Articles in PresS. Am J Physiol Gastrointest Liver Physiol (December 31, 2014). doi:10.1152/ajpgi.00281.2014 Kang et al. 1 2

Interaction between hydrogen sulfide-induced sulfhydration and tyrosine nitration in the KATP channel

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complex

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Minho Kang, Atsushi Hashimoto, Aravind Gade and Hamid I. Akbarali*

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Department of Pharmacology and Toxicology, Medical College of Virginia Campus, Virginia

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Commonwealth University, Richmond, VA USA 23298

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*Running title: Protective effect of H2S in colonic inflammation.

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*

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Hamid I. Akbarali, Department of Pharmacology and Toxicology, 1112 E. Clay St., McGuire Hall 317

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Virginia Commonwealth University, Richmond VA, USA, Tel.: (804) 828-2064; Fax: (804)828-1532;

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Email: [email protected]

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Word Count: 3624

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References : 31

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Grayscale figures: 6

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Color figures: 1 (online)

To whom correspondence should be addressed:

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Copyright © 2014 by the American Physiological Society.

Kang et al. 21 22

ABSTRACT

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Hydrogen sulfide (H2S), is an endogenous gaseous mediator affecting many physiological and

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pathophysiological conditions. Enhanced expression of H2S and reactive nitrogen/oxygen (RNS/ROS)

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species during inflammation alter cellular excitability via modulation of ion channel function.

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Sulfhydration of cysteine residues and tyrosine nitration are the post-translational modifications induced

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by H2S and RNS, respectively. The objective of this study was to define the interaction between tyrosine

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nitration and cysteine sulfhydration within the KATP channel complex, a significant target in experimental

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colitis. A modified biotin switch assay was performed to determine sulfhydration of the K ATP channel

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subunits, Kir6.1 and SUR2B and nitrotyrosine measured by immunoblot.

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significantly enhanced sulfhydration of SUR2B but not Kir6.1 subunit. SIN-1 (a donor of peroxinitrite)

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induced nitration of Kir6.1 subunit but not SUR2B.

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Kir6.1 by SIN-1 in CHO cells co-transfected with the two subunits as well as in enteric glia. Two

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specific mutations within SUR2B, C24S and C1455S, prevented sulfhydration by NaHS and these

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mutations prevented NaHS- induced reduction in tyrosine nitration of Kir6.1. NaHS also reversed

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peroxynitrite-induced inhibition of smooth muscle contraction. These studies suggest that post-

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translational modifications of the two subunits of the ATP-sensitive K+ channel interact to alter channel

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function. The studies described herein demonstrate a unique mechanism by which sulfhydration of one

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subunit modifies tyrosine nitration of another subunit within the same channel complex. This interaction

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provides a mechanistic insight on the protective effects of H2S in inflammation.

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NaHS (a donor of H2S)

Pretreatment with NaHS reduced the nitration of

Kang et al. 45 46

INTRODUCTION

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Protein function can be fundamentally altered during inflammation due to multiple oxidative reactions.

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The sulfhydryl groups on cysteine residues can undergo several modifications including S-nitrosylation,

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S-palmitoylation, S-glutathionylation, and/or S-sulfhydration (28).

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cysteine residues, nitration of tyrosine by peroxynitrite (ONOO-) can also alter functional properties of

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proteins (19) (13, 14). During colonic inflammation, S-sulfhydration as well as tyrosine nitration occur

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within smooth muscle cells. Our studies in colonic smooth muscle demonstrate that S-sulfhydration of

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the KATP channel during colonic inflammation enhances KATP currents while tyrosine nitration of the L-

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type calcium channel results in reduced calcium currents (6, 11, 12, 20).

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Hydrogen sulfide (H2S) is an endogenous gas involved in several biological functions, including

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neuromodulation, smooth muscle relaxation, inflammation, insulin release and metabolic demand. H2S is

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predominantly produced in mammalian tissues from L-cysteine by the enzymes, cystathionine β-

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synthetase (CBS) and cystathionine γ-lyase (CSE) (16, 25). H2S converts cysteine residues to

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thiocysteines (ie modifies the –SH group to –SSH; S-sulfhydration) in many proteins, including actin,

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tubulin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the KATP channel (17).

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ATP-sensitive K+ (KATP) channels play an important role in cellular function observed in a variety of

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tissues, including smooth muscles, pancreatic β-cells, myocardium, and neurons (18, 29). KATP channel in

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the smooth muscle colon is a hetero-octamer consisting of two main subunits, a pore forming subunit

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(Kir6.1) and a sulfonylurea receptor subunit (SUR2B) (8). H2S S-sulfhydrates the sulfonylurea subunit,

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SUR2B, of the KATP channel in colonic smooth muscle. The S-sulfhydation of the SUR2B by H2S,

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provides an allosteric modulation of the channel resulting in enhanced potency for the K ATP channel

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opener, levcromakalim (6).

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Since the discovery of endogenous H2S, many studies have explored the role of this gas in animal models.

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In an experimental model of colonic inflammation, enhanced H2S synthesis is thought to result in faster

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In addition to modifications of

Kang et al. 70

resolution of the inflammation (22, 23). Furthermore, inhibition of KATP channel in colitis rats also

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induces significant mortality (24), indicating that H2S and KATP channels are important components in

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ulcerative colitis. The mechanism by which KATP channel is affected may be through sulfhydration of

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specific cysteine residues (17) . However, the molecular mechanisms involved in such modulation are not

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fully elucidated. Previously we showed in mouse models of colonic inflammation that oxidative stress is

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also associated with increased tyrosine nitration in smooth muscle (1). The increased expression of

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tyrosine nitrated calcium channels in inflamed colon prevents the binding and regulation of the calcium

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channel by c-src kinase (21) (11).

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In the present study, we explore the interaction of S-sulfhydration and tyrosine nitration within the KATP

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channel complex. Here we demonstrate that sulfhydration of SUR2B subunit prevents nitration of the

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pore forming Kir6.1 subunit of the channel. This type of interaction provides mechanistic insight into the

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protective effects of H2S in colonic inflammation.

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Kang et al. 96 97 98 99 100

MATERIALS AND METHODS All animal protocols conformed to the guidelines and approval by the Virginia Commonwealth University Institutional Animal Care and Use Committee.

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Cell culture and transient transfection- Chinese hamster ovary (CHO) cells were purchased from the

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American Type Cell Collection (ATCC). CHO cells were cultured in Dulbecco’s modified Eagle’s

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medium/F12 medium supplemented with 0.2 mg/ml Geneticin and 10% fetal bovine serum (Invitrogen).

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Cells were grown in 6-well plates overnight and transfected with Kir6.1, SUR2B, EGFP or combination

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of plasmid DNAs or with Cav1.2b and β2 subunit by Genejammer (Stratagene) as described by the

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manufacturer. Untransfected CHO cells were maintained in the same media. After 24~48hrs incubation

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protein samples were isolated and were quantified using a protein assay kit (Pierce). Cells were

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maintained under standard cell culture conditions at 37°C and 5% CO2 in a humid environment.

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Immunoblots- For measuring the extent of tyrosine nitration on cells, samples were treated with 100

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M SIN-1 Chloride (Cayman Chemical) for 20 min or 150 M sodium peroxynitrite (ONOO-, Cayman

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Chemicals) twice at 5-min intervals. Protein samples were prepared as previously described (9) and were

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examined for nitration using anti-Nitrotyrosine (NY) (EMD Chemicals Cat# 487923-50UG

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RRID:AB_212231) antibody after immunoprecipitation with anti-Kir6.1 (Santa Cruz Biotechnology Cat#

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sc-11225 RRID:AB_2130774) or anti-SUR2B antibody (Santa Cruz Biotechnology Cat# sc-5793

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RRID:AB_2219773). Standard protocols for Western blots were performed using site-specific antibodies.

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Protein samples were run on 5% and 10% SDS-PAGE and transferred on nitrocellulose membranes (Bio-

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Rad). Membranes were blocked with 3% nonfat milk blocking buffer with 0.1% Tween 20. Specific

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antibodies were used for blotting at concentration of 1:200 to 1:1000. The membranes were then

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incubated with secondary antibodies at concentration of 1:1,000 to 1:5,000 and visualized using the

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Odyssey Infrared Imaging System (LI-COR Biosciences).

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Kang et al. 121

In-cell western (ICW) blot assay- Concentration-response measurements were performed by ICW

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blot assay. Enteric Glial Cell line (EGCs) were obtained from the American Type Tissue Collection

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(ATCC: CRL 2690) and cultured and maintained in Dulbecco’s modified Eagle’s medium/F12 medium

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supplemented with 0.2 mg/ml Geneticin and 10% fetal bovine serum (Invitrogen). EGC’s are derived

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from rat jejenum myenteric plexus. EGCs were stimulated by adding peroxynitrite (ONOO-) at the

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indicated doses for 15 min at 37°C. For effect of NaHS on the tyrosine nitration, the cells were pretreated

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with 1 mM NaHS for 15 min at 37°C. ONOO- was removed and cells were fixed (4% formaldehyde in

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1× PBS, 20 min at 25°C), permeabilized (0.1% Triton X-100 in 1× PBS, four washes for 5 min each at

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25°C with rotation) and blocked (0.5× Odyssey Blocking Buffer (OBB) (Licor Biosciences, Lincoln, NE),

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1 h at 25°C with rotation). Primary antibodies, anti-Nitrotyrosine (NY) or anti-GFAP (Santa Cruz

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Biotechnology, Inc. Cat# sc-9065 RRID:AB_641022), were diluted 1:100 in 0.5× OBB (50 μl per well)

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and allowed to incubate overnight at 4°C. Cells were then washed 3× with 1× PBS-Tween (0.1%) for 5

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min at 25°C with rotation, followed by the addition of secondary antibody (diluted 1:500 in 0.5× OBB)

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for 1 h at 25°C. Following three additional wash cycles, wells were aspirated dry and scanned using the

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Licor Odyssey Scanner (Licor Biosciences). Data were normalized and presented as mean ± SEM of

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IR700 responses with reference to controls (100% standard).

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Modified biotin switch assay- The biotin switch assay was carried out as described (6). In brief, cells

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treated with or without 1 mM NaHS (Cayman chemicals) were homogenized in HEN buffer [250 mM

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Hepes-NaOH (pH 7.7), 1 mM EDTA, 2.5% SDS, and 0.1 mM neocuproine] supplemented with 100uM

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deferoxamine. Protein samples (250 ug) were added to blocking buffer (HEN buffer adjusted to 2.5%

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SDS and 20 mM MMTS) at 50°C for 20 min with frequent vortexing. After acetone precipitation, the

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proteins were resuspended in HENS buffer (HEN buffer adjusted to 1% SDS). Biotin-HPDP (4  in

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dimethyl sulfoxide was added to the suspension. After 3 hours incubation, biotinylated proteins were

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precipitated by streptavidin-agarose beads, which were then washed with HENS buffer. The biotinylated

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proteins were eluted by SDS-PAGE sample buffer and subjected to Western blot analysis with specific

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Kang et al. 146

antibodies to each protein. For quantitation of protein sulfhydration, samples were run on blots alongside

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total lysates. Anti-SUR2B and anti-Kir6.1 were used at 1:1000 dilutions.

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Site-Directed Mutagenesis- The mutants were constructed using the QuikChange Site-Directed

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Mutagenesis System (Stratagene) according to the manufacturer’s procedures. Murine Kir6.1-cDNA

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(Open Biosysems), inserted into pcDNA3 (Invitrogen) was used as the template. Primers were designed

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by Vector NTI program (Invitrogen), containing the desired mutation in the middle region (Table 1).

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Sequences that are underlined indicate site mutated and change in the amino acid [cysteine (C) to Serine

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(S)]. The presence of the desired mutation was confirmed by DNA sequencing of the relevant DNA

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region.

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Immunohistochemistry - Immunohistochemical staining was performed by standard protocols. CHO

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cells were transfected with SUR2B or C1455S SUR2B mutant, with Kir6.1, and treated with 100 uM

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SIN-1 Chloride for 20 min, 1 mM NaHS for 15 min, or combination of both. After washing in PBS, cells

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were fixed in 4% formaldehyde in PBS for 15 min at room temperature and then washed in PBS three

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times for 5 min each. After formaldehyde fixation, cells were incubated for 10 min in PBS/0.3% Triton

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X-100 (PBST) following by three time washing in PBS. Cells were then blocked for 60 min with 10%

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non-immuno goat serum. Subsequently, samples were incubated with anti-Nitrotyrosine antibody (1:200)

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in 1% non-immuno goat serum for overnight at 4ºC. Cells were washed in PBS three times, and then were

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treated with the Alexa Flour 568 conjugated goat polyclonal secondary antibody at 1:1000 dilutions in

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PBST for 1 hour at room temperature in the dark. Images were collected using an Olympus FV300

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confocal microscope (Olympus America Inc, Center Valley, PA, USA) at the appropriate excitation

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wavelength.

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Patch-clamp recording of ATP-sensitive K+ current- Whole-cell K+ currents were recorded in

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transfected cells using standard methods as previously described (6-8) . After obtaining seal, cells were

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voltage clamped and K+ currents were generated by progressive 10 mV depolarizing steps (500 ms

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duration, 5 s intervals) from a holding potential of -60 mV. KATP currents were measured in a high K+

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bathing solution (140 mM) and dialyzed with low ATP (0.1 mM) in the internal solution. This protocol 7

Kang et al. 172

allows for inward currents due to ATP-sensitive K+ channel activation by levcromakalim (Tocris

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Bioscience).

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Isometric tension recording- All animal protocols confirmed to the guidelines and approval by the

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Virginia Commonwealth University Institutional Animal Care and Use Committee. Tissue preparation

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was performed as described previously (21). Approximately 1.5-cm strips of mouse ileum were

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suspended in the longitudinal direction in an organ bath containing calcium-free Krebs’ solution (118 mM

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NaCl, 4.6 mM KCl, 1.3 mM NaH2PO4, 1.2 mM MgSO4, 25 mM NaHCO3, and 11 mM glucose), bubbled

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continuously with carbogen (95% O2 and 5% CO2) at 37°C under a resting tension of 1 gram and

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equilibrated for a period of 1 h. Isometric contractions were recorded by a force transducer (model GR-

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FT03; Radnoti, Monrovia, CA) connected to a personal computer using Acqknowledge 382 software

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program (BIOPAC Systems, Santa Barbara, CA). After equilibration in Krebs’ solution, tissues were

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incubated for 45 min in calcium-free high-potassium solution (80 mM) in which equimolar NaCl was

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replaced by KCl and changed every 15 min. Tissues were pre-incubated for 20 min either in the presence

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or absence of NaHS (100 uM). Tissues were washed with Krebs solution after the 20 min pre-incubation,

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and cumulative concentration-dependent contraction responses to CaCl2 (1μM to 3 mM) were performed.

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The results were expressed as percentages of the maximal response for CaCl2 alone, and the curves were

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statistically compared.

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Statistical analysis- All experiments were performed and repeated at least three times. Results are

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presented as the mean ± S.E. for the number of cells (n). Sigmaplot 12.5 software was used for detection

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of differences in paired or unpaired t-tests as appropriate, and EC50 values were calculated using 4

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parameter logistic nonlinear regression model in Sigmaplot. A p value of less than 0.05 was considered

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significant.

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Kang et al. 194

RESULTS

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DTT reduces sulfhydration of SUR2B- Recently we reported that in cells co-transfected with Kir6.1

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and SUR2B and in freshly isolated mouse colonic smooth muscle cells, NaH2S induces sulfhydration of

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SUR2B as cysteine thiols (-SH) react with H2S to form hydropersulfides (-SSH) (6). Figure 1 shows the

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effect of the reducing agent, dithiothreitol (DTT), on KATP channel sulfhydration in CHO cells transfected

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with Kir6.1 and SUR2B. Cell lysates were isolated and treated with 1 mM NaHS, 1 mM DTT, or the

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combination and subjected to the modified biotin switch assay with antibody against SUR2B to detect

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KATP channel sulfhydration. There was basal sulfhydration of SUR2B (0.20±0.025), and this sulfhydration

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was significantly enhanced with NaHS treatment (0.35±0.07) (Fig.1A). There was no significant effect of

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DTT on the basal sulfhydration of SUR2B subunit. However, treatment with DTT significantly reversed

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H2S mediated sulfhydration (0.21±0.05). H2S did not induce sulfhydration of Kir6.1 subunit of the

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channels (6) (not shown). Thus, SUR2B sulfhydration is a covalent modification involving sulfhydryl

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groups. The presence of functional KATP channels upon co-transfection of Kir6.1 and SUR2B was

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confirmed by whole-cell voltage-clamp in CHO cells. Levcromakalim-induced currents were measured at

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a holding potential of -60 mV in the presence of high K+ solution with low internal ATP to isolate the

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KATP currents as described previously (6). Levcromakalim induced currents were significantly inhibited

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by the channel blocker, Glibenclamide (Fig.1B).

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Cysteine residues affect sulfhydration- In order to determine the specific cysteine residues within

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SUR2B that are sulfhydrated, a preliminary analysis of the possible cysteine residues was carried out

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using the program PSIPRED, and the mutants constructed using the QuikChange Site-Directed

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Mutagenesis System. The following cysteine residues were identified as most likely to be involved, based

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on the preliminary analysis: C6, C24, C263, C706, C818, C1410 and C1455. CHO cells were transfected

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with Kir6.1 and SUR2B or mutants of SUR2B and subjected to the modified biotin switch assay with

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antibody against SUR2B to detect KATP channel sulfhydration following NaHS. Figure 2 shows that

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mutation of cysteine 24 (C24S) and 1455 (C1455S) reduced sulfhydration: C24S (0.12±0.023) and

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Kang et al. 219

C1455S (0.12±0.035) compared to control (0.31±0.033) (Fig.2). The other mutants did not affect the

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extent of sulfhydration of the channel.

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Sulfhydration prevents tyrosine nitration- An interaction between tyrosine nitration and H2S has been

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suggested in neuronal cells (26), in Interstitial Cells of Cajal and in processes involving

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apoptosis/necrosis and PARP-1 activation (4, 27, 30). We examined the interaction of H2S induced

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sulfhydration with tyrosine nitration in the KATP channel complex. In CHO cells co-transfected with the

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two subunits, treatment with SIN-1 and peroxynitrite resulted in nitration of Kir6.1 but not SUR2B

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(Fig.3A and 3B). Immunoprecipitation with anti-Kir6.1 followed by immunoblot with anti-Nitrotyrosine

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(NY) revealed that the extent of nitration of Kir6.1 was significantly reduced by pretreatment with NaHS

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(1 mM). NaHS treatment did not affect the basal level of nitration of Kir6.1. The reduction in nitration by

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NaHS was also evident when examined by immunohistochemistry. Cells co-transfected with Kir6.1 and

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SUR2B or the C1455S were treated with SIN-1 or NaHS or the combination. Tyrosine nitration was

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readily observed in cells treated with SIN-1 but markedly reduced in the presence of NaHS (Fig.3C). To

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further confirm that SUR2B subunit is the potential target for sulfhydration, the C1455S mutant was co-

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transfected with Kir6.1 and examined for tyrosine nitration following sulfhydration. As shown in the

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bottom panel of figure 3C, mutation of C1455 resulted in robust expression of tyrosine nitration

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indicative of the requirement of sulfhydration of C1455 for the reduction in tyrosine nitration. The effect

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of NaHS was further evaluated in enteric glia cells (EGC) by In-cell western (ICW) blot assay. Tyrosine

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nitration was evaluated over a thousand fold concentration range. We also examined the level of GFAP,

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marker for enteric glia cells, to establish the validity of cells in each well. Significant increase in total

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cellular tyrosine nitration in EGCs was observed at the concentration of 1 and 3 mM ONOO- stimulation

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(1054±101, 516±49, fold respectively, n=4) (Fig.3D). Pretreatment with NaHS reduced the level of

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nitration in the enteric glia cells to 181 ± 19 (1mM ONOO-) and 84 ± 23 (3 mM ONOO-) fold

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respectively (n=4, ***p ≤ 0.001). The expression of GFAP remained constant across the concentration

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range. These data suggest that NaHS may have the potential to inhibit tyrosine nitration formation.

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Kang et al. 244

Mutation of cysteine residues prevents nitration- The above data suggest that Kir6.1 is the potential

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target for tyrosine nitration while cysteine residues in SUR2B subunit are sulfhydrated and that

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sulfhdydration prevents tyrosine nitration. In order to test this, the cysteine residues C24 and C1455 that

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were identified as potential sulfhydration sites were mutated to serine residues. In CHO cells co-

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transfected with SUR2B 1) wild type, 2) C263S (control), 3) C24S and 4) C1455S with Kir6.1 were

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examined for prevention of nitration by NaHS. Figure 4A and 4B show that SIN-1 induced nitration of

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Kir6.1 is reduced by NaHS in wild type and in C263S SUR2B mutant but not in C24S or C1455S mutants.

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None of the mutants of SUR2B were nitrated by SIN-1 (Fig.4C). We next examined whether tyrosine

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nitration of Kir6.1 could be reversed by NaHS. Enhanced tyrosine nitration of Kir6.1 was detected with

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SIN-1 treatment (2.024±0.58), but interestingly, NaHS did not reduce post-nitration of Kir6.1 (2.11±0.26)

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(Fig.4D). These data suggest that sulfhydration of SUR2B is required for reducing nitration.

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Sulfhydration prevents Ca2+ channel nitration- Previously we have shown that L-type calcium

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channels in colonic smooth muscle cells are tyrosine nitrated during colonic inflammation, resulting in

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reduced currents and inhibition of contractions (21). Cav1.2 channels in rat cardiomyocytes have been

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suggested to be sulfhydrated (31). We therefore examined whether tyrosine nitration of Cav1.2b is

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reduced by NaHS. Figure 5 shows pretreatment with NaHS reduced the nitration of Cav1.2.

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H2S prevents ONOO- induced inhibition of CaCl2-induced contraction- In order to further evaluate

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the physiological role of H2S on tyrosine nitration, CaCl2 induced contractions of the isolated mouse

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ileum was measured in the presence and absence of ONOO-. Mouse ileum tissues were incubated in a

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depolarizing calcium-free high potassium (80 mM) physiological saline solution. A cumulative

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concentration-response to CaCl2 was established in control tissues. Pre-incubation with ONOO- (150 uM)

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for 10 min significantly reduced the calcium induced contraction and shifted the dose-response curve to

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the right (Fig.6). In tissues pre-incubated with NaHS (100 μM) for 15 mins followed by ONOO- resulted

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in a significant recovery of CaCl2 induced contractions. The EC50 values for CTL, H2S+ONOO-, and

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ONOO- were 143.3±15.5 uM, 197.3±23.5 uM, and 270.2±23.7 uM, respectively. These data demonstrate

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that the inhibition of the calcium influx under depolarizing conditions by ONOO- is reduced by NaHS. 11

Kang et al. 270 271 272 273

DISCUSSION

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Hydrogen sulfide (H2S) is emerging as a highly significant endogenous gaseous mediator and

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potential target for pharmacological manipulation (5, 15). It is known to have several intracellular targets

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of which ATP-sensitive K+ channels represent a significant target for H2S, particularly in colonic

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inflammation. Previously we have shown that the KATP channel opener levkromakalim and NaHS induced

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currents are enhanced in a TNBS model of colonic inflammation. An enhancement in the s-sulfhydration

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of the KATP channel subunit SUR2B is also seen post colonic inflammation which may allow for the

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allosteric modulation of channel. In the present study we examined the potential sites within the K ATP

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channel complex that are sulfhyrated and determined the interaction with nitration of tyrosine residues.

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We have found that 1) NaHS significantly enhanced sulfhydration of SUR2B but not Kir 6.1 subunit; 2)

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SIN – 1 (a donor of peroxinitrite) induced nitration of Kir6.1 subunit but not SUR2B; 3) Two specific

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mutations within SUR2B, C24S and C1455S prevented sulfhydration by NaHS and these mutations

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prevented NaHS induced reduction in tyrosine nitration of Kir6.1 4) NaHS also reversed peroxynitrite-

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induced inhibition of smooth muscle contraction.

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Ion channel activity can be significantly altered by the intracellular redox state maintained by

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reactive oxygen species (ROS) and reactive nitrogen species (RNS). Physiological generation of these

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compounds are highly regulated, however an imbalance occurs in pathophysiological conditions such as

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in colonic inflammation. ROS/RNS generated can directly induce post translational modifications at

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specific residues and alter the ion channel function. These include redox modification of cysteine

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residues by ROS, s-nitrosylation by nitric oxide, and/or nitration of tyrosine residues by peroxynitrite.

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Our studies demonstrate that within a single protein complex involving two subunits, there is significant

294

specificity as sulfhydration occurred in the SUR2B but not Kir 6.1, while nitration occurred in Kir 6.1 but

295

not SUR2B. Since the discovery of endogenous H2S, many studies have explored the roles of this gas in 12

Kang et al. 296

animals and humans and have shown H2S to be involved in diverse physiological and pathophysiological

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processes. However, our understanding of the pharmacology and the pathophysiological roles of H 2S in

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gastrointestinal (GI) tract is still quite rudimentary. There is growing evidence that during colonic

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inflammation, including ulcerative colitis, there is an increased production of hydrogen sulfide (H2S) in

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the GI tract (23) that may have protective effects during colitis. In the present study we demonstrate that

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sulfhydration of one subunit (SUR2B) of the ATP-sensitive K channel, prevents the tyrosine nitration in

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the other subunit (Kir6.1). We also show that H2S sulfhydrates SUR2B at C24 and C1455 residues and

303

causes the reversal of tyrosine nitration of the Kir6.1 in the KATP channel complex. This suggests that

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sulfhydration of cysteine residues of SUR2B on either sides of the membrane is required to alter the

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tyrosine nitration of Kir6.1 as C24 is localized on the extracellular side whereas C1455 is localized

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towards the intracellular side. The specific mechanism by which these cysteine residues affect modulation

307

of the KATP currents requires further work, but it is noteworthy that ATP-sensitive K+ channel activity is

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markedly enhanced in model of experimental colitis (8). In addition to the allosteric effects on channel

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function, the present studies show that sulfhydration also appears to reduce tyrosine nitration of pore-

310

forming subunit Kir6.1. Since post-nitration of Kir6.1 is not affected by NaHS, it would appear that the

311

order of exposure to the free radicals may be critical in the regulation of the ion channel activity.

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During colonic inflammation reduced muscle contractility occurs partly as a result of decreased

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calcium influx through L-type calcium channels and enhanced KATP currents (2). Calcium channels along

314

with the KATP play an important role in regulating the excitability of the smooth muscle cells. Previous

315

work has shown that nitration of the tyrosine residues within the carboxy terminus of Ca v1.2 prevents src-

316

kinase mediated tyrosine phosphorylation leading to decreased calcium currents (9-12, 20, 21). Cav1.2

317

channels can also be sulfhydrated (31).

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contractions that occur as a result of calcium influx under depolarizing conditions is reduced by NaHS.

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Thus, similar to the findings for the K+ channel, sulfhydration may also reduce tyrosine nitration in Cav1.2.

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However further works needs to done to identify the cysteine residues involved in the sulfhydration of the

321

Cav1.2. Occurrence of this interaction in KATP channels complex in transfected CHO cells and enteric

The inhibition by tyrosine nitration of calcium-induced

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Kang et al. 322

glial cells and in Cav1.2 in transfected CHO cells demonstrates the ubiquitous nature of this phenomenon.

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This unique interaction within a protein complex may provide mechanistic basis by which H 2S affords a

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protective role in colonic dysmotility during colitis. Enhanced production of endogenous H 2S and that via

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resident luminal bacteria may compete with the reactive nitrogen species produced during colonic

326

inflammation. Peroxynitrite is enhanced during colonic inflammation as a result of the uncoupling of

327

nitric oxide synthase (NOS) and enhanced expression of inducible NOS (3).

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Ion channels play a central role in the regulation of cell excitability, therefore altered regulation of ion

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channels as a consequence of protein modification is of significance not only for colonic inflammation

330

but in other disease states and may help provide novel approaches in channel-targeted therapeutics. Our

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findings that sulfhydration of KATP channel prevents the detrimental effects of tyrosine nitration within a

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single ion channel complex provides a novel basis to examine the anti-oxidant properties of H2S.

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Kang et al. 348 349

Acknowledgments – We thank Dr. Kazuharu Frutani, Osaka University for providing us with mouse

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SUR2B cDNA.

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This work was supported by NIH DK046367.

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Kang et al. 367 368

LIST OF ABBREVIATIONS

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CHO - Chinese Hamster Ovary

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DTT - dithiothreitol

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EGC - Enteric glia cells

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GAPDH - glyceraldehde-3-phosphate

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GFAP - Glial Fibrillary acidic protein

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H2S - Hydrogen sulfide

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ICW - In cell western

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KATP - ATP-sensitive K+ channel

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NY - nitrotyrosine

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ONOO- - peroxynitrite

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ROS - Reactive Oxygen Species

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RNS - Reactive Nitrogen Species

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SIN-1 - 3-Morpholinosydnonimine

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SUR2B - Sulphonylurea 2B

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Kang et al. 384

FIGURE LEGENDS

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FIGURE 1. Sulfhydration is dependent on cysteine residues. A, representative biotin switch assay of

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SUR2B sulfhydration in CHO cells transfected with Kir6.1 and SUR2B. The bar graph represents ratio of

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sulfhydration of SUR2B and total SUR2B and presented as mean ± SEM from three independent

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experiments. * represents significant difference between untreated (col 1) versus NaHS (1 mM) (col 2).

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DTT treatment also reduced the sulfhydration by NaHS (1 mM) (col 4). B) representative levcromakalim

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(LevK)-induced whole cell K+ currents recording in a single cell. The currents were recorded from CHO

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transfected cells. LevK currents were recorded in the presence of high external K+ and low intracellular

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ATP at -60 mV holding potential.

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FIGURE 2. Cysteine residues on SUR2B affect sulfhydration. Biotin switch assay of SUR2B

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sulfhydration in CHO cells transfected with Kir6.1 and SUR2B mutants. The bar graph represents ratio of

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sulfhydrated SUR2B and total SUR2B, and presented as mean ± SEM from three independent

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experiments. * presents significant difference between control (CTL) versus mutants. Both C24S and

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C1455S were significantly different from control. Bottom panel is a representative blot for WT and all

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mutants.

400 401

FIGURE 3. Tyrosine nitration is attenuated by H2S. A and B, representative immunoblot analyses of

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tyrosine nitration proteins in CHO transfected cells. Cells were lysed in immunoprecipitation assay buffer

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(RIPA), and Kir6.1(A) or SUR2B (B) was immunoprecipitated (IP) using anti-Kir6.1 or anti-SUR2B and

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immunoblotted using anti-nitrotyrosine antibody (NY). The bar graph represents ratio of nitration of Kir

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6.1 (A) and SUR2B (B) against total Kir and SUR2B, respectively (mean ± SEM from three independent

406

experiments). * represents significant difference between SIN-1 induced nitration of Kir6.1 and in the

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presence of NaHS. C) representative immunostaining for tyrosine nitration in CHO transfected cells.

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CHO cells were transfected with Kir6.1 and SUR2B or C1455S, and treated with 100 μM SIN-1 for 20

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min or 1 mM NaHS for 10 min, or combination of both. Cells were visualized by fluorescence confocal 17

Kang et al. 410

microscopy. Scale bar = 20μm. D) In-cell western of EGCs treated with the indicated doses of ONOO- for

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15 min in the absence or presence of NaHS (1 mM, 15 min) and processed for imaging (bottom panels) as

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described in methods. The image shows whole-cell fluorescence from actual wells of a 96-well plate,

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highlighting increase in tyrosine nitration signal. The signal is background subtracted and normalized to

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control. Bar graph (top panel) shows the fold induction of tyrosine nitration measured as intensity over

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the control (absence of ONOO-). Values represent the mean ± SEM. *** represents significant difference

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between ONOO- treated versus NaHS + ONOO- -treated cells.

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FIGURE 4. Tyrosine nitration of Kir6.1 and the effect of cysteine mutations of SUR2B. A and B,

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representative Immunostaining for tyrosine nitration in CHO transfected cells. Wild type or mutants of

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SUR2B were transiently expressed, immunoprecipitated (IP) using Kir6.1 antibody, and then

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immunobloted for tyrosine nitration of Kir6.1 using anti-nitrotyrosine antibody (NY). The bar graph

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represents normalized data for tyrosine nitration and presented as mean ± SEM from three independent

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experiments. Significant differences between CTL versus SIN-1 and SIN-1 vs NaHS + SIN-1 are

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represented by an asterick (*). Cells were treated with NaHS (150 uM, 15 min), SIN-1 (100 uM, 20 min)

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or both. B) Representative western blots of immunoprecipitated Kir 6.1 and immunoblotted with anti-

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nitrotyrosine. C) representative immunoblot analyses of tyrosine nitration of SUR2B proteins in CHO

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transfected cells. Wild type or mutants of SUR2B were transiently expressed, treated with SIN-1 (100 uM,

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20 min), immunoprecipitated (IP) using anti-SUR2B and immunoblotted using antibody for tyrosine

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nitration proteins (NY). D) representative SIN-1- induced tyrosine nitration of Kir6.1 is not reversed by

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NaHS post- tyrosine nitration. Kir6.1 and wild type SUR2B were transiently expressed and treated with

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SIN-1(100 uM, 20 min) followed by NaHS (150 uM, 15 min). The cell lysates were immunoprecipitated

432

(IP) using Kir6.1 antibody, and then immunoblotted for tyrosine nitration (NY). The bar graph represents

433

normalized data for tyrosine nitration and presented as mean ± SEM from three independent experiments.

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* represents significant difference between wild type and SIN-1.

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Kang et al. 436

FIGURE 5. Sulfhydration prevents Ca2+ channel nitration. Bar graph represents nitration of calcium

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channel in CHO transfected cells. Ca2+ channel (Cav1.2b) was transiently expressed, and treated with

438

NaHS (150 uM, 15 min), SIN-1(100 uM, 20 min) or NaHS followed by SIN-1. Protein was

439

immunoprecipitated (IP) using Cav1.2b antibody, and then immunoblotted for tyrosine nitration (NY).

440

The bar graph represents normalized data for tyrosine nitration and presented as mean ± SEM from three

441

independent experiments. * represents significant differences between NaHS and SIN-1.

442 443

FIGURE 6. Effect of ONOO- or H2S + ONOO- on the cumulative CaCl2-induced concentration-

444

dependent contractions in isolated mouse ileum. Maximal contractions of control group (CTL) were

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considered as 100%. The CaCl2 contractions were elicited in tissues depolarized by high-potassium (80

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mM) physiological saline solution. A, representative graph showing the cumulative CaCl2 concentration-

447

response curves by ONOO- and H2S + ONOO- in mouse ileum. B, representative tracing of CaCl2-

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induced concentration-dependent contractions in isolated ileum from mouse. Points represent mean ±

449

S.E.M. Data were analyzed by repeated measures-ANOVA followed by Bonferroni’s post-test. *, P ≤

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0.05, compare to ONOO-. #, P ≤ 0.05, ##, P ≤ 0.01 versus ONOO- compare to CTL. n = 3-4.

451 452

FIGURE7. A schematic of the proposed protective effects of H2S in colonic inflammation. Exogenous

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H2S enhances sulfhydration of two cysteine residues (24 and 1455) within SUR2B subunit of KATP

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channels which prevents tyrosine nitration within Kir6.1 subunit of KATP channel complex.

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Kang et al. 459 460 461 462

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