Ryanodine receptor type 1 (RyR1) mutations C4958S and C4961S reveal excitation-coupled calcium entry (ECCE) is independent of sarcoplasmic reticulum store depletion.
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 Ca(2+) entry channel with SOCC-like properties, has recently been presented. This form of conformational coupling, termed excitation-coupled calcium entry (ECCE) is triggered by the alpha(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 Ca(2+) 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+)>Sr(2+) approximately Ba(2+), 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 Cys(4958) and Cys(4961) 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.
Hurne
Alanna M
AM
Department of Molecular Biosciences, School of Veterinary Medicine and Center for Children's Environmental Health and Disease Prevention, University of California, Davis, California 95616, USA.
O'Brien
Jennifer J
JJ
Wingrove
Douglas
D
Cherednichenko
Gennady
G
Allen
Paul D
PD
Beam
Kurt G
KG
Pessah
Isaac N
IN
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002412:Cations / Q000378:metabolism; D003545:Cysteine / Q000235:genetics / Q000378:metabolism; D004594:Electrophysiology; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology; D018482:Muscle, Skeletal / Q000166:cytology; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics*; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*
D002412:Cations; D019837:Ryanodine Receptor Calcium Release Channel; D003545:Cysteine; D002118:Calcium