Patch Clamp Combined with Voltage/Concentration Clamp to Determine the Kinetics and Voltage Dependency of N-Methyl-d-aspartate (NMDA) Receptor Open Channel Blockers

  • Chris G. ParsonsEmail author
  • Kate E. Gilling
Part of the Methods in Molecular Biology book series (MIMB, volume 1183)


Electrophysiological techniques can be used to great effect to help determine the mechanism of action of a compound. However, many factors can compromise the resulting data and their analysis, such as the speed of solution exchange, expression of additional ion channel populations including other ligand-gated receptors and voltage-gated channels, compounds having multiple binding sites, and current desensitization and rundown. In this chapter, such problems and their solutions are discussed and illustrated using data from experiments involving the uncompetitive NMDA receptor antagonist memantine. Memantine differs from many other NMDA receptor channel blockers in that it is well tolerated and does not cause psychotomimetic effects at therapeutic doses. Various electrophysiological parameters of NMDA-induced current blockade by memantine have been proposed to be important in determining therapeutic tolerability; potency, onset and offset kinetics, and voltage dependency. These were all measured using whole cell patch clamp techniques using hippocampal neurons. Full results are shown here for memantine, and these are summarized and compared to those from similar experiments with other NMDA channel blockers. The interpretation of these results is discussed, as are theories concerning the tolerability of NMDA channel blockers, with the aim of illustrating how electrophysiological data can be used to form and support a physiological hypothesis.

Key words

NMDA Uncompetitive Concentration dependence Concentration clamp Voltage dependence Kinetics 


  1. 1.
    Reisberg B, Doody R, Stoffler A et al (2003) Memantine in moderate-to-severe Alzheimer’s disease. N Engl J Med 348:1333–1341PubMedCrossRefGoogle Scholar
  2. 2.
    Tariot PN, Farlow MR, Grossberg GT et al (2004) Memantine treatment in patients with moderate to severe Alzheimer disease already receiving donepezil: a randomized controlled trial. JAMA 291:317–324PubMedCrossRefGoogle Scholar
  3. 3.
    Parsons CG, Danysz W, Quack G (1999) Memantine is a clinically well tolerated N-methyl-D-aspartate (NMDA) receptor antagonist - a review of preclinical data. Neuropharmacology 38:735–767PubMedCrossRefGoogle Scholar
  4. 4.
    Danysz W, Parsons CG (2003) The NMDA receptor antagonist memantine as a symptomatological and neuroprotective treatment for Alzheimer’s disease preclinical evidence. Int J Geriatr Psychiatry 18:S23–S32PubMedCrossRefGoogle Scholar
  5. 5.
    Parsons CG, Gruner R, Rozental J et al (1993) Patch clamp studies on the kinetics and selectivity of N-methyl-D-aspartate receptor antagonism by memantine (1-amino-3,5-dimethyladamantan). Neuropharmacology 32:1337–1350PubMedCrossRefGoogle Scholar
  6. 6.
    Johnson JW, Kotermanski SE (2006) Mechanism of action of memantine. Curr Opin Pharmacol 6:61–67PubMedCrossRefGoogle Scholar
  7. 7.
    Rogawski MA (1993) Therapeutic potential of excitatory amino acid antagonists - channel blockers and 2,3-benzodiazepines. Trends Pharmacol Sci 14:325–331PubMedCrossRefGoogle Scholar
  8. 8.
    Parsons CG, Quack G, Bresink I et al (1995) Comparison of the potency, kinetics and voltage-dependency of a series of uncompetitive NMDA receptor antagonists in vitro with anticonvulsive and motor impairment activity in vivo. Neuropharmacology 34:1239–1258PubMedCrossRefGoogle Scholar
  9. 9.
    Parsons CG, Panchenko VA, Pinchenko VO et al (1996) Comparative patch-clamp studies with freshly dissociated rat hippocampal and striatal neurons on the NMDA receptor antagonistic effects of amantadine and memantine. Eur J Neurosci 8:446–454PubMedCrossRefGoogle Scholar
  10. 10.
    Parsons CG, Hartmann S, Spielmanns P (1998) Budipine is a low affinity, N-methyl-D-aspartate receptor antagonist: patch clamp studies in cultured striatal, hippocampal, cortical and superior colliculus neurones. Neuropharmacology 37:719–727PubMedCrossRefGoogle Scholar
  11. 11.
    Bresink I, Benke TA, Collett VJ et al (1996) Effects of memantine on recombinant rat NMDA receptors expressed in HEK 293 cells. Br J Pharmacol 119:195–204PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Blanpied TA, Boeckman FA, Aizenman E et al (1997) Trapping channel block of NMDA-activated responses by amantadine and memantine. J Neurophysiol 77:309–323PubMedGoogle Scholar
  13. 13.
    Chen HS, Lipton SA (1997) Mechanism of memantine block of NMDA-activated channels in rat retinal ganglion cells: uncompetitive antagonism. J Physiol 499:27–46PubMedCentralPubMedGoogle Scholar
  14. 14.
    Sobolevsky AI, Koshelev SG, Khodorov BI (1998) Interaction of memantine and amantadine with agonist-unbound NMDA-receptor channels in acutely isolated rat hippocampal neurons. J Physiol 512:47–60PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Sobolevsky A, Koshelev S (1998) Two blocking sites of amino-adamantane derivatives in open N-methyl-D- aspartate channels. Biophys J 74:1305–1319PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Grantyn R, Lux HD (1988) Similarity and mutual exclusion of NMDA- and proton-activated transient Na < + > -currents in rat tectal neurons. Neurosci Lett 89:198–203PubMedCrossRefGoogle Scholar
  17. 17.
    Clark GD, Clifford DB, Zorumski CF (1990) The effect of agonist concentration, membrane voltage and calcium on N-methyl-D-aspartate receptor desensitization. Neuroscience 39:787–797PubMedCrossRefGoogle Scholar
  18. 18.
    Zilberter Y, Uteshev V, Sokolova S et al (1991) Desensitization of N-methyl-D-aspartate receptors in neurons dissociated from adult rat hippocampus. Mol Pharmacol 40:337–341PubMedGoogle Scholar
  19. 19.
    Johnson JW, Ascher P (1987) Glycine potentiates the NMDA response in cultured mouse brain neurons. Nature 325:529–531PubMedCrossRefGoogle Scholar
  20. 20.
    Parsons CG, Zong XG, Lux HD (1993) Whole cell and single channel analysis of the kinetics of glycine-sensitive N-methyl-D-aspartate receptor desensitization. Br J Pharmacol 109:213–221PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Hashimoto A, Oka T (1997) Free D-aspartate and D-serine in the mammalian brain and periphery. Prog Neurobiol 52:325–353PubMedCrossRefGoogle Scholar
  22. 22.
    Hamill OP, Marty A, Neher E et al (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100PubMedCrossRefGoogle Scholar
  23. 23.
    Sigworth FJ, Affolter H, Neher E (1995) Design of the EPC-9, a computer-controlled patch-clamp amplifier. 2. Software. J Neurosci Methods 56:203–215PubMedCrossRefGoogle Scholar
  24. 24.
    Albuquerque EX, Pereira EFR, Castro NG et al (1995) Nicotinic receptor function in the mammalian central nervous system. Ann N Y Acad Sci 757:48–72PubMedCrossRefGoogle Scholar
  25. 25.
    Rogawski MA, Yamaguchi SI, Jones SM et al (1991) Anticonvulsant Activity of the Low-Affinity Uncompetitive N-Methyl-D-Aspartate Antagonist (+/-)-5-Aminocarbonyl-10,11-dihydro-5H-dibenzo < a, d > cyclohepten-5, 10-imine (ADCI) - comparison with the Structural Analogs Dizocilpine (mK-801) and Carbamazepine. J Pharmacol Exp Ther 259:30–37PubMedGoogle Scholar
  26. 26.
    Chen HSV, Pellegrini JW, Aggarwal SK et al (1992) Open-channel block of N-methyl-D-aspartate (NMDA) responses by memantine - therapeutic advantage against NMDA receptor-mediated neurotoxicity. J Neurosci 12:4427–4436PubMedGoogle Scholar
  27. 27.
    Black M, Lanthorn T, Small D et al (1996) Study of potency, kinetics of block and toxicity of NMDA receptor antagonists using fura-2. Eur J Pharmacol 317:377–381PubMedCrossRefGoogle Scholar
  28. 28.
    Frankiewicz T, Potier B, Bashir ZI et al (1996) Effects of memantine and MK-801 on NMDA-induced currents in cultured neurones and on synaptic transmission and LTP in area CA1 of rat hippocampal slices. Br J Pharmacol 117:689–697PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Clements JD, Lester RAJ, Tong G et al (1992) The time course of glutamate in the synaptic cleft. Science 258:1498–1501PubMedCrossRefGoogle Scholar
  30. 30.
    Nowak L, Bregestovski P, Ascher P et al (1984) Magnesium gates glutamate-activated channels in mouse central neurons. Nature 307:462–465PubMedCrossRefGoogle Scholar
  31. 31.
    Benveniste H, Drejer J, Schusboe A et al (1984) Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis. J Neurochem 43:1369–1374PubMedCrossRefGoogle Scholar
  32. 32.
    Andine P, Sandberg M, Bagenholm R et al (1991) Intra- and extracellular changes of amino acids in the cerebral cortex of the neonatal rat during hypoxic-ischemia. Dev Brain Res 64:115–120CrossRefGoogle Scholar
  33. 33.
    Globus MYT, Busto R, Martinez E et al (1991) Comparative effect of transient global ischemia on extracellular levels of glutamate, glycine, and gamma-aminobutyric acid in vulnerable and nonvulnerable brain regions in the rat. J Neurochem 57:470–478PubMedCrossRefGoogle Scholar
  34. 34.
    Globus MYT, Ginsberg MD, Busto R (1991) Excitotoxic index - a biochemical marker of selective vulnerability. Neurosci Lett 127:39–42PubMedCrossRefGoogle Scholar
  35. 35.
    Buisson A, Callebert J, Mathieu E et al (1992) Striatal protection induced by lesioning the substantia-nigra of rats subjected to focal ischemia. J Neurochem 59:1153–1157PubMedCrossRefGoogle Scholar
  36. 36.
    Mitani A, Andou Y, Kataoka K (1992) Selective vulnerability of hippocampal CA1 neurons cannot be explained in terms of an increase in glutamate concentration during ischemia in the gerbil: brain microdialysis study. Neuroscience 48:307–313PubMedCrossRefGoogle Scholar
  37. 37.
    Davies SN, Martin D, Millar JD et al (1988) Differences in results from in vivo and in vitro studies on the use-dependency of N-methyl-aspartate antagonism by MK-801 and other phencyclidine receptor ligands. Eur J Pharmacol 145:141–152PubMedCrossRefGoogle Scholar
  38. 38.
    Coan EJ, Irving AJ, Collingridge GL (1989) Low-frequency activation of the NMDA receptor system can prevent the induction of LTP. Neurosci Lett 105:205–210PubMedCrossRefGoogle Scholar
  39. 39.
    Frankiewicz T, Parsons CG (1999) Memantine restores long term potentiation impaired by tonic N-methyl-D-aspartate (NMDA) receptor activation following reduction of Mg2+ in hippocampal slices. Neuropharmacology 38:1253–1259PubMedCrossRefGoogle Scholar
  40. 40.
    Furukawa Y, Okada M, Akaike N et al (2000) Reduction of voltage-dependent magnesium block of N-methyl-D-aspartate receptor-mediated current by in vivo axonal injury. Neuroscience 96:385–392PubMedCrossRefGoogle Scholar
  41. 41.
    Rogawski MA, Wenk GL (2003) The neuropharmacological basis for the use of memantine in the treatment of Alzheimer’s disease. CNS Drug Rev 9:275–308PubMedCrossRefGoogle Scholar
  42. 42.
    Wang LY, Macdonald JF (1995) Modulation by magnesium of the affinity of NMDA receptors for glycine in murine hippocampal neurones. J Physiol 486:83–95PubMedCentralPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  1. 1.Pharmacology, Non-Clinical ScienceMerz Pharmaceuticals GmbHFrankfurt am MainGermany
  2. 2.Charité-UniversitätsmedizinBerlinGermany

Personalised recommendations