In vivo phosphorylation in Kv7 channels

The family of voltage-gated potassium Kv7 channels consists of Kv7.1 to Kv7.5 and four out of these five proteins are involved in human diseases, such as neonatal epilepsy. Kv7.2, Kv.7.3, and Kv.7.5 channels play a fundamental role in the control of neuronal excitability by providing slowly activating and non-inactivating outward K+ currents at subthreshold voltages, while Kv7.1 contributes to the repolarization during cardiac action potentials. The gating of Kv7 channels is tightly regulated by various mediators and neurotransmitters acting via G protein-coupled receptors (GPCRs); the underlying signaling cascades involve Ca2+/Calmodulin or depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP2). In addition, conventional phosphoprotein biochemistry and electrophysiology using kinase motif-based mutagenesis and/or kinase inhibitors has revealed that phosphorylation also control the functions of Kv7 channels. However, these approaches do not provide information about in vivo phosphorylation sites. Vice versa, for the in vivo phosphorylation sites detected so far, functional information is lacking. Therefore, this project aims to identify and functionally characterize in vivo phosphorylation sites in Kv7 channels by proteomics based on mass spectrometry (MS) and by electrophysiology, mutagenesis and immunofluorescence microscopy. This study will provide a comprehensive in vivo phosphorylation map for Kv7 channels. Furthermore, specific kinases being involved will be characterized, the consequences for the modulation of Kv7channels and neuronal excitability via GPCRs will be analyzed, the importance for channel targeting and assembly will be challenged, and potential changes in channel voltage dependence will be tested.

The neuronal voltage-gated potassium Kv7.2, Kv7.3 and Kv7.5 channels, alternatively called M-channels, play a fundamental role in the control of neuronal excitability by stabilizing the resting membrane potential and limiting the number of action potentials. The importance of these channels is referred by related diseases, such as early onset epilepsy caused by several mutations in KCNQ2 (Kv7.2) and KCNQ3 (Kv7.3) genes. For these reasons, openers of these channels have emerged as anti-epilepsy drugs. A specific membrane phospholipid, phosphatidylinositol-4,5-bisphosphate (PIP2) is a key regulator of the function of many membrane proteins including ion channels. The interaction of PIP2 with a target protein is usually determined by electrostatic interactions defined by the proteins amino acid sequence.Kv7 channels are dependent on the abundance of PIP2. The levels of which are tightly controlled by G-protein-coupled receptors (GPCRs). Kv7 channel subunits have different PIP2 affinities and the Kv7 subunit specificity of some openers or drugs seems to be related to their different PIP2 affinities. However, homologous protein sequences in putative PIP2 binding domains between Kv7 subunits hamper to explain the different PIP2 affinities in Kv7 channels.In this project, we identified a number of phosphorylation sites and their corresponding protein kinases in Kv7.2, Kv7.3 and Kv7.5 subunits. In particular, five phosphorylated serines were clustered within one of four putative PIP2-binding domains in Kv7.2 channels and dephosphorylation of these amino acid residues increased the apparent affinity of Kv7.2 channels towards PIP2. This indicated that phosphorylation of Kv7.2 channels was required to maintain its low affinity for PIP2, thereby ensuring the tight regulation of the channels via GPCRs. This also provided evidence that the PIP2 affinity could be affected by not only positively charged amino acids but also posttranslational modification, namely phosphorylation. Interestingly, Kv7.3 channels have only a single phosphorylation site within the same PIP2 binding region. This inspires a follow-up project to determine whether subunit-specific phosphorylation in this PIP2 domain might be relevant for the hundred-fold difference in apparent PIP2 affinities between Kv7.2 and Kv7.3 subunits. In addition, we identified a different number and location of protein kinase A (PKA)-responsible phosphorylation sites between Kv7.2 and Kv7.5 channels. We determined that PKA phosphorylation specifically affects Kv7.5 function but not that of Kv7.2 channels. As protein kinases are one of the most important drug targets, this may help not only to elucidate the function of these specific protein kinases in Kv7 channel function but also to their importance as a molecular target.