Heterogeneity of chloride channels in the apical membrane of isolated mitochondria-rich cells from toad skin

Abstract

The isolated epithelium of toad skin was disintegrated into single cells by treatment with collagenase and trypsine. Chloride channels of cell- attached and excised inside-out apical membrane-patches of mitochondria-rich cells were studied by the patch-clamp technique. The major population of Cl- channels constituted small 7-pS linear channels in symmetrical solutions (125 mM Cl-). In cell-attached and inside-out patches the single channel i/V-relationship could be described by electrodiffusion of Cl- with a Goldmann-Hodgkin-Katz permeability of P(Cl) = 1.2 x 10-14 - 2.6 x 10-14 cm3 · s-1. The channel exhibited voltage-independent activity and could be activated by cAMP. This channel is a likely candidate for mediating the well known cAMP-induced transepithelial Cl- conductance of the amphibian skin epithelium. Another population of Cl- channels exhibited large, highly variable conductances (upper limit conductances, 150-550 pS) and could be activated by membrane depolarization. A group of intermediate-sized Cl-- channels included: (a) channels (mean conductance, 30 pS) with linear or slightly outwardly rectifying i/V-relationships and activity occurring in distinct 'bursts,' (b) channels (conductance-range, 10-27 pS) with marked depolarization-induced activity, and (c) channels with unresolvable kinetics. The variance of current fluctuations of such 'noisy' patches exhibited a minimum close to the equilibrium-potential for Cl-. With channels occurring in only 38% of sealed patches and an even lower frequency of voltage- activated channels, the chloride conductance of the apical membrane of mitochondria-rich cells did not match quantitatively that previously estimated from macroscopic Ussing-chamber experiments. From a qualitative point of view, however, we have succeeded in demonstrating the existence of Cl- channels in the apical membrane with features comparable to macroscopic predictions, i.e., activation of channel gating by cAMP and, in a few patches, also by membrane depolarization.