Biophys J, January 2002, p. 145-155, Vol. 82, No. 1

FKBP12 Modulation of the Binding of the Skeletal Ryanodine Receptor onto the II-III Loop of the Dihydropyridine Receptor

Fiona M. O'Reilly,^* Mylène Robert,^* Istvan Jona, Csaba Szegedi, Mireille Albrieux,^* Sandrine Geib,^* Michel De Waard,^* Michel Villaz,^* and Michel Ronjat^*

 ^*Laboratoire Canaux Ioniques et Signalisation, Département de Biologie Moléculaire et Structurale, CEA-Grenoble, F-38054 Grenoble, France, and  Department of Physiology, University Medical School Debrecen, H-4012 Debrecen, Hungary

In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ of the II-III loop of the skeletal DHPR ₁-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu⁷²⁴-Pro⁷⁶⁰ antagonizes this effect. Two peptides, covering these sequences (peptide A_Sk and C_Sk, respectively) were immobilized on polystyrene beads. We demonstrate that peptide A_Sk binds to the skeletal isoform of RyR (RyR1) whereas peptide C_Sk does not. Using surface plasmon resonance detection, we show that 1) domain Thr⁶⁷¹-Leu⁶⁹⁰ is the only sequence of the II-III loop binding with RyR1 and 2) the interaction of peptide A_Sk with RyR1 is not modulated by Ca²⁺ (pCa 9-2) nor by Mg²⁺ (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC₅₀ = 10 nM) and inhibited by both rapamycin (IC₅₀ = 5 nM) and FK506. Peptide A_Sk induces a 300% increase of the opening probability of the RyR1 incorporated in lipid bilayer. Removal of FKBP12 from RyR1 completely abolishes this effect of domain A_Sk on RyR1 channel behavior. These results demonstrate a direct interaction of the RyR1 with the discrete domain of skeletal DHPR ₁-subunit corresponding to Thr⁶⁷¹-Leu⁶⁹⁰ and show that the association of FKBP12 with RyR1 specifically modulates this interaction.

Biophys J, January 2002, p. 145-155, Vol. 82, No. 1
© 2002 by the Biophysical Society   0006-3495/02/01/145/11  $2.00

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