Regions of ryanodine receptors that influence activation by the dihydropyridine receptor ß1a subunit

Robyn Rebbeck, Hermia WILLEMSE, L Groom, Marco Casarotto, Philip Board, Nicole BEARD, R Dirksen, Angela F. Dulhunty

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Abstract

Background: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR ß1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of ß1a activation of RyRs and the ß1a binding site on RyR1. Methods: We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes. Results: We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10-100 nM of the ß1a subunit. Activation of RyR2 by 10 nM ß1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for ß1a. A reduction in activation was also observed when 10 nM ß1a 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 ß1a 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 Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for ß1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of ß1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca2+ release generated during skeletal EC coupling. Conclusions: We conclude that the ß1a 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.
Original languageEnglish
Article number23
Pages (from-to)1-15
Number of pages15
JournalSkeletal Muscle
Volume5
DOIs
Publication statusPublished - 2015

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L-Type Calcium Channels
Ryanodine Receptor Calcium Release Channel
Excitation Contraction Coupling
Skeletal Muscle Fibers
Lipid Bilayers
Protein Isoforms

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Rebbeck, Robyn ; WILLEMSE, Hermia ; Groom, L ; Casarotto, Marco ; Board, Philip ; BEARD, Nicole ; Dirksen, R ; Dulhunty, Angela F. / Regions of ryanodine receptors that influence activation by the dihydropyridine receptor ß1a subunit. In: Skeletal Muscle. 2015 ; Vol. 5. pp. 1-15.
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title = "Regions of ryanodine receptors that influence activation by the dihydropyridine receptor {\ss}1a subunit",
abstract = "Background: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR {\ss}1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of {\ss}1a activation of RyRs and the {\ss}1a binding site on RyR1. Methods: We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes. Results: We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10-100 nM of the {\ss}1a subunit. Activation of RyR2 by 10 nM {\ss}1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for {\ss}1a. A reduction in activation was also observed when 10 nM {\ss}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 {\ss}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 Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for {\ss}1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of {\ss}1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 {\%} of the Ca2+ release generated during skeletal EC coupling. Conclusions: We conclude that the {\ss}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.",
keywords = "Cardiac muscle, Dihydropyridine receptor β<inf>1a</inf> subunit, Excitation-contraction coupling, Ryanodine receptor isoforms, Skeletal muscle",
author = "Robyn Rebbeck and Hermia WILLEMSE and L Groom and Marco Casarotto and Philip Board and Nicole BEARD and R Dirksen and Dulhunty, {Angela F.}",
year = "2015",
doi = "10.1186/s13395-015-0049-3",
language = "English",
volume = "5",
pages = "1--15",
journal = "Skeletal Muscle",
issn = "2044-5040",
publisher = "BioMed Central",

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Rebbeck, R, WILLEMSE, H, Groom, L, Casarotto, M, Board, P, BEARD, N, Dirksen, R & Dulhunty, AF 2015, 'Regions of ryanodine receptors that influence activation by the dihydropyridine receptor ß1a subunit', Skeletal Muscle, vol. 5, 23, pp. 1-15. https://doi.org/10.1186/s13395-015-0049-3

Regions of ryanodine receptors that influence activation by the dihydropyridine receptor ß1a subunit. / Rebbeck, Robyn; WILLEMSE, Hermia; Groom, L; Casarotto, Marco; Board, Philip; BEARD, Nicole; Dirksen, R; Dulhunty, Angela F.

In: Skeletal Muscle, Vol. 5, 23, 2015, p. 1-15.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Regions of ryanodine receptors that influence activation by the dihydropyridine receptor ß1a subunit

AU - Rebbeck, Robyn

AU - WILLEMSE, Hermia

AU - Groom, L

AU - Casarotto, Marco

AU - Board, Philip

AU - BEARD, Nicole

AU - Dirksen, R

AU - Dulhunty, Angela F.

PY - 2015

Y1 - 2015

N2 - Background: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR ß1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of ß1a activation of RyRs and the ß1a binding site on RyR1. Methods: We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes. Results: We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10-100 nM of the ß1a subunit. Activation of RyR2 by 10 nM ß1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for ß1a. A reduction in activation was also observed when 10 nM ß1a 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 ß1a 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 Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for ß1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of ß1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca2+ release generated during skeletal EC coupling. Conclusions: We conclude that the ß1a 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.

AB - Background: Although excitation-contraction (EC) coupling in skeletal muscle relies on physical activation of the skeletal ryanodine receptor (RyR1) Ca2+ release channel by dihydropyridine receptors (DHPRs), the activation pathway between the DHPR and RyR1 remains unknown. However, the pathway includes the DHPR ß1a subunit which is integral to EC coupling and activates RyR1. In this manuscript, we explore the isoform specificity of ß1a activation of RyRs and the ß1a binding site on RyR1. Methods: We used lipid bilayers to measure single channel currents and whole cell patch clamp to measure L-type Ca2+ currents and Ca2+ transients in myotubes. Results: We demonstrate that both skeletal RyR1 and cardiac RyR2 channels in phospholipid bilayers are activated by 10-100 nM of the ß1a subunit. Activation of RyR2 by 10 nM ß1a was less than that of RyR1, suggesting a reduced affinity of RyR2 for ß1a. A reduction in activation was also observed when 10 nM ß1a 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 ß1a 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 Ca2+ transient amplitude following expression in RyR1-null (dyspedic) myotubes and that the PBM is also required for ß1a subunit activation of RyR1 channels in lipid bilayers. These results suggest that the removal of ß1a subunit interaction with the PBM in RyR1 could contribute directly to ~50 % of the Ca2+ release generated during skeletal EC coupling. Conclusions: We conclude that the ß1a 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.

KW - Cardiac muscle

KW - Dihydropyridine receptor β<inf>1a</inf> subunit

KW - Excitation-contraction coupling

KW - Ryanodine receptor isoforms

KW - Skeletal muscle

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