Characterization of a phosphorylation‐activated Cl‐selective channel in isolated Necturus enterocytes.

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Abstract
1. The cell-attached and excised inside-out configurations of the patch-clamp technique were employed to probe isolated enterocytes of Necturus maculosus for the presence of Cl--selective channels. 2. Chloride-selective channels were rarely observed unless cells were previously stimulated by agonists that raise cyclic AMP. In cell-attached patches forskolin (20 .mu.m) or dibutyryl cyclic AMP (2 mm) evoked single-channel activity that reversed, depending on the cell, between 9 and 27 mV positive to the spontaneous membrane potential. This is close to the Cl- equilibrium potential in these cells; the single-channel current-voltage relationship was linear with a unitary slope conductance between 17 and 25 pS (pipettes filled with 100 mm-NaCl). 3. Large depolarizing voltage steps also activated Cl- channels in excised inside-out membrane patches that were previously quiescent. This mode of activation produced a distinctive single-channel current-voltage relationship with strong outward rectification at depolarizing membrane potentials. Single-channel cord conductance at negative potentials was 15-18 pS and increased to 45 pS at +100 mV. 4. Altering the Cl- concentration in the bathing solution of excised inside-out patches displaced the observed reversal potential (Erev) to values predicted for Cl- equilibrium potential. Replacement of K+ for Na+ was without effect. 5. The effect of different anions upon Erev was used to determine the channel anion selectivity in excised inside-out patches. The permeability sequence was SCN- > I- > Br- > Cl- > F- > HCO3- > gluconate which corresponds to Eisenman''s sequence 1. Neither ionic size nor diffusion rates determine the permeation of ions through the channel. 6. In channels activated by depolarization the open probability (Po) was insensitive to changes in the Ca2+ concentration (> 10-8-10-3 M) bathing the cytoplasmic face of excised inside-out patches. Depolarization was also without marked effect on Po. 7. Chloride channels in excised inside-out patches were inhibited by stilbene and diphenylamine-2-carboxylate derivatives. 4-Acetamido-4''-isothiocyanatostilbene-2,2-disulphonic acid (SITS, 5 .times. 10-5 M) and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB, 1 .times. 10-5 M) caused an irreversible ''flickery'' blockade without altering single-channel current. 3''5-Dichlorodiphenylamine-2-carboxylic acid (DDPC, 5 .times. 10-5 M) reduced the currents at every voltage without apparent effects on the gating properties of the channel. 8. Channel activity could be evoked in excised inside-out membrane patches by exposure to the purified catalytic subunit of cyclic AMP-dependent protein kinase in the presence of ATP. Channel phosphorylation therefore appears to be a prerequisite for activation. The single-channel current-voltage relationship of the kinase-A-activated channel resembled that evoled by forskolin in cell-attached patches and that produced by strong depolarization but in this case, only at negative potentials. 9. The present study demonstrates the presence of Cl- selective channels in epithelial cells of the small intestine. These channels may underly the apical membrane Cl- conductance observed in Necturus enterocytes and might be responsible for salt secretion.