CLIC2-RyR1 interaction and structural characterization by cryo-electron microscopy.
Chloride intracellular channel 2 (CLIC2), a newly discovered small protein distantly related to the glutathione transferase (GST) structural family, is highly expressed in cardiac and skeletal muscle, although its physiological function in these tissues has not been established. In the present study, [3H]ryanodine binding, Ca2+ efflux from skeletal sarcoplasmic reticulum (SR) vesicles, single channel recording, and cryo-electron microscopy were employed to investigate whether CLIC2 can interact with skeletal ryanodine receptor (RyR1) and modulate its channel activity. We found that: (1) CLIC2 facilitated [3H]ryanodine binding to skeletal SR and purified RyR1, by increasing the binding affinity of ryanodine for its receptor without significantly changing the apparent maximal binding capacity; (2) CLIC2 reduced the maximal Ca2+ efflux rate from skeletal SR vesicles; (3) CLIC2 decreased the open probability of RyR1 channel, through increasing the mean closed time of the channel; (4) CLIC2 bound to a region between domains 5 and 6 in the clamp-shaped region of RyR1; (5) and in the same clamp region, domains 9 and 10 became separated after CLIC2 binding, indicating CLIC2 induced a conformational change of RyR1. These data suggest that CLIC2 can interact with RyR1 and modulate its channel activity. We propose that CLIC2 functions as an intrinsic stabilizer of the closed state of RyR channels.
Meng
Xing
X
Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.
Wang
Guoliang
G
Viero
Cedric
C
Wang
Qiongling
Q
Mi
Wei
W
Su
Xiao-Dong
XD
Wagenknecht
Terence
T
Williams
Alan J
AJ
Liu
Zheng
Z
Yin
Chang-Cheng
CC
D000818:Animals; D002118:Calcium / Q000378:metabolism; D018118:Chloride Channels / Q000378:metabolism* / Q000648:ultrastructure*; D020285:Cryoelectron Microscopy; D018360:Crystallography, X-Ray; D015640:Ion Channel Gating; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism / Q000648:ultrastructure; D013499:Surface Properties; D014316:Tritium
D018118:Chloride Channels; D019837:Ryanodine Receptor Calcium Release Channel; D014316:Tritium; D002118:Calcium