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The Journal of Neuroscience, July 26, 2006, 26(30):7950-7961; doi:10.1523/JNEUROSCI.2138-06.2006

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Cellular/Molecular
Probing the Regulation of M (Kv7) Potassium Channels in Intact Neurons with Membrane-Targeted Peptides

Jon Robbins,^1,2 Stephen J. Marsh,¹ and David A. Brown¹

¹Department of Pharmacology, University College London, London WC1E 6BT, United Kingdom, and ²Wolfson Centre for Age Related Disease, King’s College London, London SE1 1UL, United Kingdom

Correspondence should be addressed to Prof. David A. Brown at the above address. d.a.brown{at}ucl.ac.uk

M-type (Kv7) potassium channels are closed by Gq/11 G-protein-coupled receptors. Several membrane- or channel-associated molecules have been suggested to contribute to this effect, including depletion of phosphatidylinositol-4,5-bisphosphate (PIP₂) and activation of Ca²⁺/calmodulin and protein kinase C. To facilitate further study of these pathways in intact neurons, we have devised novel membrane-targeted probes that can be applied from the outside of the neuron, by attaching a palmitoyl group to site-directed peptides ("palpeptides") (cf. Covic et al., 2002a,b). A palpeptide incorporating the 10-residue C terminus of Gq/11 reduced Gq/11-mediated M-current inhibition in sympathetic neurons by the muscarinic acetylcholine receptor (mAChR) agonist oxotremorine-M but not Go-mediated inhibition of the N-type Ca²⁺ current by norepinephrine. Instead, the latter was inhibited by the corresponding Go palpeptide. A PIP₂ palpeptide, based on the putative PIP₂ binding domain of the Kv7.2 channel, inhibited M current (IC₅₀ = 1.5 µM) and enhanced inhibition by oxotremorine-M. Inhibition could not be attributed to activation of mAChRs, calcium influx, or block of M channels but was antagonized by intracellular diC₈-PIP₂ (dioctanoyl-phosphatidylinositol-4,5-bisphosphate), suggesting that it disrupted PIP₂–M channel gating. A fluorescently tagged PIP₂ palpeptide was highly targeted to the plasma membrane but did not accumulate in the cytoplasm. We suggest that these palpeptides are anchored in the plasma membrane via the palmitoyl group, such that the peptide moiety can interact with target molecules on the inner face of the membrane. The G-protein-replicating palpeptides were sequence specific and probably compete with the receptor for the cognate G-protein. The PIP₂ palpeptide was not sequence specific so probably interacts electrostatically with anionic PIP₂ head groups.

Key words: M current; Kv7 channels; KCNQ; muscarinic acetylcholine receptor; palmitoylated peptides; phosphatidylinositol-4,5-bisphosphate; superior cervical ganglion cells; G-protein

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Received Nov. 18, 2005; revised June 20, 2006; accepted June 20, 2006.

Correspondence should be addressed to Prof. David A. Brown at the above address. d.a.brown{at}ucl.ac.uk

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