Jason L. Scragg

Carbon Monoxide Inhibits L-type Ca2+ Channels via Redox Modulation of Key Cysteine Residues by Mitochondrial Reactive Oxygen Species

Conditions of stress, such as myocardial infarction, stimulate up-regulation of heme oxygenase (HO-1) to provide cardioprotection. Here, we show that CO, a product of heme catabolism by HO-1, directly inhibits native rat cardiomyocyte L-type Ca2+ currents and the recombinant α1C subunit of the human cardiac L-type Ca2+ channel. CO (applied via a recognized CO donor molecule or as the dissolved gas) caused reversible, voltage-independent channel inhibition, which was dependent on the presence of a spliced insert in the cytoplasmic C-terminal region of the channel. Sequential molecular dissection and point mutagenesis identified three key cysteine residues within the proximal 31 amino acids of the splice insert required for CO sensitivity. CO-mediated inhibition was independent of nitric oxide and protein kinase G but was prevented by antioxidants and the reducing agent, dithiothreitol. Inhibition of NADPH oxidase and xanthine oxidase did not affect the inhibitory actions of CO. Instead, inhibitors of complex III (but not complex I) of the mitochondrial electron transport chain and a mitochondrially targeted antioxidant (Mito Q) fully prevented the effects of CO. Our data indicate that the cardioprotective effects of HO-1 activity may be attributable to an inhibitory action of CO on cardiac L-type Ca2+ channels. Inhibition arises from the ability of CO to promote generation of reactive oxygen species from complex III of mitochondria. This in turn leads to redox modulation of any or all of three critical cysteine residues in the channels cytoplasmic C-terminal tail, resulting in channel inhibition.

Hypoxia and Neurodegeneration

Periods of chronic hypoxia, which can arise from numerous cardiorespiratory disorders, predispose individuals to the development of dementias, particularly Alzheimers disease (AD). AD is characterized in part by the increased production of amyloid β peptide (Aβ), which forms the extracellular plaques by which the disease can be identified post mortem. Numerous studies have now shown that hypoxia, even in vitro, can increase production of Aβ in different cell types. Evidence has been produced to indicate hypoxia alters both expression of the Aβ precursor, APP, and also the expression of the secretase enzymes, which cleave Aβ from APP. Other studies implicate reduced Aβ degradation as a possible means by which hypoxia increases Aβ levels. Such variability may be attributable to cell‐specific responses to hypoxia. Further evidence indicates that some, but not all of the cellular adaptations to chronic hypoxia (including alteration of Ca2+ homeostasis) require Aβ formation. However, other aspects of hypoxic remodeling of cell function appear to occur independently of this process. The molecular and cellular responses to hypoxia contribute to our understanding of the clinical association of hypoxia and increased incidence of AD. However, it remains to be determined whether inhibition of one or more of the effects of hypoxia may be of benefit in arresting the development of this neurodegenerative disease.

Modulation of Ion Channels by Hydrogen Sulfide

SIGNIFICANCE Evidence of the ability of the gasotransmitter hydrogen sulfide (H(2)S) to serve as a regulator of many physiological functions, including control of blood pressure, regulation of cardiac function, protection of neurons, and cardiomyocytes against apoptosis, and in pain sensation is accumulating. However, the mechanisms accounting for its many actions are not yet well understood. RECENT ADVANCES Following the pioneering studies of the regulation of N-methyl-d-aspartate receptors and ATP-sensitive K(+) channels by H(2)S, data continue to emerge indicating that H(2)S modulates other ion channel types. This article reviews the numerous, yet diverse, types of ion channels now reported to be regulated by H(2)S. CRITICAL ISSUES Currently, a critical issue within this field is to determine the mechanisms by which H(2)S regulates ion channels, as well as other target proteins. Mechanisms to account for regulation include direct channel protein sulfhydration, channel redox modulation, effects mediated by interactions with other gasotransmitters (carbon monoxide and nitric oxide), and indirect effects, such as modulation of channel-regulating kinases. Through such modulation of ion channels, novel roles for H(2)S are emerging as important factors in both physiological and pathological processes. FUTURE DIRECTIONS Increasing current awareness and understanding of the roles and mechanisms of action of ion channel regulation by H(2)S will open opportunities for therapeutic intervention with clear clinical benefits, and inform future therapies. In addition, more sensitive methods for detecting relevant physiological concentrations of H(2)S will allow for clarification of specific ion channel regulation with reference to physiological or pathophysiological settings.

Carbon monoxide protects against oxidant-induced apoptosis via inhibition of Kv2.1.

Oxidative stress induces neuronal apoptosis and is implicated in cerebral ischemia, head trauma, and age‐related neurodegenerative diseases. An early step in this process is the loss of intracellular K+ via channels, and evidence indicates that Kv2.1 is of particular importance in this regard, being rapidly inserted into the plasma membrane in response to apoptotic stimuli. An additional feature of neuronal oxidative stress is the up‐regulation of the inducible enzyme heme oxygenase‐1 (HO‐1), which catabolizes heme to generate biliverdin, Fe2+, and carbon monoxide (CO). CO provides neuronal protection against stresses such as stroke and excitotoxicity, although the underlying mechanisms are not yet elucidated. Here, we demonstrate that CO reversibly inhibits Kv2.1. Channel inhibition by CO involves reactive oxygen species and protein kinase G activity. Overexpression of Kv2.1 in HEK293 cells increases their vulnerability to oxidant‐induced apoptosis, and this is reversed by CO. In hippocampal neurons, CO selectively inhibits Kv2.1, reverses the dramatic oxidant‐induced increase in k+ current density, and provides marked protection against oxidant‐induced apoptosis. Our results provide a novel mechanism to account for the neuroprotective effects of CO against oxidative apoptosis, which has potential for therapeutic exploitation to provide neuronal protection in situations of oxidative stress.—Dallas, M. L., Boyle, J. P., Milligan, C. J., Sayer, R., Kerrigan, T. L., McKinstry, C., Lu, P., Mankouri, J., Harris, M., Scragg, J. L., Pearson, H. A., Peers, C. Carbon monoxide protects against oxidant‐induced apoptosis via inhibition of Kv2.1. FASEB J. 25, 1519–1530 (2011). www.fasebj.org

Alzheimer's amyloid peptides mediate hypoxic up-regulation of L-type Ca2+ channels.

We examined the effects of chronic hypoxia on recombinant human L‐type Ca2+ channel α1C subunits stably expressed in HEK 293 cells, using whole‐cell patch‐clamp recordings. Current density was dramatically increased following 24 h exposure to chronic hypoxia (CH), and membrane channel protein levels were enhanced. CH also increased the levels of Alzheimers amyloid β peptides (AβPs), determined immunocytochemically. Pharmacological prevention of AβP production (via exposure to inhibitors of secretase enzymes that are required to cleave AβP from its precursor protein) prevented hypoxic augmentation of currents, as did inhibition of vesicular trafficking with bafilomycin A1. The enhancing effect of AβPs or CH were abolished following incubation with the monoclonal 3D6 antibody, raised against the extracellular N′ terminus of AβP. Immunolocalization and immunoprecipitation studies provided compelling evidence that AβPs physically associated with the α1C subunit, and this association was promoted by hypoxia. These data suggest an important role for AβPs in mediating the increase in Ca2+ channel activity following CH and show that AβPs act post‐transcriptionally to promote α1C subunit insertion into (and/or retention within) the plasma membrane. Such an action will likely contribute to the Ca2+ dyshomeostasis of Alzheimers disease and may contribute to the mechanisms underlying the known increased incidence of this neurodegenerative disease following hypoxic episodes.

Modulation of hTREK-1 by carbon monoxide

TREK-1 is a background K+ channel important in the regulation of neuronal excitability. Here, we demonstrate that recombinant human TREK-1 is activated by low concentrations of carbon monoxide (CO) and nitric oxide (NO), applied via their respective donor molecules. Related channels hTASK-1 and hTASK-3 were unaffected by CO. Effects of both CO and NO were prevented by preincubation of cells with the protein kinase G inhibitor, Rp-8-Br-PET-cGMPS. The effects of CO were independent of NO formation. At higher concentrations, both NO and CO were inhibitory. As both NO and CO are important neuronal gasotransmitters and TREK is crucial in regulating neuronal excitability, our results provide a novel means by which these gases may modulate neuronal activity.

Carbon Monoxide Mediates the Anti-apoptotic Effects of Heme Oxygenase-1 in Medulloblastoma DAOY Cells via K+ Channel Inhibition

Background: Heme oxygenase-1 (HO-1) is constitutively expressed in many cancers which are highly resistant to apoptosis. Results: CO, a product of HO-1, inhibits K+ channels in the medulloblastoma cell line DAOY and protects against apoptosis. Conclusion: HO-1 increases resistance to apoptosis in cancer cells via CO generation. Significance: targeting HO-1 expression may increase the effectiveness of cancer therapies. Tumor cell survival and proliferation is attributable in part to suppression of apoptotic pathways, yet the mechanisms by which cancer cells resist apoptosis are not fully understood. Many cancer cells constitutively express heme oxygenase-1 (HO-1), which catabolizes heme to generate biliverdin, Fe2+, and carbon monoxide (CO). These breakdown products may play a role in the ability of cancer cells to suppress apoptotic signals. K+ channels also play a crucial role in apoptosis, permitting K+ efflux which is required to initiate caspase activation. Here, we demonstrate that HO-1 is constitutively expressed in human medulloblastoma tissue, and can be induced in the medulloblastoma cell line DAOY either chemically or by hypoxia. Induction of HO-1 markedly increases the resistance of DAOY cells to oxidant-induced apoptosis. This effect was mimicked by exogenous application of the heme degradation product CO. Furthermore we demonstrate the presence of the pro-apoptotic K+ channel, Kv2.1, in both human medulloblastoma tissue and DAOY cells. CO inhibited the voltage-gated K+ currents in DAOY cells, and largely reversed the oxidant-induced increase in K+ channel activity. p38 MAPK inhibition prevented the oxidant-induced increase of K+ channel activity in DAOY cells, and enhanced their resistance to apoptosis. Our findings suggest that CO-mediated inhibition of K+ channels represents an important mechanism by which HO-1 can increase the resistance to apoptosis of medulloblastoma cells, and support the idea that HO-1 inhibition may enhance the effectiveness of current chemo- and radiotherapies.

Diverse mechanisms underlying the regulation of ion channels by carbon monoxide

Carbon monoxide (CO) is firmly established as an important, physiological signalling molecule as well as a potent toxin. Through its ability to bind metal‐containing proteins, it is known to interfere with a number of intracellular signalling pathways, and such actions can account for its physiological and pathological effects. In particular, CO can modulate the intracellular production of reactive oxygen species, NO and cGMP levels, as well as regulate MAPK signalling. In this review, we consider ion channels as more recently discovered effectors of CO signalling. CO is now known to regulate a growing number of different ion channel types, and detailed studies of the underlying mechanisms of action are revealing unexpected findings. For example, there are clear areas of contention surrounding its ability to increase the activity of high conductance, Ca2+‐sensitive K+ channels. More recent studies have revealed the ability of CO to inhibit T‐type Ca2+ channels and have unveiled a novel signalling pathway underlying tonic regulation of this channel. It is clear that the investigation of ion channels as effectors of CO signalling is in its infancy, and much more work is required to fully understand both the physiological and the toxic actions of this gas. Only then can its emerging use as a therapeutic tool be fully and safely exploited.

Hydrogen sulfide inhibits Cav3.2 T-type Ca2+ channels

The importance of H2S as a physiological signaling molecule continues to develop, and ion channels are emerging as a major family of target proteins through which H2S exerts many actions. The purpose of the present study was to investigate its effects on T‐type Ca2+ channels. Using patch‐clamp electrophysiology, we demonstrate that the H2S donor, NaHS (10 μM–1 mM) selectively inhibits Cav3.2 T‐type channels heterologously expressed in HEK293 cells, whereas Cav3.1 and Cav3.3 channels were unaffected. The sensitivity of Cav3.2 channels to H2S required the presence of the redox‐sensitive extracellular residue H191, which is also required for tonic binding of Zn2+ to this channel. Chelation of Zn2+ with N,N,N‘,N‘‐tetra‐2‐picolylethylenediamine prevented channel inhibition by H2S and also reversed H2S inhibition when applied after H2S exposure, suggesting that H2S may act via increasing the affinity of the channel for extracellular Zn2+ binding. Inhibition of native T‐type channels in 3 cell lines correlated with expression of Cav3.2 and not Cav3.1 channels. Notably, H2S also inhibited native T‐type (primarily Cav3.2) channels in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H2S regulation, the T‐type Ca2+ channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.—Elies, J., Scragg, J. L., Huang, S., Dallas, M. L., Huang, D., MacDougall, D., Boyle, J. P., Gamper, N., Peers, C., Hydrogen sulfide inhibits Cav3.2 T‐type Ca2+ channels. FASEB J. 28, 5376–5387 (2014). www.fasebj.org

H2O2-stimulated Ca2+ influx via TRPM2 is not the sole determinant of subsequent cell death

Activation of transient receptor potential melastatin 2 (TRPM2), a non-selective, Ca2+-permeable cation channel, is implicated in cell death. Channel opening is stimulated by oxidative stress, a feature of numerous disease states. The wide expression profile of TRPM2 renders it a potentially significant therapeutic target in a variety of pathological settings including cardiovascular and neurodegenerative diseases. HEK293 cells transfected with human TRPM2 (HEK293/hTRPM2) were more vulnerable to H2O2-mediated cell death than untransfected controls in which H2O2-stimulated Ca2+ influx was absent. Flufenamic acid partially reduced Ca2+ influx in response to H2O2 but had no effect on viability. N-(p-Amylcinnamoyl) anthranilic acid substantially attenuated Ca2+ influx but did not alter viability. Poly(adenosine diphosphate ribose) polymerase inhibitors (N-(6-oxo-5,6-dihydro-phenanthridin-2-yl)-N,N-dimethylacetamide, 3,4-dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone and nicotinamide) reduced Ca2+ influx and provided a degree of protection but also had some protective effects in untransfected controls. These data suggest H2O2 triggers cell death in HEK293/hTRPM2 cells by a mechanism that is in part Ca2+ independent, as blockade of channel opening (evidenced by suppression of Ca2+ influx) did not correlate well with protection from cell death. Determining the underlying mechanisms of TRPM2 activation is pertinent in elucidating the relevance of this channel as a therapeutic target in neurodegenerative diseases and other pathologies associated with Ca2+ dysregulation and oxidative stress.