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. 1982 Apr 1;79(4):657–678. doi: 10.1085/jgp.79.4.657

A photoisomerizable muscarinic antagonist. Studies of binding and of conductance relaxations in frog heart

PMCID: PMC2215484  PMID: 6978380

Abstract

These experiments employ the photoisomerizable compound, 3,3'-bis- [alpha-(trimethylammonium)methyl]azobenzene (Bis-Q), to study the response to muscarinic agents in frog myocardium. In homogenates from the heart, trans-Bis-Q blocks the binding of [3H]-N-methylscopolamine to muscarinic receptors. In voltage-clamped atrial trabeculae, trans- Bis-Q blocks the agonist-induced potassium conductance. The equilibrium dose-response curve for carbachol is shifted to the right, suggesting competitive blockade. Both the biochemical and electrophysiological data yield a dissociation constant of 4-5 microM for trans-Bis-Q; the cis configuration is severalfold less potent as a muscarinic blocker. Voltage-clamped preparations were exposed simultaneously to carbachol and Bis-Q and were subjected to appropriately filtered flashes (less than 1 ms duration) from a xenon flashlamp. Trans leads to cis and cis leads to trans photoisomerizations cause small (less than 20%) increases and decreases, respectively, in the agonist-induced current. The relaxation follows an S-shaped time course, including an initial delay or period of zero slope. The entire waveform is described by [1 - exp(-kt)]n. At 23 degrees C, k is approximately 3 s-1 and n is 2. Neither k nor n is affected when: (a) [Bis-Q] is varied between 5 and 100 microM; (b) [carbachol] is varied between 1 and 50 microM; (c) carbachol is replaced by other agonists (muscarine, acetylcholine, or acetyl-beta-methylcholine); or (d) the voltage is varied between the normal resting potential and a depolarization of 80 mV. However, in the range of 13-30 degrees C, k increases with temperature; the Q10 is between 2 and 2.5. In the same range, n does not change significantly. Like other investigators, we conclude that the activation kinetics of the muscarinic K+ conductance are not determined by ligand-receptor binding, but rather by a subsequent sequence of two (or more) steps with a high activation energy.

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Selected References

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  1. ARUNLAKSHANA O., SCHILD H. O. Some quantitative uses of drug antagonists. Br J Pharmacol Chemother. 1959 Mar;14(1):48–58. doi: 10.1111/j.1476-5381.1959.tb00928.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong C. M. Potassium pores of nerve and muscle membranes. Membranes. 1975;3:325–358. [PubMed] [Google Scholar]
  3. Armstrong D. L., Lester H. A. The kinetics of tubocurarine action and restricted diffusion within the synaptic cleft. J Physiol. 1979 Sep;294:365–386. doi: 10.1113/jphysiol.1979.sp012935. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bartels E., Wassermann N. H., Erlanger B. F. Photochromic activators of the acetylcholine receptor. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1820–1823. doi: 10.1073/pnas.68.8.1820. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Berrie C. P., Birdsall N. J., Burgen A. S., Hulme E. C. Guanine nucleotides modulate muscarinic receptor binding in the heart. Biochem Biophys Res Commun. 1979 Apr 27;87(4):1000–1005. doi: 10.1016/s0006-291x(79)80006-6. [DOI] [PubMed] [Google Scholar]
  6. Bieth J., Wassermann N., Vratsanos S. M., Erlanger B. F. Photoregulation of biological activity by photochromic reagents, IV. A model for diurnal variation of enzymic activity. Proc Natl Acad Sci U S A. 1970 Jul;66(3):850–854. doi: 10.1073/pnas.66.3.850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bolton T. B. Mechanisms of action of transmitters and other substances on smooth muscle. Physiol Rev. 1979 Jul;59(3):606–718. doi: 10.1152/physrev.1979.59.3.606. [DOI] [PubMed] [Google Scholar]
  8. Brown D. A., Adams P. R. Muscarinic suppression of a novel voltage-sensitive K+ current in a vertebrate neurone. Nature. 1980 Feb 14;283(5748):673–676. doi: 10.1038/283673a0. [DOI] [PubMed] [Google Scholar]
  9. DEL CASTILLO J., KATZ B. Production of membrane potential changes in the frog's heart by inhibitory nerve impulses. Nature. 1955 Jun 11;175(4467):1035–1035. doi: 10.1038/1751035a0. [DOI] [PubMed] [Google Scholar]
  10. DiFrancesco D., Noma A., Trautwein W. Separation of current induced by potassium accumulation from acetylcholine-induced relaxation current in the rabbit S-A node. Pflugers Arch. 1980 Sep;387(2):83–90. doi: 10.1007/BF00584257. [DOI] [PubMed] [Google Scholar]
  11. Garnier D., Nargeot J., Ojeda C., Rougier O. The action of acetylcholine on background conductance in frog atrial trabeculae. J Physiol. 1978 Jan;274:381–396. doi: 10.1113/jphysiol.1978.sp012154. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. George W. J., Wilkerson R. D., Kadowitz P. J. Influence of acetylcholine on contractile force and cyclic nucleotide levels in the isolated perfused rat heart. J Pharmacol Exp Ther. 1973 Jan;184(1):228–235. [PubMed] [Google Scholar]
  13. Giles W., Noble S. J. Changes in membrane currents in bullfrog atrium produced by acetylcholine. J Physiol. 1976 Sep;261(1):103–123. doi: 10.1113/jphysiol.1976.sp011550. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. HODGKIN A. L., HUXLEY A. F. A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol. 1952 Aug;117(4):500–544. doi: 10.1113/jphysiol.1952.sp004764. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hartzell H. C. Distribution of muscarinic acetylcholine receptors and presynaptic nerve terminals in amphibian heart. J Cell Biol. 1980 Jul;86(1):6–20. doi: 10.1083/jcb.86.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hartzell H. C., Kuffler S. W., Stickgold R., Yoshikami D. Synaptic excitation and inhibition resulting from direct action of acetylcholine on two types of chemoreceptors on individual amphibian parasympathetic neurones. J Physiol. 1977 Oct;271(3):817–846. doi: 10.1113/jphysiol.1977.sp012027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hill-Smith I., Purves R. D. Synaptic delay in the heart: an ionophoretic study. J Physiol. 1978 Jun;279:31–54. doi: 10.1113/jphysiol.1978.sp012329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hulme E. C., Birdsall N. J., Burgen A. S., Mehta P. The binding of antagonists to brain muscarinic receptors. Mol Pharmacol. 1978 Sep;14(5):737–750. [PubMed] [Google Scholar]
  19. Jakobs K. H., Aktories K., Schultz G. GTP-dependent inhibition of cardiac adenylate cyclase by muscarinic cholinergic agonists. Naunyn Schmiedebergs Arch Pharmacol. 1979 Dec;310(2):113–119. doi: 10.1007/BF00500275. [DOI] [PubMed] [Google Scholar]
  20. Kehoe J. S., Marty A. Certain slow synaptic responses: their properties and possible underlying mechanisms. Annu Rev Biophys Bioeng. 1980;9:437–465. doi: 10.1146/annurev.bb.09.060180.002253. [DOI] [PubMed] [Google Scholar]
  21. Kehoe J. Ionic mechanisms of a two-component cholinergic inhibition in Aplysia neurones. J Physiol. 1972 Aug;225(1):85–114. doi: 10.1113/jphysiol.1972.sp009930. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kuba K., Koketsu K. Synaptic events in sympathetic ganglia. Prog Neurobiol. 1978;11(2):77–169. doi: 10.1016/0301-0082(78)90010-2. [DOI] [PubMed] [Google Scholar]
  23. Lester H. A. Analysis of sodium and potassium redistribution during sustained permeability increases at the innervated face of Electrophorus electroplaques. J Gen Physiol. 1978 Dec;72(6):847–862. doi: 10.1085/jgp.72.6.847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lester H. A., Chang H. W. Response of acetylcholine receptors to rapid photochemically produced increases in agonist concentration. Nature. 1977 Mar 24;266(5600):373–374. doi: 10.1038/266373a0. [DOI] [PubMed] [Google Scholar]
  25. Lester H. A., Krouse M. E., Nass M. M., Wassermann N. H., Erlanger B. F. A covalently bound photoisomerizable agonist: comparison with reversibly bound agonists at Electrophorus electroplaques. J Gen Physiol. 1980 Feb;75(2):207–232. doi: 10.1085/jgp.75.2.207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lester H. A., Krouse M. E., Nass M. M., Wassermann N. H., Erlanger B. F. Light-activated drug confirms a mechanism of ion channel blockade. Nature. 1979 Aug 9;280(5722):509–510. doi: 10.1038/280509a0. [DOI] [PubMed] [Google Scholar]
  27. Lester H. A., Nass M. M., Krouse M. E., Nerbonne J. M., Wassermann N. H., Erlanger B. F. Electrophysiological experiments with photoisomerizable cholinergic compounds: review and progress report. Ann N Y Acad Sci. 1980;346:475–490. doi: 10.1111/j.1749-6632.1980.tb22118.x. [DOI] [PubMed] [Google Scholar]
  28. MURAD F., CHI Y. M., RALL T. W., SUTHERLAND E. W. Adenyl cyclase. III. The effect of catecholamines and choline esters on the formation of adenosine 3',5'-phosphate by preparations from cardiac muscle and liver. J Biol Chem. 1962 Apr;237:1233–1238. [PubMed] [Google Scholar]
  29. Marty A., Ascher P. Slow relaxations of acetylcholine-induced potassium currents in Aplysia neurones. Nature. 1978 Aug 3;274(5670):494–497. doi: 10.1038/274494a0. [DOI] [PubMed] [Google Scholar]
  30. Michell R. H. Inositol phospholipids and cell surface receptor function. Biochim Biophys Acta. 1975 Mar 25;415(1):81–47. doi: 10.1016/0304-4157(75)90017-9. [DOI] [PubMed] [Google Scholar]
  31. Nass M. M., Lester H. A., Krouse M. E. Response of acetylcholine receptors to photoisomerizations of bound agonist molecules. Biophys J. 1978 Oct;24(1):135–160. doi: 10.1016/S0006-3495(78)85352-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Noma A., Osterrieder W., Trautwein W. The effect of external potassium on the elementary conductance of the ACh-induced potassium channel in the sino-atrial node. Pflugers Arch. 1979 Sep;381(3):263–269. doi: 10.1007/BF00583258. [DOI] [PubMed] [Google Scholar]
  33. Noma A., Peper K., Trautwein W. Acetylcholine-induced potassium current fluctuations in the rabbit sino-atrial node. Pflugers Arch. 1979 Sep;381(3):255–262. doi: 10.1007/BF00583257. [DOI] [PubMed] [Google Scholar]
  34. Noma A., Trautwein W. Relaxation of the ACh-induced potassium current in the rabbit sinoatrial node cell. Pflugers Arch. 1978 Nov 30;377(3):193–200. doi: 10.1007/BF00584272. [DOI] [PubMed] [Google Scholar]
  35. Osterrieder W., Noma A., Trautwein W. On the kinetics of the potassium channel activated by acetylcholine in the S-A node of the rabbit heart. Pflugers Arch. 1980 Jul;386(2):101–109. doi: 10.1007/BF00584196. [DOI] [PubMed] [Google Scholar]
  36. Pott L. On the time course of the acetylcholine-induced hyperpolarization in quiescent guinea-pig atria. Pflugers Arch. 1979 May 15;380(1):71–77. doi: 10.1007/BF00582615. [DOI] [PubMed] [Google Scholar]
  37. Purves R. D. Function of muscarinic and nicotinic acetylcholine receptors. Nature. 1976 May 13;261(5556):149–151. doi: 10.1038/261149a0. [DOI] [PubMed] [Google Scholar]
  38. Putney J. W., Jr, Weiss S. J., Van De Walle C. M., Haddas R. A. Is phosphatidic acid a calcium ionophore under neurohumoral control? Nature. 1980 Mar 27;284(5754):345–347. doi: 10.1038/284345a0. [DOI] [PubMed] [Google Scholar]
  39. Rosenberger L. B., Roeske W. R., Yamamura H. I. The regulation of muscarinic cholinergic receptors by guanine nucleotides in cardiac tissue. Eur J Pharmacol. 1979 Jun;56(1-2):179–180. doi: 10.1016/0014-2999(79)90451-5. [DOI] [PubMed] [Google Scholar]
  40. Rougier O., Vassort G., Stämpfli R. Voltage clamp experiments on frog atrial heart muscle fibres with the sucrose gap technique. Pflugers Arch Gesamte Physiol Menschen Tiere. 1968;301(2):91–108. doi: 10.1007/BF00362729. [DOI] [PubMed] [Google Scholar]
  41. Salmon D. M., Honeyman T. W. Proposed mechanism of cholinergic action in smooth muscle. Nature. 1980 Mar 27;284(5754):344–345. doi: 10.1038/284344a0. [DOI] [PubMed] [Google Scholar]
  42. Sheridan R. E., Lester H. A. Rates and equilibria at the acetylcholine receptor of Electrophorus electroplaques: a study of neurally evoked postsynaptic currents and of voltage-jump relaxations. J Gen Physiol. 1977 Aug;70(2):187–219. [PMC free article] [PubMed] [Google Scholar]
  43. Sheridan R. E., Lester H. A. Relaxation measurements on the acetylcholine receptor. Proc Natl Acad Sci U S A. 1975 Sep;72(9):3496–3500. doi: 10.1073/pnas.72.9.3496. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. TRAUTWEIN W., DUDEL J. Zum Mechanismus der Membranwirkung des Acetylcholin an der Herzmuskelfaser. Pflugers Arch. 1958;266(3):324–334. doi: 10.1007/BF00416781. [DOI] [PubMed] [Google Scholar]
  45. Wassermann N. H., Bartels E., Erlanger B. F. Conformational properties of the acetylcholine receptor as revealed by studies with constrained depolarizing ligands. Proc Natl Acad Sci U S A. 1979 Jan;76(1):256–259. doi: 10.1073/pnas.76.1.256. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Watanabe A. M., Besch H. R., Jr Interaction between cyclic adenosine monophosphate and cyclic gunaosine monophosphate in guinea pig ventricular myocardium. Circ Res. 1975 Sep;37(3):309–317. doi: 10.1161/01.res.37.3.309. [DOI] [PubMed] [Google Scholar]
  47. Wei J. W., Sulakhe P. V. Agonist-antagonist interactions with rat atrial muscarinic cholinergic receptor sites: differential regulation by guanine nucleotides. Eur J Pharmacol. 1979 Sep 1;58(1):91–92. doi: 10.1016/0014-2999(79)90346-7. [DOI] [PubMed] [Google Scholar]

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