cGMP-dependent protein kinase regulation of a chloride channel in T84 cells

Chloride channels at the apical membrane of intestinal epithelial cells are involved in the excessive fluid secretion in diarrhea and diminished secretion in cystic fibrosis (CF). Diarrhea induced by heat-stable toxin from Escherichia coli is associated with elevated guanosine 3',5'-cyclic monophosphate (cGMP) in intestinal epithelial cells, but it is unknown whether chloride secretion is regulated by cGMP directly or via cGMP- dependent protein kinase (PKG). Single-channel recordings (inside-out excised patches) from the apical membrane of T84 cells reveal a 10-pS chloride channel with a linear current-voltage relationship, which is opened when an endogenous membrane-bound PKG is activated with ATP (1 mM) and cGMP (100 μM). Soluble PKG (200 nM) isolated from bovine lung, added to the intracellular face of patches, also opens this channel. No activation occurs with Ringer solution alone or only ATP or cGMP. Addition of nonhydrolyzable forms of ATP (AMP-PNP, 1 mM) or a combination of ATP, cGMP, plus H-8 (5 μM), an inhibitor of PKG, also does not stimulate the channel. The catalytic subunit of adenosine 3',5'-cyclic monophosphate-dependent protein kinase (PKA, 200 nM, with 1 mM ATP) activates a channel with similar characteristics. The 10 pS channel has a P(Na)/P(Cl) ratio of 0.06, an anion selectivity of Br^- (1.2) > Cl^- (1.0) > I^- (0.8) > F^- (0.4), and a low affinity for the chloride channel blockers, 4,4-dinitrostilbene-2,2- disulfonic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid. This channel, which is distinctive from the outwardly rectifying chloride channel, has properties similar to the low conductance chloride channel expressed when the CF gene is transfected into nonepithelial cells.