title: Relationship between membrane phosphatidylinositol-4,5-bisphosphate and receptor-mediated inhibition of native neuronal M channels.
abstract: The relationship between receptor-induced membrane phosphatidylinositol-4'5'-bisphosphate (PIP2) hydrolysis and M-current inhibition was assessed in single-dissociated rat sympathetic neurons by simultaneous or parallel recording of membrane current and membrane-to-cytosol translocation of the fluorescent PIP2/inositol 1,4,5-trisphosphate (IP3)-binding peptide green fluorescent protein-tagged pleckstrin homology domain of phospholipase C (GFP-PLCdelta-PH). The muscarinic receptor agonist oxotremorine-M produced parallel time- and concentration-dependent M-current inhibition and GFP-PLCdelta-PH translocation; bradykinin also produced parallel time-dependent inhibition and translocation. Phosphatidylinositol-4-phosphate-5-kinase (PI5-K) overexpression reduced both M-current inhibition and GFP-PLCdelta-PH translocation by both oxotremorine-M and bradykinin. These effects were partly reversed by wortmannin, which inhibits phosphatidylinositol-4-kinase (PI4-K). PI5-K overexpression also reduced the inhibitory action of oxotremorine-M on PIP2-gated G-protein-gated inward rectifier (Kir3.1/3.2) channels; bradykinin did not inhibit these channels. Overexpression of neuronal calcium sensor-1 protein (NCS-1), which increases PI4-K activity, did not affect responses to oxotremorine-M but reduced both fluorescence translocation and M-current inhibition by bradykinin. Using an intracellular IP3 membrane fluorescence-displacement assay, initial mean concentrations of membrane [PIP2] were estimated at 261 microm (95% confidence limit; 192-381 microm), rising to 693 microm (417-1153 microm) in neurons overexpressing PI5-K. Changes in membrane [PIP2] during application of oxotremorine-M were calculated from fluorescence data. The results, taken in conjunction with previous data for KCNQ2/3 (Kv7.2/Kv7.3) channel gating by PIP2 (Zhang et al., 2003), accorded with the hypothesis that the inhibitory action of oxotremorine-M on M current resulted from depletion of PIP2. The effects of bradykinin require additional components of action, which might involve IP3-induced Ca2+ release and consequent M-channel inhibition (as proposed previously) and stimulation of PIP2 synthesis by Ca2+-dependent activation of NCS-1.
authors.affiliation: 1Ion Channel Pharmacology Group, IPC 388, Pfizer Global Research and Development, Sandwich Laboratories, Sandwich, Kent CT13 9NJ, United Kingdom.
authors.forename: Joanna S
authors.initials: JS
authors.lastname: Winks
authors.forename: Simon
authors.initials: S
authors.lastname: Hughes
authors.forename: Alexander K
authors.initials: AK
authors.lastname: Filippov
authors.forename: Lucine
authors.initials: L
authors.lastname: Tatulian
authors.forename: Fe C
authors.initials: FC
authors.lastname: Abogadie
authors.forename: David A
authors.initials: DA
authors.lastname: Brown
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Marsh
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001920:Bradykinin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002462:Cell Membrane / Q000187:drug effects / Q000502:physiology*; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004558:Electric Stimulation / Q000379:methods; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D005456:Fluorescent Dyes / Q000378:metabolism; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D007150:Immunohistochemistry / Q000379:methods; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008297:Male; D018613:Microscopy, Confocal / Q000379:methods; D018721:Muscarinic Agonists / Q000494:pharmacology; D009154:Mutation / Q000502:physiology; D009433:Neural Inhibition / Q000187:drug effects / Q000502:physiology*; D051580:Neuronal Calcium-Sensor Proteins; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D009479:Neuropeptides / Q000378:metabolism; D010095:Oxotremorine / Q000031:analogs & derivatives / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000502:physiology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D054803:Phospholipase C delta; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017783:Superior Cervical Ganglion / Q000166:cytology; D014162:Transfection / Q000379:methods; D010738:Type C Phospholipases / Q000235:genetics / Q000378:metabolism
chemical_list: D002135:Calcium-Binding Proteins; D004963:Estrenes; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D007527:Isoenzymes; D018721:Muscarinic Agonists; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; D019269:Phosphatidylinositol 4,5-Diphosphate; D010726:Phosphodiesterase Inhibitors; D011760:Pyrrolidinones; D011976:Receptors, Muscarinic; C101464:frequenin calcium sensor proteins; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; D049452:Green Fluorescent Proteins; D010095:Oxotremorine; C042743:oxotremorine M; D010738:Type C Phospholipases; D054803:Phospholipase C delta; D001920:Bradykinin; D002118:Calcium