Probing the regulation of M (Kv7) potassium channels in intact neurons with membrane-targeted peptides
| Autor: | Jon Robbins, David A. Brown, Stephen J. Marsh |
|---|---|
| Rok vydání: | 2006 |
| Předmět: |
Calmodulin
G protein Journal Club Superior Cervical Ganglion Receptors G-Protein-Coupled Feedback Cell membrane Rats Sprague-Dawley chemistry.chemical_compound GTP-Binding Proteins M current Muscarinic acetylcholine receptor medicine Humans Animals Cells Cultured Neurons biology KCNQ Potassium Channels General Neuroscience Cell Membrane Articles Potassium channel Rats medicine.anatomical_structure Phosphatidylinositol 4 5-bisphosphate chemistry Biochemistry Molecular Probes biology.protein Biophysics GTP-Binding Protein alpha Subunits Gq-G11 lipids (amino acids peptides and proteins) Peptides Ion Channel Gating Intracellular Signal Transduction |
| Zdroj: | The Journal of neuroscience : the official journal of the Society for Neuroscience. 26(30) |
| ISSN: | 1529-2401 |
| Popis: | 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 Gαq/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 PIP2palpeptide, based on the putative PIP2binding 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 PIP2palpeptide 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 PIP2palpeptide was not sequence specific so probably interacts electrostatically with anionic PIP2head groups. |
| Databáze: | OpenAIRE |
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