Redox- and Calmodulin-dependent S-Nitrosylation of the KCNQ1 Channel

Abstract

S-Nitrosylation is a nitric oxide (NO) 2 -induced post-translational modification in which a cysteinyl thiol (R-SH) is converted to a nitrosothiol (1-3) and acts as a regulatory mechanism of various classes of proteins, including ion channels, such as the skeletal muscle type ryanodine receptor (ryanodine receptor type 1) channel (4, 5), the N-methyl-D-aspartate receptor channel (6, 7), the cardiac L-type Ca 2ϩ channel (8), and the cardiac Na ϩ channel (9). We have previously reported that NO derived from endothelial NO synthase activates ion currents through the cardiac slowly activating delayed rectifier potassium channel (I Ks ) composed of the pore-forming ␣-subunit KCNQ1 and the auxiliary ␤-subunit KCNE1. The NO-dependent regulation of the I Ks channel plays a pivotal role in regulation of cardiac membrane potential by intracellular Ca 2ϩ (10) and by sex hormones (11-13). Because the NO-dependent I Ks activation was inhibited by an inhibitor of soluble guanylate cyclase, 1H-(1,2,4)oxadiazolo(4,3-a)quinoxlin-1-1 (ODQ), with only a limited magnitude but was robustly inhibited by a thiol-alkylating reagent, N-ethylmaleimide, and reversed by a reducing reagent, dithiothreitol, soluble guanylate cyclase-independent, the protein S-nitrosylation mechanism is posited to be mainly involved (14). However, the following issues remain to be addressed: (i) Is the I Ks channel S-nitrosylated? (ii) If so, then what is the target of S-nitrosylation between the ␣-subunit KCNQ1 and the ␤-subunit KCNE1? (iii) Among multiple Cys residues, which Cys is a target of S-nitrosylation? and (iv) How does NO specifically recognize the target Cys? In the present study, we used the biotin-switch assay and functional patch clamp experiment to answer these questions. Our data show that KCNQ1 is a target of S-nitrosylation, and the presence of a redox motif contributes to making the Cys at 445 in the C terminus of KCNQ1 a preferential target of S-nitrosylation.

Transnitrosylating Nitric Oxide Species Directly Activate Type I Protein Kinase A, Providing a Novel Adenylate Cyclase-independent Cross-talk to β-Adrenergic-like Signaling

“…Precisely how OONO Ϫ interferes with PKA-mediated phosphorylations at a molecular level is unclear, but a variety of thiol-and tyrosine-targeted modifications may be responsible. With regard to protein S-nitrosylation, several muscle proteins that are integral to regulated contractile function are modulated in this way, including the ryanodine receptor (32), potassium channels (33), the L-type calcium channel, and SERCA (34).…”

SNO-ing at the Nociceptive Synapse?

“…In particular, S-nitrosylation affects the functions of glutamate receptors (Lipton et al, 1993;Kim et al, 1999), receptor trafficking molecules Selvakumar et al, 2009), transcription factors Reynaert et al, 2004;Li et al, 2007;Tsang et al, 2009), proinflammatory enzymes (Gu et al, 2002), heat-shock proteins (Martínez-Ruiz et al, 2005), proteases (Mannick et al, 1999(Mannick et al, , 2001, cytoskeletal (Lu et al, 2009) and synaptosomal (Palmer et al, 2008) proteins, and ion channels (Evans and Bielefeldt, 2000;Yoshida et al, 2006;Jian et al, 2007;Asada et al, 2009) (Figs. 5 and 6).…”

S-Nitrosothiols and theS-Nitrosoproteome of the Cardiovascular System

“…prolongation of the QT interval in the inherited long QT syndrome (LQTS) (i.e., LQTS due to a missense mutation of the SNTA1 gene [A390V-SNTA1]) (138), whereas S-nitrosylation of the I Ks channel at Cys445 shortens the cardiac action potential to modulate chronotropic responses to autonomic stimulation (1,3,53). In nNOS deficient rats, decreased bioavailable NO is associated with impaired S-nitrosylation of the voltage-dependent L-type Ca + 2 channel that destabilizes the cardiomyocyte membrane potential and decreases the threshold for ventricular arrhythmias (13).…”