The CamKKβ Inhibitor STO609 Causes Artefacts in Calcium Imaging and Selectively Inhibits BKCa in Mouse Carotid Body Type I Cells

  • Jennifer G. Jurcsisn
  • Richard L. Pye
  • Jon Ali
  • Barbara L. Barr
  • Christopher N. WyattEmail author
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 860)


It has previously been reported that AMP-activated protein kinase (AMPK) may be critical for hypoxic chemotransduction in carotid body type I cells. This study sought to determine the importance of the regulatory upstream kinase of AMPK, CamKKβ, in the acute response to hypoxia in isolated mouse type I cells.

Initial data indicated several previously unreported artefacts associated with using the CamKKβ inhibitor STO609 and Ca2+ imaging techniques. Most importantly Fura-2 and X-Rhod1 imaging revealed that STO609 quenched emission fluorescence even in the absence of intracellular Ca2+ ([Ca2+]i). Furthermore, STO609 (100 μM) rapidly inhibited outward macroscopic currents and this inhibition was abolished in the presence of the selective BKCa inhibitor paxilline.

Taken together these data suggest that ST0609 should be used with caution during Ca2+ imaging studies as it can directly interact with Ca2+ binding dyes. The rapid inhibitory effect of STO609 on BKCa was unexpected as the majority of studies using this compound required an incubation of approximately 10 min to inhibit the kinase. Furthermore, as AMPK activation inhibits BKCa,inhibiting AMPK’s upstream kinases would, if anything, be predicted to have the opposite effect on BKCa. Future work will determine if the inhibition of BKCa is via CamKKβ or via an off target action of STO609 on the channel itself.


Carotid body Type I cells CamKKβ STO609 



This work was supported by NIH-1RO1HL091836


  1. Burlon DC, Jordan HL, Wyatt CN (2009) Presynaptic regulation of isolated neonatal rat carotid body type I cells by histamine. Respir Physiol Neurobiol 168(3):218–223PubMedCrossRefGoogle Scholar
  2. Carling D, Sanders MJ, Woods A (2008) The regulation of AMP-activated protein kinase by upstream kinases. Int J Obes (Lond) 32(4):55–59CrossRefGoogle Scholar
  3. Fogarty S, Hawley SA, Green KA, Saner N, Mustard KJ, Hardie DG (2010) Calmodulin-dependent protein kinase kinase-beta activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP. Biochem J 426(1):109–118PubMedCrossRefPubMedCentralGoogle Scholar
  4. Hardie DG (2008) AMPK: a key regulator of energy balance in the single cell and the whole organism. Int J Obes (Lond) 32:S7–S12CrossRefGoogle Scholar
  5. Hawley SA, Boudeau J, Reid JL, Mustard KJ, Udd L, Ma¨kela¨ TP et al (2003) Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade. J Biol 2:28PubMedCrossRefPubMedCentralGoogle Scholar
  6. Hawley SA, Pan DA, Mustard KJ, Ross L, Bain J, Edelman AM et al (2005) Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase. Cell Metab 2:9–19PubMedCrossRefGoogle Scholar
  7. Kukimoto-Niino M et al (2011) Crystal structure of the Ca2+/calmodulin-dependent protein kinase kinase in complex with the inhibitor STO-609. J Biol Chem 286:22570–22579PubMedCrossRefPubMedCentralGoogle Scholar
  8. Momcilovic M, Hong SP, Carlson M (2006) Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro. J Biol Chem 281(35):25336–25343PubMedCrossRefGoogle Scholar
  9. Monteiro P, Gilot D, Langouet S, Fardel O (2008) Activation of the aryl hydrocarbon receptor by the calcium/calmodulin-dependent protein kinase kinase inhibitor 7-Oxo-7H-benzimidazo[2,1-a]benz[de]isoquinoline-3-carboxylic acid (STO-609). Drug Metab Dispos 36(12):2556–2563PubMedCrossRefGoogle Scholar
  10. Ross FA, Rafferty JN, Dallas ML, Ogunbayo O, Ikematsu N, McClafferty H, Tian L, Widmer H, Rowe ICM, Wyatt CN, Shipston MJ, Peers C, Hardie DG, Evans AM (2011) Selective expression in carotid body type I cells of a single splice variant of the large conductance calcium- and voltage-activated potassium channel confers regulation by AMP-activated protein kinase. J Biol Chem 286(14):11929–11936PubMedCrossRefPubMedCentralGoogle Scholar
  11. Schmitt JM, Guire ES, Saneyoshi T, Soderling TR (2005) Calmodulin-dependent kinase kinase/calmodulin kinase I activity gates extracellular-regulated kinase-dependent long-term potentiation. J Neurosci 25(5):1281–1290PubMedCrossRefGoogle Scholar
  12. Tamás P, Hawley SA, Clarke RG, Mustard KJ, Green K, Hardie DG, Cantrell DA (2006) Regulation of the energy sensor AMP-activated protein kinase by antigen receptor and Ca2+ in T lymphocytes. J Exp Med 203(7):1665–1670PubMedCrossRefPubMedCentralGoogle Scholar
  13. Tokumitsu H, Inuzuka H, Ishikawa Y, Ikeda M, Saji I, Kobayashi R (2002) STO-609, a specific inhibitor of the Ca(2+)/calmodulin-dependent protein kinase kinase. J Biol Chem 277:15813–15818PubMedCrossRefGoogle Scholar
  14. Wayman GA, Kaech S, Grant WF, Davare M, Impey S, Tokumitsu H, Nozaki N, Banker G, Soderling TR (2004) Regulation of axonal extension and growth cone motility by calmodulin-dependent protein kinase I. J Neurosci 24(15):3786–3794PubMedCrossRefGoogle Scholar
  15. Woods A, Johnstone SR, Dickerson K, Leiper FC, Fryer LG, Neumann D et al (2003) LKB1 is the upstream kinase in the AMP-activated protein kinase cascade. Curr Biol 13:2004–2008PubMedCrossRefGoogle Scholar
  16. Woods A, Dickerson K, Heath R, Hong SP, Momcilovic M, Johnstone SR et al (2005) Ca2+/calmodulin-dependent protein kinase kinase-beta acts upstream of AMP-activated protein kinase in mammalian cells. Cell Metab 2:21–33PubMedCrossRefGoogle Scholar
  17. Wyatt CN, Evans AM (2007) AMP-activated protein kinase and chemotransduction in the carotid body. Respir Physiol Neurobiol 157:22–29PubMedCrossRefGoogle Scholar

Copyright information

© Springer International Publishing Switzerland 2015

Authors and Affiliations

  • Jennifer G. Jurcsisn
    • 1
  • Richard L. Pye
    • 1
  • Jon Ali
    • 1
  • Barbara L. Barr
    • 1
  • Christopher N. Wyatt
    • 1
    Email author
  1. 1.Department of Neuroscience, Cell Biology and PhysiologyWright State UniversityDaytonUSA

Personalised recommendations