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Ryanodine receptor binding to FKBP12 is modulated by channel activation state

Jonathan-Lee Jones1, Deborah F Reynolds, F Anthony Lai

  • 1Wales Heart Research Institute, Department of Cardiology, School of Medicine, Cardiff University, Heath Park, Cardiff, CF14 4XN, UK.

Journal of Cell Science
|September 24, 2005
PubMed

Insights

FK506 binding protein 12 (FKBP12) binding to the ryanodine receptor (RyR) Ca2+ channel is state-dependent. FKBP12 binds RyR more strongly when the channel is closed, a finding crucial for understanding RyR channel regulation.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Physiology

Background:

  • Ryanodine receptors (RyRs) are intracellular calcium release channels critical for muscle contraction and cellular signaling.
  • FK506 binding protein 12 (FKBP12) is a known modulator of RyR channel gating.
  • Understanding the RyR-FKBP12 interaction is key to elucidating calcium channel regulation.

Purpose of the Study:

  • To investigate the interdependence between RyR and FKBP12 binding.
  • To determine how RyR channel gating (open/closed states) affects FKBP12 binding affinity.
  • To evaluate the utility of surface plasmon resonance for studying RyR-FKBP12 kinetics.

Main Methods:

  • Utilized ELISA sandwich assays to assess FKBP12 binding under varying free Ca2+ concentrations and in the presence of ATP.
  • Employed [3H]ryanodine binding assays to confirm RyR channel gating states induced by different agents (EGTA, Ca2+, ruthenium red, caffeine).
  • Applied surface plasmon resonance (SPR) to measure equilibrium binding kinetics (KA, kd) of RyR1 and FKBP12 under defined channel states.

Main Results:

  • FKBP12 binding to RyR was significantly lower at 1-10 microM free Ca2+ compared to conditions favoring channel closure (1 mM EGTA, 1 mM Ca2+).
  • ATP enhanced FKBP12 binding, while ruthenium red increased it, suggesting modulation of channel conformation.
  • [3H]ryanodine binding confirmed that EGTA, Ca2+, and ruthenium red closed the channel, whereas free Ca2+, Ca2+ + ATP, and caffeine opened it.
  • SPR analysis revealed a significantly higher affinity constant (KA) for FKBP12 binding to the closed RyR channel compared to the open state, primarily due to a decreased dissociation rate constant (kd).

Conclusions:

  • FKBP12 exhibits state-dependent binding to RyR, with higher affinity for the closed channel conformation.
  • The binding affinity is modulated by Ca2+ levels, ATP, and specific pharmacological agents.
  • Surface plasmon resonance is a robust technique for quantifying differences in RyR1-FKBP12 binding kinetics across channel states.

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