INTRODUCTION C A 2+ -RELEASE/RYANODINE RECEPTOR CHANNELS (RyR channels) are massive integral membrane proteins (~2.3 MDa) present in endo/sarcoplasmic reticulum membranes as homotetramers (9). Ca 2+ release through these channels has a central role in Ca 2+ signaling. Through Ca 2+ -induced Ca 2+ re- lease (CICR), RyR channels can amplify Ca 2+ entry signals and can also efficiently propagate Ca 2+ signals to other cell locations or organelles, such as mitochondria or nuclei (35). These properties place RyR channels at the center of several important processes elicited by cellular Ca 2+ signals. Some of them occur in all cells, such as apoptosis and necrosis, whereas others are cell-specific and include events as diverse as contraction, secretion, fertilization, and neuronal plastic- ity. Consequently, RyR channel function is extensively regu- lated by small ions and molecules (e.g., Ca 2+ , Mg 2+ , or ade- nine nucleotides) and protein–protein interactions (e.g., calmodulin) (10, 25, 31). Additionally, RyR channels are also regulated by phosphorylation/dephosphorylation events (28) and by redox modifications, as detailed below. Mammalian RyR1 channels are likely to act as cellular redox sensors. They contain a few cysteine residues that react with pharmacological or endogenous redox-active molecules at physiological pH (6, 8, 32, 40, 46, 47). Moreover, RyR1 redox modifications have a profound effect on RyR1-medi- ated Ca 2+ release and single-channel activity (14, 17, 27). Ad- ditionally, the RyR1 protein is susceptible to S-nitrosylation 870 1 FONDAP Center for Molecular Studies of the Cell, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile. 2 Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, TX. Effects of S-Glutathionylation and S-Nitrosylation on Calmodulin Binding to Triads and FKBP12 Binding to Type 1 Calcium Release Channels PAULA ARACENA, 1,2 WEI TANG, 2 SUSAN L. HAMILTON, 2 and CECILIA HIDALGO 1 ABSTRACT This study shows that the combination of glutathione (GSH) plus hydrogen peroxide (H 2 O 2 ) promotes the S- glutathionylation of ryanodine receptor type 1 (RyR1) Ca 2+ release channels, and confirms their joint S-glu- tathionylation and S-nitrosylation by S-nitrosoglutathione (GSNO). In addition, we show that 35 S-labeled 12- kDa FK506-binding protein ([ 35 S]FKBP12) bound with a K d of 13.1 nM to RyR1 present in triads or heavy sarcoplasmic reticulum vesicles; RyR1 S-nitrosylation by NOR-3 or GSNO, but not S-glutathionylation, spe- cifically increased by four- to fivefold this K d value. RyR1 redox modifications also increased the K d of [ 35 S]calmodulin binding to triads without affecting B max . RyR1 S-glutathionylation (induced by GSH plus H 2 O 2 ) or RyR1 S-nitrosylation (produced by NOR-3) increased by approximately six- or twofold, respectively, the K d of apocalmodulin (apoCaM) or Ca 2+ -calmodulin (CaCaM) binding to triads. Likewise, the combined S- glutathionylation and S-nitrosylation of RyR1 induced by GSNO increased by fourfold the K d of CaCaM binding to triads and abolished apoCaM binding. As both FKBP12 and CaCaM inhibit RyR1, decreased FKBP12 binding to RyR1 and/or decreased CaCaM binding to either RyR1 or dihydropyridine receptor in triad preparations may cause the reported enhanced activation of Ca 2+ -induced Ca 2+ release kinetics medi- ated by S-glutathionylation/S-nitrosylation. We discuss possible consequences of these redox modifications on RyR1-mediated Ca 2+ release in physiological or pathological conditions. Antioxid. Redox Signal. 7, 870–881. Forum Original Research Communication ANTIOXIDANTS & REDOX SIGNALING Volume 7, Numbers 7 & 8, 2005 © Mary Ann Liebert, Inc.