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

We used the patch-clamp technique to analyse the open/close kinetics of sin gle, outwardly rectifying, intermediate-conductance (ORIC) Cl- channels fro m cultured epithelial cells under control conditions and in presence of dif ferent inhibitors. As observed previously in excised inside/out patches und er control conditions, the switching kinetics were characterized by one ope n-state time constant (tau(o)approximate to 30 ms) and three closed-state t ime constants (tau(c1)approximate to 0.2 ms, tau(c2)approximate to 2 ms and tau(c3)approximate to 60 ms). Aldosterone, six further steroids and two al dosterone antagonists inhibited channel open probability (NPo) concentratio n dependently with the potency at 10 mu mol/l increasing in the sequence: h ydrocortisone, corticosterone, P-oestradiol, cortisone, aldosterone, testos terone, progesterone, canrenone, spironolactone. Although all substances de creased tau(o), neither the steroids nor the aldosterone antagonists affect ed tau(c1), tau(c2), or tau(c3) or induced additional transitions with addi tional time constants. Instead, the steroids increased the prevalence of ta u(c2) in the dwell-time histograms and the aldosterone antagonists increase d the prevalence of tau(c3), both in a concentration-dependent manner. Thes e observations may be explained by a model in which one open state leads to one of three closed states with rate constants alpha, beta and gamma, and in which beta or gamma increase under the influence of steroids or aldoster one antagonists, respectively. Cytosol, which contains a Cl- channel inhibi tor of unknown molecular structure, (Krick et al., Pflugers Arch 418:491, 1 991) was also tested, but the results did not conform to the blocker mechan isms described above. This shows that there are even further modes of chann el inhibition and argues against the cytosolic Cl- channel inhibitor being a steroid.