PIP(2)-dependent inhibition of M-type (Kv7.2/7.3) potassium channels: direct on-line assessment of PIP(2) depletion by Gq-coupled receptors in single living neurons.
The open state of M(Kv7.2/7.3) potassium channels is maintained by membrane phosphatidylinositol-4,5-bisphosphate (PI(4,5)P(2)). They can be closed on stimulating receptors that induce PI(4,5)P(2) hydrolysis. In sympathetic neurons, closure induced by stimulating M1-muscarinic acetylcholine receptors (mAChRs) has been attributed to depletion of PI(4,5)P(2), whereas closure by bradykinin B(2)-receptors (B2-BKRs) appears to result from formation of IP(3) and release of Ca(2+), implying that BKR stimulation does not deplete PI(4,5)P(2). We have used a fluorescently tagged PI(4,5)P(2)-binding construct, the C-domain of the protein tubby, mutated to increase sensitivity to PI(4,5)P(2) changes (tubby-R332H-cYFP), to provide an on-line read-out of PI(4,5)P(2) changes in single living sympathetic neurons after receptor stimulation. We find that the mAChR agonist, oxotremorine-M (oxo-M), produces a near-complete translocation of tubby-R332H-cYFP into the cytoplasm, whereas bradykinin (BK) produced about one third as much translocation. However, translocation by BK was increased to equal that produced by oxo-M when synthesis of PI(4,5)P(2) was inhibited by wortmannin. Further, wortmannin 'rescued' M-current inhibition by BK after Ca(2+)-dependent inhibition was reduced by thapsigargin. These results provide the first direct support for the view that BK accelerates PI(4,5)P(2) synthesis in these neurons, and show that the mechanism of BKR-induced inhibition can be switched from Ca(2+) dependent to PI(4,5)P(2) dependent when PI(4,5)P(2) synthesis is inhibited.
Hughes
Simon
S
Department of Pharmacology, University College London, London, WC1E 6BT, UK.
Marsh
Stephen J
SJ
Tinker
Andrew
A
Brown
David A
DA
D000818:Animals; D001920:Bradykinin / Q000494:pharmacology; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D018076:DNA, Complementary / Q000096:biosynthesis / Q000235:genetics; D051658:KCNQ2 Potassium Channel / Q000187:drug effects* / Q000378:metabolism*; D018721:Muscarinic Agonists / Q000494:pharmacology; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism / Q000502:physiology*; D026902:Potassium Channel Blockers; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000187:drug effects*; D011976:Receptors, Muscarinic / Q000187:drug effects; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017783:Superior Cervical Ganglion / Q000166:cytology / Q000187:drug effects; D014178:Translocation, Genetic
D018076:DNA, Complementary; D051658:KCNQ2 Potassium Channel; D018721:Muscarinic Agonists; D019269:Phosphatidylinositol 4,5-Diphosphate; D026902:Potassium Channel Blockers; D043562:Receptors, G-Protein-Coupled; D011976:Receptors, Muscarinic; D001920:Bradykinin