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 (PIP(2)) 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 PIP(2) and mutation of PKA sites reduces the affinity of ROMK1 for PIP(2). In this study we investigate the molecular mechanism for PKC regulation of ROMK and report that mutants of ROMK1 with reduced PIP(2) 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 PIP(2) to the cytoplasmic face of excised inside-out membranes restores channel activity. PMA reduces PIP(2) content in oocyte membrane and calphostin-C prevents the reduction. These results suggest that reduction of membrane PIP(2) 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.
Zeng
Wei-Zhong
WZ
Department of Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-8856, USA.
Li
Xin-Ji
XJ
Hilgemann
Donald W
DW
Huang
Chou-Long
CL
D000818:Animals; D015640:Ion Channel Gating / Q000502:physiology*; D007685:Kidney Tubules, Collecting / Q000502:physiology*; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D010766:Phosphorylation; D015221:Potassium Channels / Q000502:physiology*; D024661:Potassium Channels, Inwardly Rectifying; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D014982:Xenopus laevis
C510692:Kcnj1 protein, rat; D019269:Phosphatidylinositol 4,5-Diphosphate; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying