Molecular Neurobiology

, Volume 55, Issue 5, pp 3889–3900 | Cite as

Reduced CaM Kinase II and CaM Kinase IV Activities Underlie Cognitive Deficits in NCKX2 Heterozygous Mice

  • Shigeki Moriguchi
  • Satomi Kita
  • Yasushi Yabuki
  • Ryo Inagaki
  • Hisanao Izumi
  • Yuzuru Sasaki
  • Hideaki Tagashira
  • Kyoji Horie
  • Junji Takeda
  • Takahiro IwamotoEmail author
  • Kohji FukunagaEmail author


Among five members of the K+-dependent Na+/Ca2+ exchanger (NCKX) family (NCKX1–5), only NCKX2 is highly expressed in mouse brain. NCKX2 in plasma membranes mediates cytosolic calcium excretion through electrogenic exchange of 4 Na+ for 1 Ca2+ and 1 K+. Here, we observed significantly decreased levels of NCKX2 protein and mRNA in the CA1 region of APP23 mice, a model of Alzheimer’s disease. We also found that, like APP23 mice, heterozygous NCKX2-mutant mice exhibit mildly impaired hippocampal LTP and memory acquisition, the latter based on novel object recognition and passive avoidance tasks. When we addressed underlying mechanisms, we found that both CaMKII autophosphorylation and CaMKIV phosphorylation significantly decreased in CA1 regions of NCKX2+/− relative to control mice. Likewise, phosphorylation of GluA1 (Ser-831) and CREB (Ser-133), respective downstream targets of CaMKII and CaMKIV, also significantly decreased in the CA1 region. BDNF protein and mRNA levels significantly decreased in CA1 of NCKX2+/− relative to control mice. Finally, CaN activity increased in CA1 of NCKX2+/− mice. Our findings suggest that like APP23 mice, NCKX2+/− mice may exhibit impaired learning and hippocampal LTP due to decreased CaM kinase II and CaM kinase IV activities.


K+-dependent Na+/Ca2+ exchangers Cognition Calcium/calmodulin-dependent protein kinase II Long-term potentiation Hippocampus 



Alzheimer’s disease


α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor


Brain-derived neurotrophic factor


Calcium/calmodulin-dependent protein kinase II


Calcium/calmodulin-dependent protein kinase IV




cAMP-responsive element binding protein


Dentate gyrus


Extracellular signal-regulated kinase


Field excitatory post-synaptic potentials


Green fluorescent protein


High-frequency stimulation


Long-term potentiation


K+-dependent Na+/Ca2+ exchangers


Na+/Ca2+ exchangers


Protein phosphatase 1





This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health and Welfare of Japan (KAKENHI 22390109 to K.F.; 20790398 to S.M.; 23590319 to T.I.; 25460350 to S.K.), the Uehara Memorial Foundation (K.F.) and the Smoking Research Foundation (S.M.). We also thank Novartis Pharma for providing APP23 mice.

Author Contributions

S.M., Y.Y., R.I., H.I., Y.S., K.S., H.T., and T.I. performed experiments. S.K., K.H., J.T., and T.I. provided NCKX2 antibody and NCKX2 knockout mice, and critical advice. S.M. and K.F. wrote the manuscript and designed the study.

Compliance with Ethical Standards

All animal protocols were approved by the Committee on Animal Experiments at Tohoku University.

Conflict of Interest

The authors declare that they have no competing interests.


  1. 1.
    Berridge MJ (1998) Neuronal calcium signaling. Neuron 21:13–26CrossRefPubMedGoogle Scholar
  2. 2.
    Berrie MJ, Lipp P, Bootman MD (2000) The versatility and universality of calcium signalling. Nat Rev Mol Cell Biol 1:11–21CrossRefGoogle Scholar
  3. 3.
    Augustine GJ, Santamaria F, Tanaka K (2003) Local calcium signaling in neurons. Neuron 40:331–346CrossRefPubMedGoogle Scholar
  4. 4.
    Berridge MJ, Bootman MD, Roderick HL (2003) Calcium signaling: dynamics, homeostasis and remodeling. Nat Rev Mol Cell Biol 4:517–529CrossRefPubMedGoogle Scholar
  5. 5.
    Jeon D, Yang YM, Jeong MJ et al (2003) Enhanced learning and memory in mice lacking Na+/Ca2+ exchanger 2. Neuron 38:965–976CrossRefPubMedGoogle Scholar
  6. 6.
    Li XF, Kiedrowski L, Tremblay F et al (2006) Importance of K+-dependent Na+/Ca2+-exchanger 2, NCKX2, in motor learning and memory. J Biol Chem 281:6273–6282CrossRefPubMedGoogle Scholar
  7. 7.
    Lee SH, Park KH, Ho WK et al (2007) Postnatal developmental changes in Ca2+ homeostasis in supraoptic magnocellular neurons. Cell Calcium 41:441–450CrossRefPubMedGoogle Scholar
  8. 8.
    Dong H, Light PE, French RJ et al (2001) Electrophysiological characterization and ionic stoichiometry of the rat brain K+-dependent Na+/Ca2+ exchanger, NCKX2. J Biol Chem 276:25919–25928CrossRefPubMedGoogle Scholar
  9. 9.
    Cai X, Lytton J (2004) Molecular cloning of a sixth member of the K+-dependent Na+/Ca2+ exchanger gene family, NCKX6. J Biol Chem 279:5867–5876CrossRefPubMedGoogle Scholar
  10. 10.
    Lytton J (2007) Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport. Biochem J 406:365–382CrossRefPubMedGoogle Scholar
  11. 11.
    Tsoi M, Rhee KH, Bungard D et al (1998) Molecular cloning of a novel potassium-dependent sodium-calcium exchanger from rat brain. J Biol Chem 273:4155–4162CrossRefPubMedGoogle Scholar
  12. 12.
    Lytton J, Li XF, Dong H et al (2002) K+-dependent Na+/Ca2+ exchangers in the brain. Ann N Y Acad Sci 976:382–393CrossRefPubMedGoogle Scholar
  13. 13.
    Lee KH, Ho WK, Lee SH (2013) Endocytosis of somatodendritic NCKX2 is regulated by Src family kinase-dependent tyrosine phosphorylation. Front Cell Neurosci 7:14PubMedPubMedCentralGoogle Scholar
  14. 14.
    Bliss TV, Collingridge GL (1993) A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361:31–39CrossRefPubMedGoogle Scholar
  15. 15.
    Malenka RC, Nicoll RA (1999) Long-term potentiation—a decade of progress? Science 285:1870–1874CrossRefPubMedGoogle Scholar
  16. 16.
    Fukunaga K, Stoppini L, Miyamoto E et al (1993) Long-term potentiation is associated with a increased activity of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 268:7863–7867PubMedGoogle Scholar
  17. 17.
    Fukunaga K, Muller D, Miyamoto E (1995) Increased phosphorylation of Ca2+/calmodulin-dependent protein kinase II and its endogenous substrates in the induction of long term potentiation. J Biol Chem 270:6119–6124CrossRefPubMedGoogle Scholar
  18. 18.
    Silva AJ, Paylor R, Wehner JM et al (1992) Impaired spatial learning in alpha-calcium-calmodulin kinase II mutant mice. Science 257:206–211CrossRefPubMedGoogle Scholar
  19. 19.
    Lledo PM, Hjelmstad GO, Mukherji S et al (1995) Calcium/calmodulin-dependent kinase II and long-term potentiation enhance synaptic transmission by the same mechanism. Proc Natl Acad Sci U S A 92:11175–11179CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Wang YJ, Chen GH, Hu XY et al (2005) The expression of calcium/calmodulin-dependent protein kinase II-alpha in the hippocampus of patients with Alzheimer’s disease and its links with AD-related pathology. Brain Res 1031:101–108CrossRefPubMedGoogle Scholar
  21. 21.
    Barria A, Muller D, Derkach V et al (1997) Regulatory phosphorylation of AMPA-type glutamate receptor by CaM-KII during long-term potentiation. Science 276:2042–2045CrossRefPubMedGoogle Scholar
  22. 22.
    Giese KP, Fedorov NB, Filipkowski RK et al (1998) Autophosphorylation at Thr286 of the alpha calcium-calmodulin kinase II in LTP and learning. Science 279:870–873CrossRefPubMedGoogle Scholar
  23. 23.
    McGlade-McCulloh E, Yamamoto H, Tan SE et al (1993) Phosphorylation and regulation of glutamate receptors by calcium/calmodulin-dependent protein kinase II. Nature 362:640–642CrossRefPubMedGoogle Scholar
  24. 24.
    Jensen KF, Ohmstede CA, Fisher RS et al (1991) Nuclear and axonal localization of Ca2+/calmodulin-dependent protein kinase type Gr in rat cerebellar cortex. Proc Natl Acad Sci U S A 88:2850–2853CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Miyano O, Kameshita I, Fujisawa H (1992) Purification and characterization of a brain-specific multifunctional calmodulin-dependent protein kinase from rat cerebellum. J Biol Chem 267:1198–1203PubMedGoogle Scholar
  26. 26.
    Kasahara J, Fukunaga K, Miyamoto E (2001) Activation of calcium/calmodulin-dependent protein kinase IV in long term potentiation in the rat hippocampal CA1 region. J Biol Chem 276:24044–24050CrossRefPubMedGoogle Scholar
  27. 27.
    Ho N, Liauw JA, Blaeser F et al (2000) Impaired synaptic plasticity and cAMP response element-binding protein activation in Ca2+/calmodulin-dependent protein kinase type IV/Gr-deficient mice. J Neurosci 20:6459–6472PubMedGoogle Scholar
  28. 28.
    Lee KH, Chatila TA, Ram RA et al (2009) Impaired memory of eyeblink conditioning in CaMKIV KO mice. Behav Neurosci 123:438–442CrossRefPubMedGoogle Scholar
  29. 29.
    Wei F, Qiu CS, Liauw J et al (2002) Calcium calmodulin-dependent protein kinase IV is required for fear memory. Nat Neurosci 5:573–579CrossRefPubMedGoogle Scholar
  30. 30.
    Takao K, Tanda K, Nakamura K et al (2010) Comprehensive behavioral analysis of calcium/calmodulin-dependent protein kinase IV knockout mice. PLoS One 5:e9460CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Sturchler-Pierrat C, Abramowski D, Duke M et al (1997) Two amyloid precursor protein transgenic mouse models with Alzheimer’s disease-like pathology. Proc Natl Acad Sci U S A 94:13287–13292CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Sturchler-Pierrat C, Staufenbiel M (2000) Pathogenic mechanisms of Alzheimer’s disease analyzed in the APP23 transgenic mouse model. Ann N Y Acad Sci 920:134–139CrossRefPubMedGoogle Scholar
  33. 33.
    Calhoun ME, Burgermeister P, Phinney AL et al (1999) Neuronal overexpression of mutant amyloid precursor proteins results in prominent deposition of cerebrovascular amyloid. Proc Natl Acad Sci U S A 96:14088–14093CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Moriguchi S, Ishizuka T, Yabuki Y et al (2016) Blockade of the KATP channel Kir6.2 by memantine represents a novel mechanism relevant to Alzheimer’s disease therapy. Mol Psychiatry. doi: 10.1038/mp.2016.187
  35. 35.
    Keng VW, Yae K, Hayakawa T et al (2005) Region-specific saturation germline mutagenesis in mice using the Sleeping Beauty transposon system. Nat Methods 2:763–769CrossRefPubMedGoogle Scholar
  36. 36.
    Moriguchi S, Yamamoto Y, Ikuno T et al (2011) Sigma-1 receptor stimulation by dehydroepiandrosterone ameliorates cognitive impairment through activation of CaM kinase II, protein kinase C and extracellular signal-regulated kinase in olfactory bulbectomized mice. J Neurochem 117:879–891CrossRefPubMedGoogle Scholar
  37. 37.
    Moriguchi S, Sakagami H, Yabuki Y et al (2015) Stimulation of sigma-1 receptor ameliorates depressive-like behaviors in CaMKIV null mice. Mol Neurobiol 52:1210–1222CrossRefPubMedGoogle Scholar
  38. 38.
    Moriguchi S, Yabuki Y, Fukunaga K (2012) Reduced calcium/calmodulin-dependent protein kinase II activity in the hippocampus is associated with impaired cognitive function in MPTP-treated mice. J Neurochem 120:541–551CrossRefPubMedGoogle Scholar
  39. 39.
    Fukunaga K, Horikawa K, Shibata S et al (2002) Ca2+/calmodulin-dependent protein kinase II-dependent long-term potentiation in the rat suprachiasmatic nucleus and its inhibition by melatonin. J Neurosci Res 70:799–807CrossRefPubMedGoogle Scholar
  40. 40.
    Taigen T, De Windt LJ, Lim HW et al (2000) Targeted inhibition of calcineurin prevents agonist-induced cardiomyocyte hypertrophy. Proc Natl Acad Sci U S A 97:1196–1201CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Zheng F, Zhou X, Luo Y et al (2011) Regulation of brain-derived neurotrophic factor exon IV transcription through calcium responsive elements in cortical neurons. PLoS One 6:e28441CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Kidane AH, Heinrich G, Dirks RP et al (2009) Differential neuroendocrine expression of multiple brain-derived neurotrophic factor transcripts. Endocrinology 150:1361–1368CrossRefPubMedGoogle Scholar
  43. 43.
    Soderling TR, Derkach VA (2000) Postsynaptic protein phosphorylation and LTP. Trend Neurosci 23:75–80CrossRefPubMedGoogle Scholar
  44. 44.
    Lisman J, Schulman H, Cline H (2002) The molecular basis of CaMKII function synaptic and behavioral memory. Nat Rev Neurosci 3:175–190CrossRefPubMedGoogle Scholar
  45. 45.
    Blaustein MP, Lederer WJ (1999) Sodium/calcium exchange: its physiological implications. Physiol Rev 79:763–854CrossRefPubMedGoogle Scholar
  46. 46.
    Kang H, Sun LD, Atkins CM et al (2001) An important role of neural activity-dependent CaMKIV signaling in the consolidation of long-term memory. Cell 106:771–783CrossRefPubMedGoogle Scholar
  47. 47.
    Tao X, Finkbeiner S, Arnold DB et al (1998) Ca2+ influx regulates BDNF transcription by a CREB family transcription factor-dependent mechanism. Neuron 20:709–726CrossRefPubMedGoogle Scholar
  48. 48.
    Gass P, Wolfer DP, Balschun D et al (1998) Deficits in memory tasks of mice with CREB mutations depend on gene dosage. Learn Mem 5:274–288PubMedPubMedCentralGoogle Scholar
  49. 49.
    Montkowski A, Holsboer F (1997) Intact spatial learning and memory in transgenic mice with reduced BDNF. Neuroreport 8:779–782CrossRefPubMedGoogle Scholar
  50. 50.
    Korte M, Kang H, Bonhoeffer T et al (1998) A role for BDNF in the late-phase of hippocampal long-term potentiation. Neuropharmacology 37:553–559CrossRefPubMedGoogle Scholar
  51. 51.
    Sun P, Enslen H, Myung PS et al (1994) Differential activation of CREB by Ca2+/calmodulin-dependent protein kinases type II and type IV involves phosphorylation of a site that negatively regulates activity. Genes Dev 8:2527–2539CrossRefPubMedGoogle Scholar
  52. 52.
    Schlman H, Lou LL (1989) Multifunctional Ca2+/calmodulin-dependent protein kinase: domain structure and regulation. Trends Biol Sci 14:62–66CrossRefGoogle Scholar
  53. 53.
    Klee CB (1991) Concerted regulation of protein phosphorylation and dephosphorylation by calmodulin. Neurochem Res 16:1059–1065CrossRefPubMedGoogle Scholar
  54. 54.
    Strack S, Barban MA, Wadzinski BE et al (1997) Differential inactivation of postsynaptic density-associated and soluble Ca2+/calmodulin-dependent protein kinase II by protein phosphatase 1 and 2A. J Neurochem 68:2119–2128CrossRefPubMedGoogle Scholar
  55. 55.
    Hemmings HC Jr, Greengard P, Tung HY et al (1984) DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1. Nature 310:503–505CrossRefPubMedGoogle Scholar
  56. 56.
    Morioka M, Nagahiro S, Fukunaga K et al (1997) Calcineurin in the adult rat hippocampus: different distribution in CA1 and CA3 subfields. Neuroscience 78:673–684CrossRefPubMedGoogle Scholar
  57. 57.
    Kasahara J, Fukunaga K, Miyamoto E (1999) Differential effects of a calcineurin inhibitor on glutamate-induced phosphorylation of Ca2+/calmodulin-dependent protein kinases in cultured rat hippocampal neurons. J Biol Chem 274:9061–9067CrossRefPubMedGoogle Scholar
  58. 58.
    Thayer SA, Usachev YM, Pottorf WJ (2002) Modulating Ca2+ clearance from neurons. Front Biosci 7:D1255–D1279PubMedGoogle Scholar
  59. 59.
    Lee KH, Lee JS, Lee D et al (2012) KIF21A-mediated axonal transport and selective endocytosis underlie the polarized targeting of NCKX2. J Neurosci 32:4102–4117CrossRefPubMedGoogle Scholar
  60. 60.
    Wang JH, Kelly PT (1995) Postsynaptic injection of Ca2+/CaM induces synaptic potentiation requiring CaMKII and PKC activity. Neuron 15:443–452CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Department of Pharmacology, Graduate School of Pharmaceutical SciencesTohoku UniversitySendaiJapan
  2. 2.Department of Pharmacology, Faculty of MedicineFukuoka UniversityFukuokaJapan
  3. 3.Department of Pharmacology, Faculty of Pharmaceutical SciencesTokushima Bunri UniversityTokushimaJapan
  4. 4.Department of Physiology IINara Medical UniversityNaraJapan
  5. 5.Department of Social and Environmental Medicine, Graduate School of MedicineOsaka UniversityOsakaJapan

Personalised recommendations