EVIDENCE FROM INCORPORATION EXPERIMENTS FOR AN ANIONIC CHANNEL OF SMALL-CONDUCTANCE AT THE APICAL MEMBRANE OF THE RABBIT DISTAL TUBULE

Many of the hormone-regulated ion transport processes in distal nephro n involve transcellular pathways which require a passive entry of ions at the apical membrane of the distal tubule cells. To investigate mol ecular mechanisms underlying the ionic permeability of the distal tubu le apical membrane, a study was undertaken in which vesicles prepared from apical membranes from isolated rabbit distal tubules were fused o nto a planar lipid bilayer. These experiments led to the identificatio n of several ionic channels including a Cl--permeable channel of 14 pS with a Na+ over Cl- permeability ratio, P-Na/P-Cl < 0.09. The open ch annel probability (P-0) showed a weak voltage dependency with P-0 incr easing slightly at negative potential values (intracellular (trans) re lative to extracellular (cia) for right-side-out vesicles). Channel ac tivity was inhibited by NPPB at high concentrations (>100 mu M) and by DIDS (300 mu M). A small inhibitory effect was also observed in the p resence of DPC at concentrations ranging from 200 mu M to 500 mu M. Th e presence of SO42- (32 mmol/l) in the trans solution caused a complet e inhibition of channel activity, but no modification of channel behav iour was observed with the non-selective channel blocking agent gadoli nium (Gd3+) at 100 mu M. Finally, addition of the catalytic subunit of protein kinase A into the trans chamber (60 U/ml to 80 U/ml) led to a n increase in channel activity characterized by a greater number of ac tive channels coupled to an increase of the individual channel open pr obability. The action of the protein kinase A could be cancelled by th e addition of a non specific protein phosphatase, such as alkaline pho sphatase. Our results suggest that the apical membrane of the rabbit d istal tubule contains a Cl- permeable channel of small conductance the activity of which may be modulated by hormones Linked to the adenylat e cyclase pathway.