The role of Ca2+ stores in the muscarinic inhibition of the K+ current IK(SO) in neonatal rat cerebellar granule cells.
Cerebellar granule neurons (CGNs) possess a standing outward potassium current (IK(SO)) which shares many similarities with current through the two-pore domain potassium channel TASK-1 and which is inhibited following activation of muscarinic acetylcholine receptors. The action of muscarine on IK(SO) was unaffected by the M2 receptor antagonist methoctramine (100 nM) but was blocked by the M3 antagonist zamifenacin, which, at a concentration of 100 nM, shifted the muscarine concentration-response curve to the right by around 50-fold. Surprisingly, M3 receptor activation rarely produced a detectable increase in [Ca2+]i unless preceded by depolarization of the cells with 25 mM K+. Experiments with thapsigargin and ionomycin suggested that the endoplasmic reticulum Ca2+ stores in CGNs were depleted at rest. In contrast, cerebellar glial cells in the same fields of cells possessed substantial endoplasmic reticulum Ca2+ stores at rest. Pretreatment of the cells with BAPTA AM, thapsigargin or the phospholipase C (PLC) inhibitor U-73122 all blocked the muscarine-induced Ca2+ signal but had little or no effect on muscarinic inhibition of IK(SO). Raising [Ca2+]i directly with ionomycin caused a small but significant inhibition of IK(SO). It is concluded that muscarine acts on M3 muscarinic acetylcholine receptors both to inhibit IK(SO) and to mobilize Ca2+ from intracellular stores in CGNs. While the mobilization of Ca2+ occurs through activation of PLC, this does not seem to be the primary mechanism underlying muscarinic inhibition of IK(SO).
Boyd
D F
DF
Department of Pharmacology, Medawar Building, University College London, Gower Street, London WC1E 6BT, UK.
Millar
J A
JA
Watkins
C S
CS
Mathie
A
A
D000818:Animals; D000831:Animals, Newborn; D002118:Calcium / Q000502:physiology*; D020013:Calcium Signaling / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D004149:Dioxoles / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D004553:Electric Conductivity; D004963:Estrenes / Q000494:pharmacology; D005260:Female; D008297:Male; D008564:Membrane Potentials; D018727:Muscarinic Antagonists / Q000494:pharmacology; D010880:Piperidines / Q000494:pharmacology; D011188:Potassium / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism*; D020033:Protein Isoforms / Q000502:physiology; D011760:Pyrrolidinones / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic / Q000502:physiology*; D019284:Thapsigargin / Q000494:pharmacology
D002614:Chelating Agents; D004149:Dioxoles; D004963:Estrenes; D018727:Muscarinic Antagonists; D010880:Piperidines; D015221:Potassium Channels; D020033:Protein Isoforms; D011760:Pyrrolidinones; D043587:Receptor, Muscarinic M3; D011976:Receptors, Muscarinic; C060229:1-(6-((3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D019284:Thapsigargin; D011188:Potassium; D002118:Calcium; C097932:zamifenacin