Protein Kinase A-Dependent Phosphorylation Modulates the Affinity of Kv7.5 Potassium Channels to Phosphatidylinositol 4,5-Bisphosphate to Enhance Potassium Conductance

Abstract

PKA-dependent phosphorylation of Kv7.5 potassium channels was previously found to enhance Kv7.5 currents, but the mechanism of the enhancement by PKA as well as putative PKA phosphorylation sites remained unknown. Activity of all known Kv7 channels critically depends on the presence of a minor membrane phospholipid, phosphatidylinositol 4,5-bisphosphate (PIP2). In the present study, we tested the hypothesis that PKA-dependent phosphorylation increases the affinity of Kv7.5 potassium channels to PIP2. Application of wortmannin (10 µM), an inhibitor of PI-4 kinase that decreases the level of membrane PIP2, caused time-dependent inhibition of the endogenous Kv7.5 current in A7r5 rat aortic smooth muscle cells. Activation of PKA by co-application of forskolin (activator of adenylyl cyclase, 1 µM) with IBMX (phosphodiesterase inhibitor, 500 µM) significantly attenuated the rate and total amount of wortmannin-induced inhibition of endogenous Kv7.5 current. Co-expression of ci-VSP, a voltage dependent phosphatase that dephosphorylates PIP2, with human Kv7.5 (hKv7.5) in A7r5 cells caused pronounced voltage and time-dependent inhibition of hKv7.5 current at membrane voltages positive to 0 mV. Enhancement of the hKv7.5 current observed in the presence of forskolin (10 µM) and IBMX (500 µM) occurred in parallel with the positive shift of the ci-VSP-induced voltage-dependent inhibition of the Kv7.5 current. In the presence of forskolin and IBMX, ci-VSP induced inhibition of the Kv7.5 current at voltages positive to +60 mV. To identify PKA phosphorylation sites we mutated predicted PKA phosphorylation sites S406 and S412 (located near the putative PIP2 binding domain on the C-terminus of the Kv7.5 channels) to alanine. Currents through both mutant channels Kv7.5 S406A and Kv7.5 S412A were enhanced by co-application of forskolin with IBMX to the same degree as wild type Kv7.5 current. Thus, phosphorylation of S406 and S412 cannot account for PKA-dependent Kv7.5 current enhancement. Overall, experimental results support our hypothesis that enhancement of Kv7.5 currents caused by PKA activation is associated with increased affinity of the Kv7.5 potassium channels to PIP2.