Phosphorylation of Kv7 Channels Regulates their PIP2 Sensitivity

Kv7 channels are a subfamily of voltage-gated K+ channels that play a major role in the regulation of neuronal excitability. One important factor in the function of these channels is PIP2, which is required for gating. GPCRs govern Kv7 channels by determining the levels of PIP2, on one hand, and via phosphorylation on the other hand. However, an interaction of these pathways has not been explored.By applying liquid chromatography-coupled mass spectrometry to Kv7.2 immunoprecipitates of rat brain membranes and heterologous cells, we located a cluster of phosphorylation sites in one of the PIP2-binding domains. To evaluate the effect of phosphorylation on PIP2-mediated Kv7.2 current regulation, we generated a quintuple alanine mutant of according serines (S427/436/438/446/455; A5 mutant) to mimic a dephosphorylated state. Activation of the voltage-sensitive phosphatase Dr-VSP was used to reduce PIP2 levels. Perforated patch-clamp recordings showed that the Kv7.2 A5 mutant needed longer VSP activation time for current inhibition than the wildtype channels. In vitro phosphorylation assays with the purified C-terminus of Kv7.2 revealed that various kinases are able to phosphorylate these 5 serines. After treatment of cells expressing wildtype Kv7.2 with inhibitors of PKA, p38MAPK and CDK5, activation of VSP had to be significantly longer than in untreated controls in order to achieve current inhibition.Our results reveal that the phosphorylation status of residues located within the putative PIP2 binding domain determines the phospholipid sensitivity of Kv7 channels.