Article

Redox-Sensitive Sulfenic Acid Modification Regulates Surface Expression of the Cardiovascular Voltage-Gated Potassium Channel Kv1.5

Department of Pharmacology, University of Michigan Medical School, 1301 MSRB III, 1150 W Medical Center Dr, Ann Arbor, MI 48109-5632. .
Circulation Research (Impact Factor: 11.09). 07/2012; 111(7):842-53. DOI: 10.1161/CIRCRESAHA.111.263525
Source: PubMed

ABSTRACT Rationale: Kv1.5 (KCNA5) is expressed in the heart, where it underlies the I(Kur) current that controls atrial repolarization, and in the pulmonary vasculature, where it regulates vessel contractility in response to changes in oxygen tension. Atrial fibrillation and hypoxic pulmonary hypertension are characterized by downregulation of Kv1.5 protein expression, as well as with oxidative stress. Formation of sulfenic acid on cysteine residues of proteins is an important, dynamic mechanism for protein regulation under oxidative stress. Kv1.5 is widely reported to be redox-sensitive, and the channel possesses 6 potentially redox-sensitive intracellular cysteines. We therefore hypothesized that sulfenic acid modification of the channel itself may regulate Kv1.5 in response to oxidative stress. Objective: To investigate how oxidative stress, via redox-sensitive modification of the channel with sulfenic acid, regulates trafficking and expression of Kv1.5. Methods and Results: Labeling studies with the sulfenic acid-specific probe DAz and horseradish peroxidase-streptavidin Western blotting demonstrated a global increase in sulfenic acid-modified proteins in human patients with atrial fibrillation, as well as sulfenic acid modification to Kv1.5 in the heart. Further studies showed that Kv1.5 is modified with sulfenic acid on a single COOH-terminal cysteine (C581), and the level of sulfenic acid increases in response to oxidant exposure. Using live-cell immunofluorescence and whole-cell voltage-clamping, we found that modification of this cysteine is necessary and sufficient to reduce channel surface expression, promote its internalization, and block channel recycling back to the cell surface. Moreover, Western blotting demonstrated that sulfenic acid modification is a trigger for channel degradation under prolonged oxidative stress. Conclusions: Sulfenic acid modification to proteins, which is elevated in diseased human heart, regulates Kv1.5 channel surface expression and stability under oxidative stress and diverts channel from a recycling pathway to degradation. This provides a molecular mechanism linking oxidative stress and downregulation of channel expression observed in cardiovascular diseases.

0 Followers
 · 
178 Views
 · 
0 Downloads
  • [Show abstract] [Hide abstract]
    ABSTRACT: In mammalian skeletal muscle, Ca(2+) release from the sarcoplasmic reticulum (SR) through the ryanodine receptor/Ca(2+)-release channel RyR1 can be enhanced by S-oxidation or S-nitrosylation of separate Cys residues, which are allosterically linked. S-oxidation of RyR1 is coupled to muscle oxygen tension (pO2) through O2-dependent production of hydrogen peroxide by SR-resident NADPH oxidase 4 (Nox4). In isolated SR (SR vesicles), an average of 6-8 thiols/RyR1 monomer are reversibly oxidized at high (21% O2) versus low pO2 (1% O2), but their identity among the 100 Cys/RyR1 monomer is unknown. Here we employ isotope-coded affinity tag (ICAT) labeling and mass spectrometry (yielding 93% coverage of RyR1 Cys) to identify 13 Cys subject to pO2-coupled S-oxidation in SR vesicles. Eight additional Cys are oxidized at high versus low pO2 only when NADPH levels are supplemented to enhance Nox4 activity. PO2-sensitive Cys were largely non-overlapping with those identified previously as hyperreactive by administration of exogenous reagents (3 of 21) or as S-nitrosylated. Cys subject to pO2-coupled oxidation are distributed widely within the cytoplasmic domain of RyR1, in multiple functional domains implicated in RyR1 activity-regulating interactions with the L-type Ca(2+) channel (dihydropyridine receptor) and and FK506-binding protein (FKBP12), as well as in ″hot spot″ regions containing sites of mutation implicated in malignant hyperthermia and central core disease. PO2-coupled disulfide formation was identified whereas neither S-glutathionylated nor sulfenamide-modified Cys were observed. Thus, physiological redox regulation of RyR1 by endogenously generated hydrogen peroxide is exerted through dynamic disulfide formation involving multiple Cys residues.
    Journal of Biological Chemistry 06/2013; 288(32). DOI:10.1074/jbc.M113.480228 · 4.57 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Controlled generation of reactive oxygen species (ROS) orchestrates numerous physiological signaling events (1). A major cellular target of ROS is the thiol side-chain (RSH) of cysteine (Cys), which may assume a wide range of oxidation states (i.e., -2 to +4). Within this context, Cys sulfenic (Cys-SOH) and sulfinic (Cys-SO2H) acids have emerged as important mechanisms for regulation of protein function. Although this area has been under investigation for over a decade, the scope and the biological role of sulfenic / sulfinic acid modifications have been recently expanded with the introduction of new tools for the monitoring of cysteine oxidation in vitro and directly in cells. This review discusses selected recent examples of protein sulfenylation and sulfinylation from the literature, highlighting the role of these post-translational modifications (PTMs) in cell signaling.
    Journal of Biological Chemistry 07/2013; 288(37). DOI:10.1074/jbc.R113.467738 · 4.57 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Significance: Voltage-gated K+ channels are a large family of K+-selective ion channel protein complexes that open on membrane depolarization. These K+ channels are expressed in diverse tissues and their function is vital for numerous physiological processes, in particular of neurons and muscle cells. Potentially reversible oxidative regulation of voltage-gated K+ channels by reactive species such as ROS (reactive oxygen species) represents a contributing mechanism of normal cellular plasticity and may play important roles in diverse pathologies including neurodegenerative diseases. Recent Advances: Studies using various protocols of oxidative modification, site-directed mutagenesis, and structural and kinetic modeling are providing a broader phenomenology and emerging mechanistic insights. Critical Issues: Physicochemical mechanisms of the functional consequences of oxidative modifications of voltage-gated K+ channels are only beginning to be revealed. In vivo documentation of oxidative modifications of specific amino-acid residues of various voltage-gated K+ channel proteins, including the target specificity issue, is largely absent. Future Directions: High-resolution chemical and proteomic analysis of ion channel proteins with respect to oxidative modification combined with ongoing studies on channel structure and function will provide a better understanding of how the function of voltage-gated K+ channels is tuned by ROS and the corresponding reducing enzymes to meet cellular needs.
    Antioxidants & Redox Signaling 09/2013; DOI:10.1089/ars.2013.5614 · 7.67 Impact Factor
Show more