J. Gen. Physiol., Volume 112, Number 1, July 1, 1998 19-31

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

Jakob Balslev Sørensen, and Erik Hviid Larsen

From the August Krogh Institute, University of Copenhagen, DK-2100 Copenhagen Ø, Denmark

Chloride channels in the luminal membrane of exocrine gland acini from frog skin (Rana esculenta) constituted a single homogeneous population. In cell-attached patches, channels activated upon exposure to isoproterenol, forskolin, or dibutyryl-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 was above electrochemical equilibrium in unstimulated, but not in stimulated, cells. In excised inside-out patches with 25 mM Cl on the inside, activity of small (8-pS) linear Cl-selective channels was dependent upon bath ATP (1.5 mM) and increased upon exposure to cAMP-dependent protein kinase. The channels displayed a single substate, located just below 2/3 of the full channel amplitude. Halide selectivity was identified as P_Br > P_I > P_Cl from the Goldman equation; however, the conductance sequence when either halide was permeating the channel was G_Cl > G_Br >> G_I. In inside-out patches, the channels were blocked reversibly by 5-nitro-2-(3-phenylpropylamino)benzoic acid, glibenclamide, and diphenylamine-2-carboxylic acid, whereas 4,4-diisothiocyanatostilbene-2,2-disulfonic acid blocked channel activity completely and irreversibly. Single-channel kinetics revealed one open state (mean lifetime = 158 ± 72 ms) and two closed states (lifetimes: 12 ± 4 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 regulator Cl channel, which has been localized to the submucosal skin glands in Xenopus by immunohistochemistry (Engelhardt, J.F., S.S. Smith, E. Allen, J.R. Yankaskas, D.C. Dawson, and J.M. Wilson. 1994. Am. J. Physiol. 267: C491-C500) and, when stimulated by cAMP-dependent phosphorylation, are suggested to function in chloride secretion.

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