Cyclic GMP regulation of a voltage-activated K channel in dissociated enterocytes

Abstract

Enterocytes from the intestinal epithelium of the winter flounder were isolated by collagenase digestion and incubated in flounder Ringer solution. Conventional whole-cell and amphotericin-perforated whole-cell recording techniques were used to characterize the properties of a voltage-activated K current present in dissociated cells. Resting membrane potentials and series resistances were significantly lower (from -23 to -39 mV and 29 to 13 M omega, respectively) when amphotericin was used to achieve the whole-cell configuration. When cells were placed in flounder Ringer solution, held at -80 mV and subsequently stepped to a series of depolarizing voltages (from -70 to 0 mV), an outward current was observed that exhibited inactivation at voltages above -20 mV. This current was sensitive to holding potential and was not activated when the cells were held at -40 mV or above. When cells were bathed in symmetric K Ringer solution and the same voltage protocol was applied to the cell, inward currents were observed in response to the negative intracellular potentials. Reversal potentials at two different extracellular K concentrations were consistent with K as the current-carrying ion. BaCl2 (2 mM) and CsCl (0.5 mM) both produced voltage-dependent blockade of the current when added to the bathing solution. Charybdotoxin (300 nM extracellular concentration) completely blocked the current. The IC50 for charybdotoxin was 50 nM. Cyclic GMP inhibited the voltage-activated current in flounder Ringer and in symmetric K Ringer solution. The cyclic GMP analog, 8-Br cGMP, lowered the threshold for voltage activation and potentiated inactivation of the current at voltages above -40 mV.(ABSTRACT TRUNCATED AT 250 WORDS)