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Laboratoire de Physiologie Cellulaire, URA CNRS 1121, Université Paris XI, Orsay, France.

1. Whole-cell currents were investigated in the model salt-secreting epithelium, human T84 cell line, by means of the perforated patch-clamp technique. In the control extracellular medium containing Cl-, depolarizing voltage ramps evoked current responses which peaked at 5.43 +/- 0.81 pA pF-1 at +60 mV and had a reversal potential (Erev) of -38.4 +/- 2.5 mV (n = 23). 2. Activation of the cAMP pathway with forskolin increased the current at +60 mV from 3.81 +/- 0.61 to 20.79 +/- 5.08 pA pF-1 (n = 18). In thirteen cells, Erev was initially shifted towards positive potentials (Erev of the cAMP-activated initial current was -18.2 +/- 1.2 mV) and subsequently shifted towards more negative potentials, consistent with the activation of both Cl- and K+ currents during cAMP stimulation. 3. Increasing the intracellular Ca2+ concentration, [Ca2+]i, with ionomycin (1 microM) or with acetylcholine (1 microM), increased the current at +60 mV from 7.79 +/- 1.57 to 57.50 +/- 12.10 pA pF-1 (n = 6) and from 6.36 to 34.13 pA pF-1 (n = 4), respectively. With both agonists, Erev was shifted either towards the reversal potential for potassium, EK, or towards the reversal potential for chloride, ECl, depending on the cell. 4. In the absence of chloride ions (gluconate substituted), stimulation of the Ca2+ pathway activated a time-independent outward current of large amplitude. This current exhibited inward rectification at positive voltages, reverted at -89.5 +/- 0.2 mV and was markedly reduced by charybdotoxin (10 nM), a specific blocker of Ca(2+)-activated K+ channels. When a voltage step protocol was used, increased [Ca2+]i also activated an outward current at potentials more positive than -40 mV which slowly relaxed during depolarizing steps. 5. The activation of both (i) a time-dependent inwardly rectifying charybdotoxin-sensitive K+ current, and (ii) a time-dependent slowly inactivating current was also produced by cAMP stimulation. 6. We concluded that (i) in the T84 epithelial cells, both Cl- and K+ currents are concomitantly increased by secretagogue stimulation, and (ii) two different types of K+ conductances are activated by either the cAMP or the intracellular Ca2+ secreting pathways.

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