A Role for Cysteine 3635 of RYR1 in Redox Modulation and Calmodulin Binding

Abstract

In the current work, we demonstrate that both cysteines needed for the oxidation-induced intersubunit crosslink are protected from alkylation with N-ethylmaleimide by bound calmodulin. We also show, using N-terminal amino acid sequencing together with analysis of the distribution of [ 3 H]NEM labeling with each sequencing cycle, that cysteine 3635 of RYR1 is rapidly labeled by NEM and that this labeling is blocked by bound calmodulin. We propose that cysteine 3635 is located at an intersubunit contact site that is close to or within a calmodulin binding site. These findings suggest that calmodulin and oxidation modulate RYR1 activity by regulating intersubunit interactions in a mutually exclusive manner and that these interactions involve cysteine 3635.Reactive oxygen species and nitric oxide (NO) 1 are produced by skeletal muscle even at rest, but their levels are dramatically increased by muscle activity (1-5). Both reactive oxygen species and NO alter muscle function (6), possibly by altering excitation-contraction coupling (7). One of the proteins involved in excitation-contraction coupling, the skeletal muscle Ca 2ϩ -release channel, is modulated by both oxidants (8 -10) and NO (6,11). This regulation appears to be controlled, at least in part, by the binding of calmodulin (CaM) to RYR1 (12, 13). Ca 2ϩ -free CaM is a partial agonist of RYR1, whereas Ca 2ϩ -CaM is an inhibitor (14). In addition to these direct functional effects, CaM bound to RYR1 protects the channel from oxidation-induced intersubunit cross-linking (12), and conversely, oxidation can block CaM binding to RYR1. In contrast to the effects of oxidation, alkylation of RYR1 with NEM rapidly destroys the ability of RYR1 to bind 35 S-Ca 2ϩ -free CaM (apoCaM) but does not alter its ability to bind 35 S-Ca 2ϩ -CaM. Alkylation also prevents oxidation-induced intersubunit crosslinking. Our studies suggest that there is only one CaM site per subunit of RYR1 at either high or low Ca 2ϩ (13). Some indication of the location of the CaM binding site has been obtained from examination of sites on RYR1 protected from tryptic cleavage by CaM (13). Treatment of RYR1 with trypsin rapidly destroys its ability to bind CaM, but CaM bound to RYR1 can protect its binding site from tryptic digestion. The sites protected by Ca 2ϩ -CaM or apoCaM are at amino acids 3630 and 3637, suggesting that either the binding sites for CaM in both the Ca 2ϩ -free and Ca 2ϩ -bound forms are physically close to this region of RYR1 or that the binding of both forms of CaM produces a conformational change that buries this region of RYR1. The latter possibility seems unlikely because the functional effects of apoCaM are opposite those of Ca 2ϩ -CaM. The sequence between these two sites is AVVACFR, suggesting that cysteine 3635 may be one of the cysteines that, in the absence of CaM, can form the intersubunit disulfide bond. This intersubunit contact site is likely to represent an important site for regulating RYR1 activity. In the current study, we demonstrate that cysteine 3635 is one...

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…A number of redox-sensitive cysteines have been identified in both the open and closed state of the channel and appear to be distributed across the primary structure of cytoplasmic region (Voss et al 2004;Aracena et al 2006). Several of these sites have been mapped to the clamp domains like Cys36 and Cys315 (Liu et al 2005;Amador 2009;Hamilton and Serysheva 2009;Lobo and Van Petegem 2009), whereas the Cys3635 is located in the subdomain 3 in the CaM binding site (Moore et al 1999b;Sun et al 2001). S-nitrosylation of Cys3635 has been shown to reverse the CaM inhibition on RyR1 and to activate the channel (Moore et al 1999b).…”

“…ApoCaM is a partial agonist whereas CaCaM is an inhibitor of RyR1 and SR Ca 2þ release (Rodney et al 2000). CaM binding site involves amino acids 3614 -3643 of the RyR1 rabbit sequence (Takeshima et al 1989;Moore et al 1999b;Yamaguchi et al 2003;Zhang et al 2003). Cryo-EM difference mapping of the three-dimensional structures of RyR1 with and without added CaM has suggested that the CaCaM binding site is located in subdomain 3.…”

“…Several of these sites have been mapped to the clamp domains like Cys36 and Cys315 (Liu et al 2005;Amador 2009;Hamilton and Serysheva 2009;Lobo and Van Petegem 2009), whereas the Cys3635 is located in the subdomain 3 in the CaM binding site (Moore et al 1999b;Sun et al 2001). S-nitrosylation of Cys3635 has been shown to reverse the CaM inhibition on RyR1 and to activate the channel (Moore et al 1999b). The S-nitrosylation of Cys3635 appears to occur only at physiological tissue O 2 tension ( pO 2 ; 10 mm Hg) and facilitates muscle contraction (Eu et al 2003).…”

“…RyRs are modulated (see Fig. 1) directly or indirectly by the dihydropyridine receptor (DHPR; also known as L-type Ca 2þ channel, Ca V 1.1/1.2) and by various ions, small molecules and proteins, e.g., Ca 2þ , Mg 2þ , protein kinase A (PKA), FK506 binding proteins (FKBP12 and 12.6), calmodulin (CaM), Ca 2þ /calmodulin-dependent protein kinase II (CaMKII), calsequestrin (CSQ), triadin, junctin Tanabe et al 1990;Ikemoto et al 1991;Sabbadini et al 1992;Wang and Best 1992;Brillantes et al 1994;Chen and MacLennan 1994;Yang et al 1994;Ma et al 1995;Mayrleitner et al 1995;Tripathy et al 1995;Timerman et al 1996;Nakai et al 1998;Moore et al 1999b;Rodney et al 2000). RyR1 and RyR2 are crucial for E-C coupling in both skeletal and cardiac muscle, respectively.…”

How widespread is stable protein S-nitrosylation as an end-effector of protein regulation?

“…However, prior to this, experiments in sarcoplasmic reticulum membranes from rabbit showed Cys-3635 formed a disulfide bond with a cysteine in a neighbouring subunit upon calmodulin binding. The formation of this inter-subunit disulfide bond resulted in structural changes to the tetrameric from of RyR1 and was thought to protect RyR1 from further oxidation [123].…”