­čôĺ Chiba 2008

A simulation study on the activation of cardiac CaMKII delta-isoform and its regulation by phosphatases1



  • CaMKII is activated through the binding of Ca2+-bound calmodulin (CaM) during the transient increase in the intracellular Ca2+ concentration ($[Ca^2+]_i$)
  • With a rise in the frequency of CaT2, the lifetime of activated CaMKII molecules is increased by intersubunit autophosphorylation, leading to an accumulation of the active CaMKII
  • Phosphorylated CaMKII maintains its catalytic activity even after CaT2 until it is inactivated by constitutive phosphatase activity
  • CaMKII autophosphorylation is associated with long-term potentiation in neurons (╬▒ and ╬▓ isoform)
  • Cardiomyocytes (╬┤ isoform??): also undergo autophosphorylation aswell as other regulatory mechanisms(oxidation) => Redox as well as frequency sensor
  • ╬┤ isoform has a higher affinity for CaM (Kd = 33.5 nM) compared to ╬▒ (Kd = 62.4 nM) and a higher autophosphorylation rate


Calmodulin (CaM)

  • CaM, a highly conserved protein, possesses at its C-terminal lobe two high-affinity Ca2+-binding sites with a Kd of Ôł╝1ÔÇô2 ╬╝M and at its N-terminal lobe two low-affinity sites with a Kd of Ôł╝2.6ÔÇô13 ╬╝M
  • Modelled as sequential binding of 4 calcium ions. Values for the rate constants were adapted from the original model.
  • The Hill coefficient (nH) and K0.5 for the [Ca2+]-CaMCa4 relationship are 1.9 and 26 ╬╝M
  • accurate measurement of free [CaMCa4] in vivo is difficult (membrane-bound, clustered, competition from other proteins)


  • One inactive (neither phosphorated nor bound to CaMCa4) and 3 active states
  • dissociation of CaMCa4: two pathways (CaMCa4 or Ca)
  • autophosphorylation of neighboring subunits
  • dephosphorylation: PP1
  • Kinetic model from brain-specific ╬▒ isoform of CaMKII => tranlate to ╬┤ isoform in the heart

Ca transient

  • hypothetical Ca2+ transient described by Negroni and Lascano


Analysis of CaMCa4 binding to CaMKII╬▒

  • The dependency of CaMKII╬▒ activation on [CaMCa4] (steps A1 and A2 of the model) was analyzed in the absence of ATP, i.e., no autophosphorylation
  • K0.5 (k_disso/k_asso) of 66.7 nM
  • 1:1 binding of CaMCa4 to CaMKII, i.e., nH = 1.0

Reconstruction of experiments measuring the CaMKII╬▒ autophosphorylation rate

  • A: 0.1 mM ATP and 500 ╬╝M Ca2+, 62 nM CaMKII╬▒
  • B: 0.25 mM ATP and 500 ╬╝M Ca2+, 5 nM CaMKII╬▒
  • C: 2 mM ATP and different [Ca2+] (0.1ÔÇô100 ╬╝M), 0.2 ╬╝M CaMKII was incubated with 50 ╬╝M CaM

Reconstruction of the frequency-dependent activation of CaMKII╬▒

  • (500 ╬╝M Ca2+, 100 nM CaM, and 0.25 mM ATP)

Parameter determination for the CaMKII╬┤ model

  • B: different [CaM] (1ÔÇô10,000 nM) at 0┬░C in the presence of 0.1 mM ATP and 500 ╬╝M Ca2+
  • CaMKII╬┤ isoform exhibited a higher CaM affinity, specifically, Kd = 33.5 nM versus Kd = 62.4 nM, and a faster autophosphorylation compared to the CaMKII╬▒ isoform
  • the rate constants k_disso, k_dissoCa, k_disso2, and k_dissoCa2 were decreased twofold and kcat was increased sixfold

Cumulative activation of CaMKII╬┤ by repetitive Ca2+-transients

  • B: 5 mM [ATP], 6 ╬╝M [CaM], and 0.1 ╬╝M [CaMKII╬┤]

Regulation of activated CaMKII╬┤ through phosphatases

  • CaMKII╬▒ (black) and CaMKII╬┤ (red)
  • 2 mM ATP and different [Ca2+] (0.1ÔÇô100 ╬╝M), 1 ╬╝M CaMKII and 1.25 ╬╝M PP1 were incubated with 5 ╬╝M CaM at 0┬░C
Effects of [PP1] variation on the frequency-dependent activation of CaMKII

  • a role of PPs in the dynamic adjustment of CaMKII╬┤ activity over the physiological range of the heart rate


  • a novel and simple four-state CaMKII╬┤ model was developed that includes autophosphorylation and dephosphorylation by PP1
  • Both models, CaMKII╬▒ and CaMKII╬┤, used in this study could well reproduce experimental findings regarding the steady-state dose-response relationships for activation by CaMCa4
  • And the time-dependent accumulation of activated CaMKII fraction

Significance of CaMKII autophosphorylation in the cardiac FDAR

  • Total concentrations of CaM, PP1, and CaMKII greatly affect simulation results
  • [CaM]total of 6 ╬╝M, difficult to directly measure [PP1] and [CaMKII] in experiments
    • The [CaMKII] was determined to be 0.12 ╬╝M
    • [PP1] was determined as 0.084 ╬╝M
  • The combination of 0.1 ╬╝M [CaMKII], 0.1 ╬╝M [PP1], and 6 ╬╝M [CaM]total in Fig. 8 A well reconciled the apparent dissociation of FDAR and protein phosphorylation by CaMKII suggested by Huke and Bers

Model limitations

  • For all three active states considered in the model (CaMKII_CaMCa4, CaMKIIP_CaMCa4, and CaMKIIP) the same autophosphorylation activity was assumed
  • CaMKII╬▒ autophosphorylated at Thr286 was shown to undergo further autophosphorylation at Thr305/Thr306 after dissociation of CaM, known as secondary or inhibitory autophosphorylation (capped state) not in the model
  • Experimental data for the kinetic properties of CaMKII╬┤ are still very limited

Model parameters

ParameterValue ($\alpha$)Value ($\delta$)Unit
$k_{-1}, k_{-2}$5050Hz
$k_{2}$8825088250Hz / mM
$k_3$1250012500Hz / mM
$k_{-3}, k_{-4}$12501250Hz
$k_{diss, Ca}$1.90.95Hz
$k_{cat}$ (273K)0.010.06Hz
$k_{cat}$ (303K)0.31.8Hz
$k_{cat}$ (310K)0.95.4Hz

Only kcat is temperature dependent in this model.

  1. Chiba H, Schneider NS, Matsuoka S, Noma A. A simulation study on the activation of cardiac CaMKII delta-isoform and its regulation by phosphatases. Biophys. J. 2008;95(5):2139-2149. doi:10.1529/biophysj.107.118505. ↩︎

  2. CaT : $[Ca^2+]_i$ transient ↩︎