Dataset Information

Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) ?(1S) subunit.

ABSTRACT: The skeletal muscle dihydropyridine receptor (DHPR) in the t-tubular membrane serves as the Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling, triggering Ca(2+) release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). The two proteins appear to be physically linked, and both the ?(1S) and ?(1a) subunits of the DHPR are essential for EC coupling. Within ?(1S), cytoplasmic domains of importance include the I-II loop (to which ?(1a) binds), the II-III and III-IV loops, and the C terminus. However, the spatial relationship of these domains to one another has not been established. Here, we have taken the approach of measuring FRET between fluorescent proteins inserted into pairs of ?(1S) cytoplasmic domains. Expression of these constructs in dyspedic (RyR1 null) and dysgenic (?(1S) null) myotubes was used to test for function and targeting to plasma membrane/SR junctions and to test whether the presence of RyR1 caused altered FRET. We found that in the absence of RyR1, measureable FRET occurred between the N terminus and C terminus (residue 1636), and between the II-III loop (residue 626) and both the N and C termini; the I-II loop (residue 406) showed weak FRET with the II-III loop but not with the N terminus. Association with RyR1 caused II-III loop FRET to decrease with the C terminus and increase with the N terminus and caused I-II loop FRET to increase with both the II-III loop and N terminus. Overall, RyR1 appears to cause a substantial reorientation of the cytoplasmic ?(1S) domains consistent with their becoming more closely packed.

SUBMITTER: Polster A

PROVIDER: S-EPMC3510851 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α(1S) subunit.

Polster Alexander A Ohrtman Joshua D JD Beam Kurt G KG Papadopoulos Symeon S

The Journal of biological chemistry 20121015 49

The skeletal muscle dihydropyridine receptor (DHPR) in the t-tubular membrane serves as the Ca(2+) channel and voltage sensor for excitation-contraction (EC) coupling, triggering Ca(2+) release via the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR). The two proteins appear to be physically linked, and both the α(1S) and β(1a) subunits of the DHPR are essential for EC coupling. Within α(1S), cytoplasmic domains of importance include the I-II loop (to which β(1a) binds), the I ...[more]

PMID: 23071115

Similar Datasets

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

Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) ?(1S) subunit.

Publications

Fluorescence resonance energy transfer (FRET) indicates that association with the type I ryanodine receptor (RyR1) causes reorientation of multiple cytoplasmic domains of the dihydropyridine receptor (DHPR) α(1S) subunit.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets