The family of KV7 potassium channels, particularly KV7.2, KV7.3, and KV7.5 controls neuronal excitability. Numerous neurotransmitters acting via G protein-coupled receptors signaling via Ca2+/calmodulin or depletion of membrane phosphatidylinositol-4,5-bisphosphate (PIP2) tightly regulate KV7 channel function. Moreover, the phosphorylation of KV7 channels has been proposed to play a crucial role. However, in vivo phosphorylation sites and their functional implications need to be determined.
To investigate the role of steady-state KV7 channel phosphorylation, superior cervical ganglion (SCG) neurons were pretreated for 30 min with different kinase inhibitors (GW8510: 10 µM, SB415286: 1 µM, SB203580: 10 µM, H7: 10 µM) which block CDK5, GSK3, p38 MAPK, and PKC as well as PKA, respectively. Thereafter, oxotremorine M (OxoM) or bradykinin-induced inhibition of the M-currents (primarily through KV7.2/7.3 heterotetramers) was tested.
Inhibition of CDK5 shifted the concentration-response curve for OxoM to the left, but not that of bradykinin. Similarly, GW8510 treatment of tsA201 cells, heterologously expressing KV7.2 channels and M1 receptors, caused a leftward shift of the OxoM concentration-response relation. In mass-spectrometric studies, several phosphorylated amino acid residues in the C-terminus of native and heterologously expressed KV7.2 channels were detected, 5 of them are located within the putative PIP2 binding site. CDK5 was predicted to target serines S427 and S446. In contrast to S446, mutation of S427 to alanine significantly increased KV7.2 channel sensitivity towards inhibition via M1 receptors. Additionally, treatment with GW8510 failed to cause any further effect. Nevertheless, these alanine mutations did not influence the channel-voltage dependence.
Hence, phosphorylation of C-terminal serine residue 427 determines KV7.2 modulation by M1 muscarinic, but not by B2 bradykinin receptors, suggesting that the phosphorylation state of S427 regulates the affinity of the KV7.2 C-terminus for PIP2.
The study is supported by the Austrian Science Fund (grants P23670-B09 and P19710), and the PhD programme CCHD of the Medical University of Vienna.