European Biophysics Journal

, Volume 33, Issue 3, pp 255–264 | Cite as

Phospholipid metabolism is required for M1 muscarinic inhibition of N-type calcium current in sympathetic neurons

  • Liwang Liu
  • Mandy L. Roberts
  • Ann R. RittenhouseEmail author


The signal transduction cascade mediating muscarinic receptor modulation of N-type Ca2+ channel activity by the slow pathway has remained incompletely characterized despite focused investigation. Recently we confirmed a role for the G-protein Gq and identified phospholipase C (PLC), phospholipase A2 (PLA2), and arachidonic acid (AA) as additional molecules involved in N-current inhibition in superior cervical ganglion (SCG) neurons by the slow pathway. We have further characterized this signal transduction cascade by testing whether additional molecules downstream of phosphatidylinositol-4,5-bisphosphate (PIP2) are required. The L-channel antagonist nimodipine was bath-applied to block L-current. Pretreating cells with pertussis toxin (PTX) minimized M2/M4 muscarinic receptor inhibition of N-current by the membrane-delimited pathway. Consistent with our previous studies, pharmacologically antagonizing M1 muscarinic receptors (M1Rs), Gqα, PLC, PLA2, and AA minimized N-current inhibition by the muscarinic agonist oxotremorine-M (Oxo-M). When cells were left untreated with PTX, leaving the membrane-delimited pathway intact and the same antagonists retested, Oxo-M decreased whole cell currents. Moreover, inhibited currents displayed slowed activation kinetics, indicating intact N-current inhibition by the membrane-delimited pathway. These findings indicate that the antagonists used to block the slow pathway acted selectively. PLA2 cleaves AA from phospholipids, generating additional metabolites. We tested whether the metabolite lysophosphatidic acid (LPA) mimicked the inhibitory actions of Oxo-M. In contrast to AA, applying LPA did not inhibit whole cell currents. Taken together, these findings suggest that the slow pathway requires M1Rs, Gqα, PLC, PIP2, PLA2, and AA for N-current inhibition.


Arachidonic acid Muscarinic receptors N-current inhibition Oxotremorine-M Superior cervical ganglion neurons 



arachidonic acid


1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid


bovine serum albumin




7,7-dimethyleicosadienoic acid


5,8,11,14-eicosatetraynoic acid


FPL 64176




L-type calcium channel


L-type calcium current


lysophosphatidic acid


M1 muscarinic receptor


N-type calcium channel


N-type calcium current






oleoyloxyethyl phosphorylcholine


oxotremorine methiodide




phospholipase C


phospholipase A2


pertussis toxin


superior cervical ganglion



Robert Carraway helped with developing the protocol for Western blot analysis. We thank Claire Baldwin, John F. Heneghan, and Maggie Lee for critically reading various versions of the manuscript and helping with the editing. This project was funded by an Established Investigator Award from the American Heart Association (ARR) and by a grant from NINDS (NS34195).


  1. Barrett CF, Liu L, Rittenhouse AR (2001) Arachidonic acid reversibly enhances N-type calcium current at an extracellular site. Am J Physiol 280:C1306–C1318Google Scholar
  2. Beech DJ, Bernheim L, Mathie A, Hille B (1991) Intracellular Ca2+ buffers disrupt muscarinic suppression of Ca2+ current and M current in rat sympathetic neurons. Proc Natl Acad Sci USA 88:652–656PubMedGoogle Scholar
  3. Beech DJ, Berheim L, Hille B (1992) Pertussis toxin and voltage dependence distinguish multiple pathways modulating calcium channels of rat sympathetic neurons. Neuron 8:97–106PubMedGoogle Scholar
  4. Bernheim L, Beech DJ, Hille B (1991) A diffusible second messenger mediates one of the pathways coupling receptors to calcium channels in rat sympathetic neurons. Neuron 6:859–867PubMedGoogle Scholar
  5. Bernheim L, Mathie A, Hille B (1992) Characterization of muscarinic receptor subtypes inhibiting Ca2+ current and M current in rat sympathetic neurons. Proc Natl Acad Sci USA 89:9544–9548PubMedGoogle Scholar
  6. Bernstein G, Blank JL, Smrcka AV, Higashijima T, Sternweis PC, Exton JH, Ross EM (1992) Reconstitution of agonist-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis using purified m1 muscarinic receptor, Gq/11, and phospholipase C-beta 1. J Biol Chem 267:8081–8088PubMedGoogle Scholar
  7. Biddlecome GH, Berstein G, Ross EM (1996) Regulation of phospholipase C-beta 1 by Gq and m1 muscarinic cholinergic receptor. Steady-state balance of receptor-mediated activation and GTPase-activating protein-promoted deactivation. J Biol Chem 271:7999–8007CrossRefPubMedGoogle Scholar
  8. Buck MA, Fraser CM (1990) Muscarinic acetylcholine receptor subtypes which selectively couple to phospholipase C: pharmacological and biochemical properties. Biochem Biophys Res Commun 173:666–672PubMedGoogle Scholar
  9. Caulfield MP, Birdsall NJM (1998) International Union of Pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50:279–290PubMedGoogle Scholar
  10. Delmas P, Abogadie FC, Dayrell M, Haley JE, Milligan G, Caulfield MP, Brown DA, Buckley NJ (1998) G-proteins and G-protein subunits mediating cholinergic inhibition of N-type calcium currents in sympathetic neurons. Eur J Neurosci 10:1654–1666CrossRefPubMedGoogle Scholar
  11. Delmas P, Abogadie FC, Buckley NJ, Brown DA (2000) Calcium channel gating and modulation by transmitters depend on cellular compartmentalization. Nat Neurosci 3:670–678CrossRefPubMedGoogle Scholar
  12. Furukawa T, Nukada T, Mori Y, Wakamori M, Fujita Y, Ishida H, Fukuda K, Kato S, Yoshii M (1998a) Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. I. Molecular determination. J Biol Chem 273:17585–17594CrossRefPubMedGoogle Scholar
  13. Furukawa T, Miura R, Mori Y, Strobeck M, Suzuki K, Ogihara Y, Asano T, Morishita R, Hashii M, Higashida H, Yoshii M, Nukada T (1998b) Differential interactions of the C terminus and the cytoplasmic I-II loop of neuronal Ca2+ channels with G-protein alpha and beta gamma subunits. II. Evidence for direct binding. J Biol Chem 273:17595–17603CrossRefPubMedGoogle Scholar
  14. Gurwitz D, Haring R, Heldman E, Fraser CM, Manor D, Fisher A (1994) Discrete activation of transduction of pathways associated with acetylcholine M1 receptor by several muscarinic ligands. Eur J Pharmacol 267:21–31PubMedGoogle Scholar
  15. Haley JE, Delmas P, Offermanns S, Abogadie FC, Simon MI, Buckley NJ, Brown DA (2000) Muscarinic inhibition of calcium current and M current in Gαq-deficient mice. J Neurosci 20:3973–3979PubMedGoogle Scholar
  16. Hilgemann DW, Ball R (1996) Regulation of cardiac Na+, Ca2+ exchange and KATP potassium channels by PIP2. Science 273:956–959PubMedGoogle Scholar
  17. Hille B (1994) Modulation of ion-channel function by G-protein-coupled receptors. Trends Neurosci 17:531–536PubMedGoogle Scholar
  18. Ikeda SR (1991) Double-pulse calcium channel current facilitation in adult rat sympathetic neurones. J Physiol (London) 439:181–214Google Scholar
  19. Jarvis SE, Zamponi GW (2001) Interactions between presynaptic Ca2+ channels, cytoplasmic messengers and proteins of the synaptic vesicle release complex. Trends Pharmacol Sci 22:519–525CrossRefPubMedGoogle Scholar
  20. Jelsema CL, Axelrod J (1987) Stimulation of phospholipase A2 activity in bovine rod outer segments by the beta gamma subunits of transducin and its inhibition by the alpha subunit. Proc Natl Acad Sci USA 84:3623–3627PubMedGoogle Scholar
  21. Kramer RM, Checani GF, Deykin D (1987) Stimulation of Ca2+-activated human platelet phopholipase A2 by diacylglycerol. Biochem J 248:779–783PubMedGoogle Scholar
  22. Liu L, Rittenhouse AR (2000) Effects of arachidonic acid on unitary calcium currents in rat sympathetic neurons. J Physiol (London) 525:391–404Google Scholar
  23. Liu L, Rittenhouse AR (2003a) Pharmacological discrimination between muscarinic receptor signal transduction cascades with bethanechol chloride. Br J Pharmacol 138:1259–1270CrossRefPubMedGoogle Scholar
  24. Liu L, Rittenhouse AR (2003b) Arachidonic acid mediates muscarinic inhibition and enhancement of N-type Ca2+ current in sympathetic neurons. Proc Natl Acad Sci USA 100:295–300CrossRefPubMedGoogle Scholar
  25. Liu L, Barret CF, Rittenhouse AR (2001) Arachidonic acid both inhibits and enhances whole cell calcium currents in rat sympathetic neurons. Am J Physiol 280:C1293–C1305Google Scholar
  26. Liu L, Gonzalez PK, Barret CF, Rittenhouse AR (2003) The calcium channel ligand FPL 64176 enhances L-type but inhibits N-type neuronal calcium currents. Neuropharmacology 45:281–292CrossRefPubMedGoogle Scholar
  27. Lopes CMB, Zhang H, Rohacs T, Jin T, Yang J, Logothetis DE (2002) Alterations in conserved Kir channel-PIP2 interactions underlie channelopathies. Neuron 34:933–944PubMedGoogle Scholar
  28. Mathie A, Bernheim L, Hille B (1992) Inhibition of N- and L-type calcium channels by muscarinic receptor activation in rat sympathetic neurons. Neuron 8:907–914PubMedGoogle Scholar
  29. Mukai H, Munekata E, Higashijima T (1992) G protein antagonists. A novel hydrophobic peptide competes with receptor for G protein binding. J Biol Chem 267:16237–16243PubMedGoogle Scholar
  30. Petit-Jacques J, Hartzell HC (1996) Effect of arachidonic acid on the L-type calcium current in frog cardiac myocytes. J Physiol (London) 493:67–81Google Scholar
  31. Plummer MR, Logothetis DE, Hess P (1989) Elementary properties and pharmacological sensitivities of calcium channels in mammalian peripheral neurons. Neuron 2:1453–1463PubMedGoogle Scholar
  32. Plummer MR, Rittenhouse AR, Kanevsky M, Hess P (1991) Neurotransmitter modulation of calcium channels in rat sympathetic neurons. J Neurosci 11:2339–2348PubMedGoogle Scholar
  33. Richards MH, van Giersbergen PLM (1995) Human muscarinic receptors expressed in A9L and CHO cells: activation by full and partial agonists. Br J Pharmacol 114:1241–1249PubMedGoogle Scholar
  34. Shapiro MS, Loose MD, Hamilton SE, Nathanson NM, Gomeza J, Wess J, Hille B (1999) Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca2+ channels by using knockout mice. Proc Natl Acad Sci USA 96:10899–10904CrossRefPubMedGoogle Scholar
  35. Shapiro MS, Gomeza J, Hamilton SE, Hille B, Loose MD, Nathanson NM, Roche JP, Wess J (2001) Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons. Life Sci 68:2481–2487CrossRefPubMedGoogle Scholar
  36. Spector AA (1975) Fatty acid binding to plasma albumin. J Lipid Res 16:165–179PubMedGoogle Scholar
  37. Suh BC, Hille B (2002) Recovery from muscarinic modulation of M current channels requires phosphatidylinositol 4,5-bisphosphate synthesis. Neuron 35:507–520PubMedGoogle Scholar
  38. Swarthout JT, Walling HW (2000) Lysophosphatidic acid: receptors, signaling and survival. Cell Mol Life Sci 57:1978–1985PubMedGoogle Scholar
  39. Tence M, Cordier J, Premont J, Glowinski J (1994) Muscarinic cholinergic agonists stimulate arachidonic acid release from mouse striatal neurons in primary culture. J Pharmacol Exp Ther 269:646–653PubMedGoogle Scholar
  40. Tsunoda Y, Owyang C (1995) The regulatory site of functional GTP binding protein coupled to the high affinity cholecystokinin receptor and phospholipase A2 pathway is on the G beta subunit of Gq protein in pancreatic acini. Biochem Biophys Res Commun 211:648–655CrossRefPubMedGoogle Scholar
  41. Wu L, Bauer CS, Zhen XG, Xie C, Yang J (2002) Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419:947–952CrossRefPubMedGoogle Scholar
  42. Zhang H, Craciun LC, Mirshahi T, Rohacs T, Lopes CM, Jin T, Logothetis DE (2003) PIP(2) activates KCNQ channels, and its hydrolysis underlies receptor-mediated inhibition of M currents. Neuron 37:963–975PubMedGoogle Scholar
  43. Zhou J, Shapiro MS, Hille B (1997) Speed of Ca2+ channel modulation by neurotransmitters in rat sympathetic neurons. J Neurophysiol 77:2040–2048PubMedGoogle Scholar

Copyright information

© EBSA 2004

Authors and Affiliations

  • Liwang Liu
    • 1
  • Mandy L. Roberts
    • 1
  • Ann R. Rittenhouse
    • 1
    Email author
  1. 1.Department of PhysiologyUniversity of Massachusetts Medical SchoolWorcesterUSA

Personalised recommendations