Abstract
Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca2+ entry channel with SOCC-like properties, has recently been presented (Cherednichenko, G., Hurne, A. M., Fessenden, J. D., Lee, E. H., Allen, P. D., Beam, K. G., and Pessah, I. N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 15793-15798). This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the α 1s-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca2+ channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca 2+>Sr2+∼Ba2+, and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys4958 and Cys4961 within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 36994-37004 |
| Number of pages | 11 |
| Journal | Journal of Biological Chemistry |
| Volume | 280 |
| Issue number | 44 |
| DOIs | |
| State | Published - Nov 4 2005 |
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ASJC Scopus subject areas
- Biochemistry
Cite this
Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion. / Hurne, Alanna M.; O'Brien, Jennifer J.; Wingrove, Douglas; Cherednichenko, Gennady; Allen, Paul D.; Beam, Kurt G.; Pessah, Isaac N.
In: Journal of Biological Chemistry, Vol. 280, No. 44, 04.11.2005, p. 36994-37004.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion
AU - Hurne, Alanna M.
AU - O'Brien, Jennifer J.
AU - Wingrove, Douglas
AU - Cherednichenko, Gennady
AU - Allen, Paul D.
AU - Beam, Kurt G.
AU - Pessah, Isaac N
PY - 2005/11/4
Y1 - 2005/11/4
N2 - Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca2+ entry channel with SOCC-like properties, has recently been presented (Cherednichenko, G., Hurne, A. M., Fessenden, J. D., Lee, E. H., Allen, P. D., Beam, K. G., and Pessah, I. N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 15793-15798). This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the α 1s-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca2+ channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca 2+>Sr2+∼Ba2+, and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys4958 and Cys4961 within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.
AB - Bi-directional signaling between ryanodine receptor type 1 (RyR1) and dihydropyridine receptor (DHPR) in skeletal muscle serves as a prominent example of conformational coupling. Evidence for a physiological mechanism that upon depolarization of myotubes tightly couples three calcium channels, DHPR, RyR1, and a Ca2+ entry channel with SOCC-like properties, has recently been presented (Cherednichenko, G., Hurne, A. M., Fessenden, J. D., Lee, E. H., Allen, P. D., Beam, K. G., and Pessah, I. N. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 15793-15798). This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the α 1s-DHPR voltage sensor and is highly dependent on RyR1 conformation. In this report, we substitute RyR1 cysteines 4958 or 4961 within the TXCFICG motif, common to all ER/SR Ca2+ channels, with serine. When expressed in skeletal myotubes, C4958S- and C4961S-RyR1 properly target and restore L-type current via the DHPR. However, these mutants do not respond to RyR activators and do not support skeletal type EC coupling. Nonetheless, depolarization of cells expressing C4958S- or C4961S-RyR1 triggers calcium entry via ECCE that resembles that for wild-type RyR1, except for substantially slowed inactivation and deactivation kinetics. ECCE in these cells is completely independent of store depletion, displays a cation selectivity of Ca 2+>Sr2+∼Ba2+, and is fully inhibited by SKF-96365 or 2-APB. Mutation of other non-CXXC motif cysteines within the RyR1 transmembrane assembly (C3635S, C4876S, and C4882S) did not replicate the phenotype observed with C4958S- and C4961S-RyR1. This study demonstrates the essential role of Cys4958 and Cys4961 within an invariant CXXC motif for stabilizing conformations of RyR1 that influence both its function as a release channel and its interaction with ECCE channels.
UR - http://www.scopus.com/inward/record.url?scp=27744546285&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=27744546285&partnerID=8YFLogxK
U2 - 10.1074/jbc.M506441200
DO - 10.1074/jbc.M506441200
M3 - Article
C2 - 16120606
AN - SCOPUS:27744546285
VL - 280
SP - 36994
EP - 37004
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
SN - 0021-9258
IS - 44
ER -