Micromolar concentrations of steroids and of aldosterone antagonists inhibit the outwardly rectifying chloride channel with different kinetics

Abstract.

We used the patch-clamp technique to analyse the open/close kinetics of single, outwardly rectifying, intermediate-conductance (ORIC) Cl^– channels from cultured epithelial cells under control conditions and in presence of different inhibitors. As observed previously in excised inside/out patches under control conditions, the switching kinetics were characterized by one open-state time constant (τ_o≈30 ms) and three closed-state time constants (τ_c1≈0.2 ms, τ_c2≈2 ms and τ_c3≈60 ms). Aldosterone, six further steroids and two aldosterone antagonists inhibited channel open probability (NP _o) concentration dependently with the potency at 10 µmol/l increasing in the sequence: hydrocortisone, corticosterone, β-oestradiol, cortisone, aldosterone, testosterone, progesterone, canrenone, spironolactone. Although all substances decreased τ_o, neither the steroids nor the aldosterone antagonists affected τ_c1, τ_c2 or τ_c3 or induced additional transitions with additional time constants. Instead, the steroids increased the prevalence of τ_c2 in the dwell-time histograms and the aldosterone antagonists increased the prevalence of τ_c3, both in a concentration-dependent manner. These observations may be explained by a model in which one open state leads to one of three closed states with rate constants α, β and γ, and in which β or γ increase under the influence of steroids or aldosterone antagonists, respectively. Cytosol, which contains a Cl^– channel inhibitor of unknown molecular structure, (Krick et al., Pflügers Arch 418:491, 1991) was also tested, but the results did not conform to the blocker mechanisms described above. This shows that there are even further modes of channel inhibition and argues against the cytosolic Cl^– channel inhibitor being a steroid.