Protein kinase C inhibits ROMK1 channel activity via a phosphatidylinositol 4,5-bisphosphate-dependent mechanism

The activity of apical K⁺ channels in cortical collecting duct (CCD) is stimulated and inhibited by protein kinase A (PKA) and C (PKC), respectively. Direct interaction between phosphatidylinositol 4,5-bisphosphate (PIP₂) and the cloned CCD K⁺ channel, ROMK1, is critical for channel opening. We have found previously that phosphorylation of ROMK1 by PKA increases affinity of the channel for PIP₂ and mutation of PKA sites reduces the affinity of ROMK1 for PIP₂. In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP₂ affinity exhibit an increased sensitivity to inhibition by phorbol myristate acetate (PMA). The effect of PMA can be prevented by pretreatment with calphostin-C. Activation of PKC by carbachol in Xenopus oocytes co-expressing M1 muscarinic receptors also causes inhibition of the channels. Calphostin-C prevents carbachol-induced inhibition, suggesting that activation of PKC is necessary for inhibition of the channels. PMA reduces open probability of the channel in cell-attached patch clamp recordings. After inhibition by PMA in cell-attached recordings, application of PIP₂ to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP₂ content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP₂ content contributes to the inhibition of ROMK1 channels by PKC. This mechanism may underscore the inhibition of K⁺ secretion in CCD by hormones that activate PKC.