Regulation of a voltage-dependent, calcium-activated K conductance by cyclic GMP in dissociated flounder enterocytes

Enterocytes from the winter flounder (Pseudopleuronectes americanus) were isolated by collagenase digestion and maintained in flounder Ringer'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 treated with 1 μmol·l^-1 ionomycin exhibited outward currents when cells were stepped through a series of voltages from-60 to +110 mV. The reversal potential of this current in flounder Ringer's solution was-55 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 solution the reversal potential shifted to zero mV and no inhibition of current 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. (1991)] 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 cell. Extracellular addition of 2 mmol·l^-1 Ba²⁺ to cells bathed in symmetric K Ringer's solution resulted in nearly complete inhibition of outward current. Charybdotoxin produced only minor effects on this current. Addition of 8-Br cGMP to the bathing solution also inhibited outward current and this effect could be partially reversed following washout of 8-Br cGMP from the bathing solution. The results of this study indicated that a Ca²⁺-activated K conductance in winter flounder enterocytes is potentially inhibited by agents that increase intracellular cGMP. A similar effect of cGMP on a delayed rectifier K channel in flounder enterocytes was previously demonstrated.