Regulation of ROMK1 Channel by Protein Kinase A via a Phosphatidylinositol 4,5-Bisphosphate-Dependent Mechanism

ROMK inward-rectifier K+ channels control renal K+ secretion. The activity of ROMK is regulated by protein kinase A (PKA), but the molecular mechanism for regulation is unknown. Having found that direct interaction with membrane phosphatidylinositol 4,5-bisphosphate (PIP2) is essential for channel activation, we investigate here the role of PIP2 in regulation of ROMK1 by PKA. By using adenosine-5′-[γ -thio]triphosphate) (ATP[γ S]) as the substrate, we found that PKA does not directly activate ROMK1 channels in membranes that are devoid of PIP2. Rather, phosphorylation by PKA + ATP[γ S] lowers the concentration of PIP2 necessary for activation of the channels. In solution-binding assays, anti-PIP2 antibodies bind PIP2 and prevent PIP2-channel interaction. In inside-out membrane patches, antibodies inhibit the activity of the channels. PKA treatment then decreases the sensitivity of ROMK1 for inhibition by the antibodies, indicating an enhanced interaction between PIP2 and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP2 interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP2-channel interaction.