Kinetics of FKBP12.6 binding to ryanodine receptors in permeabilized cardiac myocytes and effects on Ca sparks

Tao Guo, Razvan L. Cornea, Sabine Huke, Emmanuel Camors, Yi Yang, Eckard Picht, Bradley R. Fruen, Donald M Bers

Research output: Contribution to journalArticle

103 Scopus citations

Abstract

Rationale: FK506-binding proteins FKBP12.6 and FKBP12 are associated with cardiac ryanodine receptors (RyR2), and cAMP-dependent protein kinase A (PKA)-dependent phosphorylation of RyR2 was proposed to interrupt FKBP12.6-RyR2 association and activate RyR2. However, the function of FKBP12.6/12 and role of PKA phosphorylation in cardiac myocytes are controversial. OBJECTIVE: To directly measure in situ binding of FKBP12.6/12 to RyR2 in ventricular myocytes, with simultaneous Ca sparks measurements as a RyR2 functional index. Methods and results: We used permeabilized rat and mouse ventricular myocytes, and fluorescently-labeled FKBP12.6/12. Both FKBP12.6 and FKBP12 concentrate at Z-lines, consistent with RyR2 and Ca spark initiation sites. However, only FKBP12.6 inhibits resting RyR2 activity. Assessment of fluorescent FKBP binding in myocyte revealed a high FKBP12.6-RyR2 affinity (Kd=0.7±0.1 nmol/L) and much lower FKBP12-RyR2 affinity (Kd=206±70 nmol/L). Fluorescence recovery after photobleach confirmed this Kd difference and showed that it is mediated by koff. RyR2 phosphorylation by PKA did not alter binding kinetics or affinity of FKBP12.6/12 for RyR2. Using quantitative immunoblots, we determined endogenous [FKBP12] in intact myocytes is â‰̂1 μmol/L (similar to [RyR]), whereas [FKBP12.6] is ≤150 nmol/L. Conclusions: Only 10% to 20% of endogenous myocyte RyR2s have FKBP12.6 associated, but virtually all myocyte FKBP12.6 is RyR2-bound (because of very high affinity). FKBP12.6 but not FKBP12 inhibits basal RyR2 activity. PKA-dependent RyR2 phosphorylation has no significant effect on binding of either FKBP12 or 12.6 to RyR2 in myocytes.

Original languageEnglish (US)
Pages (from-to)1743-1752
Number of pages10
JournalCirculation Research
Volume106
Issue number11
DOIs
StatePublished - Jun 11 2010

Keywords

  • Binding properties
  • Ca sparks
  • FKBP12
  • FKBP12.6
  • Rapamycin
  • RyR2

ASJC Scopus subject areas

  • Physiology
  • Cardiology and Cardiovascular Medicine

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