Dataset Information

FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor.

ABSTRACT: In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr(671)-Leu(690) of the II-III loop of the skeletal DHPR alpha(1)-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu(724)-Pro(760) 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 Thr(671)-Leu(690) 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 Ca(2+) (pCa 9-2) nor by Mg(2+) (up to 10 mM). In contrast, this interaction is strongly potentiated by the immunophilin FKBP12 (EC(50) = 10 nM) and inhibited by both rapamycin (IC(50) = 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 alpha(1)-subunit corresponding to Thr(671)-Leu(690) and show that the association of FKBP12 with RyR1 specifically modulates this interaction.

SUBMITTER: O'Reilly FM

PROVIDER: S-EPMC1302456 | biostudies-other | 2002 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Publications

FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor.

O'Reilly Fiona M FM Robert Mylène M Jona Istvan I Szegedi Csaba C Albrieux Mireille M Geib Sandrine S De Waard Michel M Villaz Michel M Ronjat Michel M

Biophysical journal 20020101 1 Pt 1

In skeletal muscle, excitation-contraction coupling involves a functional interaction between the ryanodine receptor (RyR) and the dihydropyridine receptor (DHPR). The domain corresponding to Thr(671)-Leu(690) of the II-III loop of the skeletal DHPR alpha(1)-subunit is able to regulate RyR properties and calcium release from sarcoplasmic reticulum, whereas the domain corresponding to Glu(724)-Pro(760) antagonizes this effect. Two peptides, covering these sequences (peptide A(Sk) and C(Sk), respe ...[more]

PMID: 11751303

Similar Datasets

Project description:The beta-subunit of the dihydropyridine receptor (DHPR) enhances the Ca(2+) channel and voltage-sensing functions of the DHPR. In skeletal myotubes, there is additional modulation of DHPR functions imposed by the presence of ryanodine receptor type-1 (RyR1). Here, we examined the participation of the beta-subunit in the expression of L-type Ca(2+) current and charge movements in RyR1 knock-out (KO), beta1 KO, and double beta1/RyR1 KO myotubes generated by mating heterozygous beta1 KO and RyR1 KO mice. Primary myotube cultures of each genotype were transfected with various beta-isoforms and then whole-cell voltage-clamped for measurements of Ca(2+) and gating currents. Overexpression of the endogenous skeletal beta1a isoform resulted in a low-density Ca(2+) current either in RyR1 KO (36 +/- 9 pS/pF) or in beta1/RyR1 KO (34 +/- 7 pS/pF) myotubes. However, the heterologous beta2a variant with a double cysteine motif in the N-terminus (C3, C4), recovered a Ca(2+) current that was entirely wild-type in density in RyR1 KO (195 +/- 16 pS/pF) and was significantly enhanced in double beta1/RyR1 KO (115 +/- 18 pS/pF) myotubes. Other variants tested from the four beta gene families (beta1a, beta1b, beta1c, beta3, and beta4) were unable to enhance Ca(2+) current expression in RyR1 KO myotubes. In contrast, intramembrane charge movements in beta2a-expressing beta1a/RyR1 KO myotubes were significantly lower than in beta1a-expressing beta1a/RyR1 KO myotubes, and the same tendency was observed in the RyR1 KO myotube. Thus, beta2a had a preferential ability to recover Ca(2+) current, whereas beta1a had a preferential ability to rescue charge movements. Elimination of the double cysteine motif (beta2a C3,4S) eliminated the RyR1-independent Ca(2+) current expression. Furthermore, Ca(2+) current enhancement was observed with a beta2a variant lacking the double cysteine motif and fused to the surface membrane glycoprotein CD8. Thus, tethering the beta2a variant to the myotube surface activated the DHPR Ca(2+) current and bypassed the requirement for RyR1. The data suggest that the Ca(2+) current expressed by the native skeletal DHPR complex has an inherently low density due to inhibitory interactions within the DHPR and that the beta1a-subunit is critically involved in process.

Project description:Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca(2+) release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR ?1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of ?1a activation of RyRs and the ?1a binding site on RyR1.We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca(2+) currents and Ca(2+) transients in myotubes.We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10-100 nM of the ?1a subunit. Activation of RyR2 by 10 nM ?1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for ?1a. A reduction in activation was also observed when 10 nM ?1a was added to the alternatively spliced (ASI(-)) isoform of RyR1, which lacks ASI residues (A3481-Q3485). It is notable that the equivalent region of RyR2 also lacks four of five ASI residues, suggesting that the absence of these residues may contribute to the reduced 10 nM ?1a activation observed for both RyR2 and ASI(-)RyR1 compared to ASI(+)RyR1. We also investigated the influence of a polybasic motif (PBM) of RyR1 (K3495KKRRDGR3502) that is located immediately downstream from the ASI residues and has been implicated in EC coupling. We confirmed that neutralizing the basic residues in the PBM (RyR1 K-Q) results in an ~50 % reduction in Ca(2+) transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for ?1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of ?1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca(2+) release generated during skeletal EC coupling.We conclude that the ?1a subunit likely binds to a region that is largely conserved in RyR1 and RyR2 and that this region is influenced by the presence of the ASI residues and the PBM in RyR1.

Dataset Information

FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor.

Publications

FKBP12 modulation of the binding of the skeletal ryanodine receptor onto the II-III loop of the dihydropyridine receptor.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets