Probing the regulation of M(Kv7) potassium channels in intact neurons with membrane-targeted peptides

M-type (Kv7) potassium channels are closed by Gq/11G-protein-coupled receptors. Several membrane- or channel-associated molecules have been suggested to contribute to this effect, including depletion of phosphatidylinositol-4,5-bisphosphate (PIP(2)) and activation of Ca(2+)/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 G alpha q/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 Ca(2+) current by norepinephrine. Instead, the latter was inhibited by the corresponding Go palpeptide. A PIP(2) palpeptide, based on the putative PIP(2) binding domain of the Kv7.2 channel, inhibited M current (IC(50) = similar to 1.5 mu M) 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 diC(8)-PIP(2) (dioctanoyl-phosphatidylinositol-4,5-bisphosphate),suggesting that it disrupted PIP(2)-M channel gating. A fluorescently tagged PIP(2) 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 PIP(2) palpeptide was not sequence specific so probably interacts electrostatically with anionic PIP(2) head groups.