REGULATION OF A VOLTAGE-DEPENDENT, CALCIUM-ACTIVATED K-CONDUCTANCE BYCYCLIC-GMP IN DISSOCIATED FLOUNDER ENTEROCYTES

Enterocytes from the winter flounder (Pseudopleuronectes americanus) w ere isolated by collagenase digestion and maintained in flounder Ringe r's solution. Whole cell currents were studied using the amphotericin- perforated whole-cell patch clamp technique. The mean resting membrane potential and capacitance values or dissociated cells were -45 +/- 7 mV and 5 +/- 0.4 pF, respectively. Enterocytes held at -20 mV and trea ted with 1 mu mol.1(-1) ionomycin exhibited outward currents when cell s were stepped through a series of voltages from -60 to +110 mV. The r eversal potential of this current in flounder Ringer's solution was -5 5 mV and the voltage at which half-maximal activation occurred was +20 mV. Voltage-dependent inhibition of outward current was observed at 60 mV and above. When cells were bathed in symmetric K Ringer's soluti on the reversal potential shifted to zero mV and no inhibition of curr ent was observed at voltages between -60 and 140 mV. When the holding potential of the cell was changed from -20 to -80 mV and stepped from -60 to +110 mV, a second [previously characterized, O'Grady et al. (19 98)] K current with delayed-rectifier properties was identified. This observation demonstrated that the delayed rectifier K channel and the Ca2+-activated K channel described in this study exist in the same cel l. Extracellular addition of 2 mmol 1(-1) Ba2+ to cells bathed in symm etric K Ringer's solution resulted in nearly complete inhibition of ou tward current. Charybdotoxin produced only minor effects on this curre nt. Addition of 8-Br cGMP to the bathing solution also inhibited outwa rd current and this effect could be partially reversed following washo ut of 8-Br cGMP from the bathing solution. The results of this study i ndicated that a Ca2+-activated K conductance in winter flounder entero cytes is potentially inhibited by agents that increase intracellular c GMP. A similar effect of cGMP on a delayed rectifier K channel in flou nder enterocytes was previously demonstrated.