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This article is part of the supplement: 18th Scientific Symposium of the Austrian Pharmacological Society (APHAR)

Open Access Meeting abstract

Functional implications of KV7 channel phosphorylation

Isabella Salzer1, Wei-Quiang Chen2, Helmut Kubista1, Gert Lubec2, Stefan Boehm1 and Jae-Won Yang3*

Author Affiliations

1 Department of Neurophysiology and Neuropharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria

2 Department of Pediatrics, Medical University of Vienna, 1090 Vienna, Austria

3 Institute of Pharmacology, Center for Physiology and Pharmacology, Medical University of Vienna, 1090 Vienna, Austria

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BMC Pharmacology and Toxicology 2012, 13(Suppl 1):A46  doi:10.1186/2050-6511-13-S1-A46


The electronic version of this article is the complete one and can be found online at: http://www.biomedcentral.com/2050-6511/13/S1/A46


Published:17 September 2012

© 2012 Salzer et al; licensee BioMed Central Ltd.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Background

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.

Methods

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.

Results

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.

Conclusions

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.

Acknowledgements

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.