Phosphorylation regulates the sensitivity of voltage-gated Kv7.2 channels towards phosphatidylinositol-4,5-bisphosphate

Key points: Phosphatidylinositol-4,5-bisphosphate (PIP₂) is a key regulator of many membrane proteins, including voltage-gated Kv7.2 channels. In this study, we identified the residues in five phosphorylation sites and their corresponding protein kinases, the former being clustered within one of four putative PIP₂-binding domains in Kv7.2. Dephosphorylation of these residues reduced the sensitivity of Kv7.2 channels towards PIP₂. Dephosphorylation of Kv7.2 affected channel inhibition via M₁ muscarinic receptors, but not via bradykinin receptors. Our data indicated that phosphorylation of the Kv7.2 channel was necessary to maintain its low affinity for PIP₂, thereby ensuring the tight regulation of the channel via G protein-coupled receptors. Abstract: The function of numerous ion channels is tightly controlled by G protein-coupled receptors (GPCRs). The underlying signalling mechanisms may involve phosphorylation of channel proteins and participation of phosphatidylinositol-4,5-bisphosphate (PIP₂). Although the roles of both mechanisms have been investigated extensively, thus far only little has been reported on their interaction in channel modulation. GPCRs govern Kv7 channels, the latter playing a major role in the regulation of neuronal excitability by determining the levels of PIP₂ and through phosphorylation. Using liquid chromatography-coupled mass spectrometry for Kv7.2 immunoprecipitates of rat brain membranes and transfected cells, we mapped a cluster of five phosphorylation sites in one of the PIP2-binding domains. To evaluate the effect of phosphorylation on PIP₂-mediated Kv7.2 channel regulation, a quintuple alanine mutant of these serines (S427/S436/S438/S446/S455; A⁵ mutant) was generated to mimic the dephosphorylated state. Currents passing through these mutated channels were less sensitive towards PIP₂ depletion via the voltage-sensitive phosphatase Dr-VSP than were wild-type channels. In vitro phosphorylation assays with the purified C-terminus of Kv7.2 revealed that CDK5, p38 MAPK, CaMKIIα and PKA were able to phosphorylate the five serines. Inhibition of these protein kinases reduced the sensitivity of wild-type but not mutant Kv7.2 channels towards PIP₂ depletion via Dr-VSP. In superior cervical ganglion neurons, the protein kinase inhibitors attenuated Kv7 current regulation via M₁ receptors, but left unaltered the control by B2 receptors. Our results revealed that the phosphorylation status of serines located within a putative PIP₂-binding domain determined the phospholipid sensitivity of Kv7.2 channels and supported GPCR-mediated channel regulation.