concentrations permitted adjustment of [Cl]cell to different levels. For a given Cl- concentration (in the range of 11.7 to 117 mM) on both sides of a depolarized apical membrane, this structure exhibits a high Cl- permeability, P(Cl)apical. On the other hand, for the same range of [Cl]cell but with [Cl]o = 0, P(Cl)apical is reduced to negligible values. These observations indicate that when the apical membrane is depolarized P(Cl)apical is modulated by [Cl]o; in the absence of external Cl- ions, intracellular Cl- is not sufficient to activate P(Cl)apical. Computer simulation shows that the fast Cl- currents induced across the apical membrane by sudden shifts of [Cl]o from a control equilibrium value strictly follow the laws of electrodiffusion. For each experimental group, the computer-generated Isc versus [( Cl]cell - [Cl]o) curve which best fits the experimental data can only be obtained by a unique pair of P(Cl)apical and Rb (resistance of the basolateral membrane), thus allowing the calculation of these parameters. The electrodiffusional behavior of the net Cl- flux across the apical membrane supports the channel nature of the apical Cl- pathways in the Cl(-)-transporting cells. Cl- ions contribute significantly to the overall conductance of the basolateral membrane even in the presence of a high K concentration in the internal solution.

Fast and slow voltage modulation of apical Cl- permeability in toad skin at high [K+]

“…With the apical membrane depolarized by transepithelial short-circuit and high-[K + ] Ringer, apical Cl -channels should be in a state of quasi-maximal Cl -permeability activation (9,10,19). This probably explains why there is no significant increase of current after the fast initial current spike following a negative pulse.…”

“…As mentioned above, low cytoplasmic Cl -concentration leads to inactivation of apical P Cl (10), and conditions associated ] cell is presumably high. This favors apical P Cl activation, as demonstrated by a large current response to pulsing the skin voltage to either sign.…”

“…It is known that hyperpolarization of toad skin epithelium leads to a slow activation of a Cl -conductive pathway (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12). Models based on slow voltage activation of apical Cl -channels have been proposed in order to explain this observation (5,7,9).…”

“…Also, there is ample evidence that Cl -ions move through a specialized cell compartment consisting of the mitochondrion-rich cells (MR-cells) (1,2,(13)(14)(15). Evidence for voltage modulation was investigated by us (10,16; Procopio J, unpublished data) with the conclusion that Cl -permeability in the apical membrane of the Cl -transporting cells is not exclusively dependent on voltage, but requires additional presence of Cl -ions in either the external solution or in the cytoplasm (10,(16)(17)(18). The exact role of cytoplasmic Cl -ions in the modulation of apical Cl -permeability is still unknown.…”