PATCH-CLAMP ON THE LUMINAL MEMBRANE OF EXOCRINE GLAND ACINI FROM FROG-SKIN (RANA-ESCULENTA) REVEALS THE PRESENCE OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR-LIKE CL- CHANNELS ACTIVATED BY CYCLIC-AMP

Chloride channels in the luminal membrane of exocrine gland acini fr o m fr og skin (Rana esculenta) constituted a single homogeneous populat ion. In cell-attached patches, channels activated upon exposure to iso proterenol, forskolin, or dibutyrl-cAMP and isobutyl-1-methyl-xanthine rectified in the outward direction with a conductance of 10.0 +/- 0.4 pS for outgoing currents. Channels in stimulated cells reversed at 0 mV applied potential, whereas channels in unstimulated cells reversed at depolarized potentials (28.1 +/- 6.7 mV), indicating that Cl- Mas a bove electrochemical equilibrium in unstimulated, but not in stimulate d, cells. In excised inside-out patches with 25 mM Cl- on the inside, activity of small (8-pS) linear Cl--selective channels was dependent u pon bath ATP (1.5 mM) and increased upon exposure to cAMP-dependent pr otein kinase. The channels displayed a single substate, located just b elow 2/3 of the full channel amplitude. Halide selectivity was identif ied as P-Br > P-1 > P-Cl from the Goldman equation; however, the condu ctance sequence when either halide was permeating the channel was G(Cl ) > G(Br) much greater than G(I). In inside-out patches, the channels were blocked reversibly by 5-nitro-2-(3-phenyl-propylamino) benzoic ac id, glibenclamide, and diphenylamine-3-carboxylic acid, whereas 4, 4-d iisothiocyanatostilbene-2,2-disulfonic acid blocked channel activity c ompletely and irreversibly. Single-channel kinetics revealed one open state (mean lifetime = 158 +/- 72 ms) and two closed states (lifetimes : 12 +/- 1 and 224 +/- 31 ms, respectively). Power density spectra had a double-lorentzian form with corner frequencies 0.85 +/- 0.11 and 27 .9 +/- 2.9 Hz, respectively. These channels are considered homologous to the cystic fibrosis transmembrane conductance regula tor Cl- channe l, which has been localized to the submucosal skin glands in Xenopus b y immunohistochemistry (Engelhardt, J.F., S.S. Smith, E. Allen, J.R. Y ankaskas, D.C. Dawson, and J.M. Wilson. 1994. Ant. J. Physiol. 267: C4 91-C500) and, when stimulated by cAMP-dependent phosphorylation, are s uggested to function in chloride secretion.