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 (PIP(2)) is essential for channel activation, we investigate here the role of PIP(2) in regulation of ROMK1 by PKA. By using adenosine-5′-[γ-thio]triphosphate) (ATP[γS]) as the substrate, we found that PKA does not directly activate ROMK1 channels in membranes that are devoid of PIP(2). Rather, phosphorylation by PKA + ATP[γS] lowers the concentration of PIP(2) necessary for activation of the channels. In solution-binding assays, anti-PIP(2) antibodies bind PIP(2) and prevent PIP(2)–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 PIP(2) and the phosphorylated channels. Conversely, mutation of the PKA phosphorylation sites in ROMK1 decreases PIP(2) interaction with the channels. Thus, PKA activates ROMK1 channels by enhancing PIP(2)–channel interaction.