Transmembrane Redox Sensor of Ryanodine Receptor Complex

Abstract

A change in cytosolic Ca 2ϩ concentration serves as a signal for modulating a wide range of cellular activities (1-3). A major mechanism for increasing cytosolic Ca 2ϩ includes release of Ca 2ϩ from internal stores (endoplasmic or sarcoplasmic reticulum, ER or SR) 1 via a genetic superfamily of Ca 2ϩ release channels including inositol 1,4,5-trisphosphate receptors (IP 3 R) and ryanodine receptors (RyR) (4 -6). A prominent functional property of all of these channels is exquisite sensitivity to reduction and oxidation by sulfhydryl reagents (7-12). The functional consequences of sulfhydryl modification of RyRs include phases of activation and inhibition, revealing that multiple classes of sulfhydryl groups residing on Cys residues of all three isoforms of RyR channel complexes are important for native functioning and subject to chemical modification (11,12). However, defining a role for sulfhydryl redox chemistry in RyR function has been controversial since the initial suggestion that sulfhydryl oxidation is a key step in channel activation (13). A plausible physiological role for redox control of ER/SR Ca 2ϩ release channels and its attendant mechanism has remained elusive.It is known that glutathione (GSH) and glutathione disulfide (GSSG) constitute the major redox buffer system of skeletal muscle and many non-muscle cells (14, 15). In the typical mammalian cell, the ratio of [GSH]/[GSSG] in the cytosol is Ն30:1, thereby maintaining very reduced redox potential (RP) of approximately Ϫ220 mV (16). By contrast, the RP of the ER lumen is significantly more oxidized (approximately Ϫ180 mV) and is maintained with a 3:1 to 1:1 ratio of [GSH]/[GSSG] (16, 17). Thus, the typical microsomal membrane within which the RyR and IP 3 R reside is normally subject to a large transmembrane RP difference of 40 -50 mV with the lumen much more oxidized than the cytosol (16,17).To study redox regulation of RyR channel activity, the bilayer lipid membrane (BLM) preparation affords precise control of the redox state on both the cytoplasmic (cis) and luminal (trans) faces of the reconstituted channel by adjustment of the [GSH]/[GSSG] ratio to form varied redox potentials. In the present work, we provide direct evidence that RyR1 channel activity follows transmembrane redox potential. Chemical labeling studies with CPM indicate previously identified hyperreactive sulfhydryl moieties within the RyR1 complex (18,19) constitute an essential component of a unique transmembrane redox sensor. EXPERIMENTAL PROCEDURESPreparation of SR Membranes--Sarcoplasmic reticulum membrane vesicles were prepared from back and hind limb skeletal muscles of New Zealand White rabbits according to the method of Saito et al. (20) with some modifications. During the SR preparation, GSH and GSSG were included in the homogenization buffer, and the glutathione RP was made to Ϫ220 mV, which mimics the typical cytoplasmic RP in vivo. The preparations were stored in 10% sucrose, 10 mM Hepes, pH 7.4, at Ϫ80°C until needed.GSH and GSSG Stock Solutions-GSH was dissolve...

Protein Folding and Quality Control in the ER

“…3B) (Gorlach et al 2006). The ryanodine receptor is a redox sensitive Ca 2þ channel in the membrane of the sarcoplasmic reticulum (Zable et al 1997;Feng et al 2000). ERp44, which senses the redox state as well as luminal pH and Ca 2þ concentration, interacts with the luminal loop of IP3R type 1 (IP3R1) and directly inhibits its activity (Higo et al 2005).…”

A Transverse Tubule NADPH Oxidase Activity Stimulates Calcium Release from Isolated Triads via Ryanodine Receptor Type 1 S -Glutathionylation

“…Skeletal RyR1 channels have a few cysteine residues highly responsive to oxidative modification at physiological pH (65), a property that makes them likely candi- dates to act as intracellular redox sensors (66,67). In vitro experiments indicate that oxidation/alkylation, S-nitrosylation (38,68), or S-glutathionylation (38,62) of RyR1 hyperreactive SH residues enhances channel activity, whereas reduction to their free sulfhydryl state has the opposite effect (67)(68)(69)(70)(71)(72). In particular, incubation of isolated SR vesicles with superoxide anion or H 2 O 2 promotes RyR activation (42,(73)(74)(75).…”

Identification of Cysteines Involved in S-Nitrosylation, S-Glutathionylation, and Oxidation to Disulfides in Ryanodine Receptor Type 1

“…Hyper-reactive sulfhydryl groups associated with RyR1 have a well defined redox potential that is sensitive to the cellular environment (14,53). Feng et al (14) suggested that channel activators decrease the redox potential leading to modification of FIGURE 9.…”