ROLE OF CELL-VOLUME AND PROTEIN-KINASE-C IN REGULATION OF A CL- CONDUCTANCE IN SINGLE PROXIMAL TUBULE CELLS OF RANA-TEMPORARIA

1. The whole-cell patch clamp technique was used to investigate Cl- currents in single proximal tubule cells isolated from kidneys of Rana temporaria. 2. Immediately following establishment of the whole-cell clamp, the Cl- conductance (g(Cl)) of the cells was low. However, with 2 mM ATP in the pipette there was a time-dependent activation of g(Cl). Such activation was inhibited when the bath contained a hypertonic Ringer solution. 3. The Cl- conductance was not directly dependent on cell volume; g(Cl) increased with hypotonic shock and decreased with hypertonic shock, but only in the presence of BTP. 4. Activation of g(Cl) by ATP was dependent on extracellular Ca2+; however, the conductance was not directly Ca2+ sensitive. Activation was inhibited by Gd3+, which also had a direct inhibitory action on g(Cl). 5. Inhibition of protein kinase C (PKC), by 10 mu M PKC pseudo-substrate (PKC-ps), completely abolished the ATP-dependent activation of g(Cl) while stimulation of PKC, by the PKC activator 4 beta-phorbol 12-myristate, 13-acetate (PMA), increased the degree of activation typically observed with ATP. 6. We propose that g(Cl) is activated by PKC-mediated phosphorylation and plays a role in volume regulation of the cells.