Jacobo Elies

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

Carbon monoxide inhibition of Cav3.2 T-type Ca2+ channels reveals tonic modulation by thioredoxin

T‐type Ca2+ channels play diverse roles in tissues such as sensory neurons, vascular smooth muscle, and cancers, where increased expression of the cytoprotective enzyme, heme oxygenase‐1 (HO‐1) is often found. Here, we report regulation of T‐type Ca2+ channels by carbon monoxide (CO) a HO‐1 by‐product. CO (applied as CORM‐2) caused a concentrationdependent, poorly reversible inhibition of all T‐type channel isoforms (Cav3.1‐3.3, IC50 ~3 μM) expressed in HEK293 cells, and native T‐type channels in NG108‐15 cells and primary rat sensory neurons. No recognized CO‐sensitive signaling pathway could account for the CO inhibition of Cav3.2. Instead, CO sensitivity was mediated by an extracellular redox‐sensitive site, which was also highly sensitive to thioredoxin (Trx). Trx depletion (using auranofin, 2‐5 μM) reduced Cav3.2 currents and their CO sensitivity by > 50% but increased sensitivity to dithiothreitol ~3‐fold. By contrast, Cav3.1 and Cav3.3 channels, and their sensitivity to CO, were unaffected in identical experiments. Our data propose a novel signaling pathway in which Trx acts as a tonic, endogenous regulator of Cav3.2 channels, while HO‐1‐derived CO disrupts this regulation, causing channel inhibition. CO modulation of T‐type channels has widespread implications for diverse physiological and pathophysiological mechanisms, such as excitability, contractility, and proliferation.—Boycott, H. E., Dallas, M. L., Elies, J., Pettinger, L., Boyle, J. P., Scragg, J. L., Gamper, N., Peers, C., Carbon monoxide inhibition of Cav3.2 T‐type Ca2+ channels reveals tonic modulation by thioredoxin. FASEBJ. 27, 3395‐3407 (2013). www.fasebj.org

Heme oxygenase-1 regulates cell proliferation via carbon monoxide-mediated inhibition of T-type Ca2+ channels

Induction of the antioxidant enzyme heme oxygenase-1 (HO-1) affords cellular protection and suppresses proliferation of vascular smooth muscle cells (VSMCs) associated with a variety of pathological cardiovascular conditions including myocardial infarction and vascular injury. However, the underlying mechanisms are not fully understood. Over-expression of Cav3.2 T-type Ca2+ channels in HEK293 cells raised basal [Ca2+]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca2+]i levels were reduced to levels seen in non-transfected cells either by induction of HO-1 or exposure of cells to the HO-1 product, carbon monoxide (CO) (applied as the CO releasing molecule, CORM-3). In the aortic VSMC line A7r5, proliferation was also inhibited by induction of HO-1 or by exposure of cells to CO, and patch-clamp recordings indicated that CO inhibited T-type (as well as L-type) Ca2+ currents in these cells. Finally, in human saphenous vein smooth muscle cells, proliferation was reduced by T-type channel inhibition or by HO-1 induction or CO exposure. The effects of T-type channel blockade and HO-1 induction were non-additive. Collectively, these data indicate that HO-1 regulates proliferation via CO-mediated inhibition of T-type Ca2+ channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

Inhibition of the Cardiac Na+ Channel Nav1.5 by Carbon Monoxide

Background: CO poisoning causes cardiac arrhythmias, in part via modulation of the cardiac Na+ channel, Nav1.5. Results: CO inhibition of peak recombinant Nav1.5 current occurs via nitric oxide formation and is also dependent on channel redox state. Conclusion: CO inhibits peak recombinant Nav1.5 current via a mechanism distinct from activation of the late Na+ current. Significance: CO may induce Brugada-like arrhythmias via inhibition of peak Na+ current. Sublethal carbon monoxide (CO) exposure is frequently associated with myocardial arrhythmias, and our recent studies have demonstrated that these may be attributable to modulation of cardiac Na+ channels, causing an increase in the late current and an inhibition of the peak current. Using a recombinant expression system, we demonstrate that CO inhibits peak human Nav1.5 current amplitude without activation of the late Na+ current observed in native tissue. Inhibition was associated with a hyperpolarizing shift in the steady-state inactivation properties of the channels and was unaffected by modification of channel gating induced by anemone toxin (rATX-II). Systematic pharmacological assessment indicated that no recognized CO-sensitive intracellular signaling pathways appeared to mediate CO inhibition of Nav1.5. Inhibition was, however, markedly suppressed by inhibition of NO formation, but NO donors did not mimic or occlude channel inhibition by CO, indicating that NO alone did not account for the actions of CO. Exposure of cells to DTT immediately before CO exposure also dramatically reduced the magnitude of current inhibition. Similarly, l-cysteine and N-ethylmaleimide significantly attenuated the inhibition caused by CO. In the presence of DTT and the NO inhibitor Nω-nitro-l-arginine methyl ester hydrochloride, the ability of CO to inhibit Nav1.5 was almost fully prevented. Our data indicate that inhibition of peak Na+ current (which can lead to Brugada syndrome-like arrhythmias) occurs via a mechanism distinct from induction of the late current, requires NO formation, and is dependent on channel redox state.

Regulation of the T‐type Ca2+ channel Cav3.2 by hydrogen sulfide: emerging controversies concerning the role of H2S in nociception

Ion channels represent a large and growing family of target proteins regulated by gasotransmitters such as nitric oxide, carbon monoxide and, as described more recently, hydrogen sulfide. Indeed, many of the biological actions of these gases can be accounted for by their ability to modulate ion channel activity. Here, we report recent evidence that H2S is a modulator of low voltage‐activated T‐type Ca2+ channels, and discriminates between the different subtypes of T‐type Ca2+ channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H2S augments Cav3.2 currents, an observation which has led to the suggestion that H2S exerts its pro‐nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H2S is seen to inhibit Cav3.2. This inhibitory action requires the presence of the redox‐sensitive, extracellular region of the channel which is responsible for tonic metal ion binding and which particularly distinguishes this channel isoform from Cav3.1 and 3.3. Further studies indicate that H2S may act in a novel manner to alter channel activity by potentiating the zinc sensitivity/affinity of this binding site. This review discusses the different reports of H2S modulation of T‐type Ca2+ channels, and how such varying effects may impact on nociception given the role of this channel in sensory activity. This subject remains controversial, and future studies are required before the impact of T‐type Ca2+ channel modulation by H2S might be exploited as a novel approach to pain management.

Inhibition of T-type Ca2+ channels by hydrogen sulfide

T-type Ca(2+) channels are a distinct family of low voltage-activated Ca(2+) channels which serve many roles in different tissues. Several studies have implicated them, for example, in the adaptive responses to chronic hypoxia in the cardiovascular and endocrine systems. Hydrogen sulfide (H(2)S) was more recently discovered as an important signalling molecule involved in many functions, including O(2) sensing. Since ion channels are emerging as an important family of target proteins for modulation by H(2)S, and both T-type Ca(2+) channels and H(2)S are involved in cellular responses to hypoxia, we have investigated whether recombinant and native T-type Ca(2+) channels are a target for modulation by H(2)S. Using patch-clamp electrophysiology, we demonstrate that the H(2)S donor, NaHS, selectively inhibits Cav3.2 T-type Ca(2+) channels heterologously expressed in HEK293 cells, whilst Cav3.1 and Cav3.3 channels were unaffected. 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 Zn(2+) to this channel. Chelation of Zn(2+) using TPEN prevented channel inhibition by H(2)S. H2S also selectively inhibited native T-type channels (primarily Cav3.2) in sensory dorsal root ganglion neurons. Our data demonstrate a novel target for H(2)S regulation, the T-type Ca(2+) channel Cav3.2. Results have important implications for the proposed pro-nociceptive effects of this gasotransmitter. Implications for the control of cellular responses to hypoxia await further study.

Scleroderma fibroblasts suppress angiogenesis via TGF-β/caveolin-1 dependent secretion of pigment epithelium-derived factor

Objectives Systemic sclerosis (SSc) is characterised by tissue fibrosis and vasculopathy with defective angiogenesis. Transforming growth factor beta (TGF-β) plays a major role in tissue fibrosis, including downregulation of caveolin-1 (Cav-1); however, its role in defective angiogenesis is less clear. Pigment epithelium-derived factor (PEDF), a major antiangiogenic factor, is abundantly secreted by SSc fibroblasts. Here, we investigated the effect of TGF-β and Cav-1 on PEDF expression and the role of PEDF in the ability of SSc fibroblasts to modulate angiogenesis. Methods PEDF and Cav-1 expression in fibroblasts and endothelial cells were evaluated by means of immunohistochemistry on human and mouse skin biopsies. PEDF and Cav-1 were silenced in cultured SSc and control fibroblasts using lentiviral short-hairpin RNAs. Organotypic fibroblast–endothelial cell co-cultures and matrigel assays were employed to assess angiogenesis. Results PEDF is highly expressed in myofibroblasts and reticular fibroblasts with low Cav-1 expression in SSc skin biopsies, and it is induced by TGF-β in vitro. SSc fibroblasts suppress angiogenesis in an organotypic model. This model is reproduced by silencing Cav-1 in normal dermal fibroblasts. Conversely, silencing PEDF in SSc fibroblasts rescues their antiangiogenic phenotype. Consistently, transgenic mice with TGF-β receptor hyperactivation show lower Cav-1 and higher PEDF expression levels in skin biopsies accompanied by reduced blood vessel density. Conclusions Our data reveal a new pathway by which TGF-β suppresses angiogenesis in SSc, through decreased fibroblast Cav-1 expression and subsequent PEDF secretion. This pathway may present a promising target for new therapeutic interventions in SSc.

Novel ways to regulate T-type Ca2+ channels

T-type (CaV3) Ca 2C channels are distinguished from other voltage-gated Ca2C channels by their rapid activation and inactivation, slow deactivation, smaller single channel conductances and very negative activation threshold (as low as ¡60 mV). CaV3 are encoded by CACNA1G, CACNA1H and CACNA1I genes which give rise to pore-forming a subunits (CaV3.1–3.3 respectively). These channels serve surprisingly diverse roles; in central neurons they are responsible for pacemaker activity and low threshold spikes, contributing to “rebound” bursts of spikes following a hyperpolarizing postsynaptic potential. They also display a significant window current (i.e. are tonically active) at potentials close to resting membrane potential (RMP), so can contribute to setting the RMP. In the peripheral nervous system, CaV3 are expressed in several types of sensory neurons, including a subpopulation of small, capsaicin-sensitive (presumed nociceptive) DRG neurons where they influence excitability and so play an important role in nociception. Also due to their window currents, CaV3.1 and 3.2 strongly influence cell proliferation, as has been studied in vascular smooth muscle and different types of cancers (see). In sensory neurons CaV3.2 is the dominant T-type channel form and may even be the exclusive form in some mechanosensitive neurons. Their control of burst firing in DRG neurons indicates they are of central importance to nociception since stimulus intensity correlates with burst frequency. Native nociceptive and recombinant CaV3 currents are enhanced by reducing agents (which induce hyperalgesia) and inhibited by the oxidising agents. This sensitivity to redox modulation of Cav3.2 is specific among the Cav3 isoforms, and arises because of the presence of an extracellular, high affinity binding site for trace metals (Zn2C, Ni2C) formed by interacting regions of the S1-S2 and S3-S4 loops within domain I of the channel protein. Mutation of the histidine residue H191 (in the S3-S4 region; Fig. 1) abolishes high affinity current inhibition by these metals and markedly reduces redox sensitivity. Since this site is clearly important in nociception, it represents an attractive site for therapeutic development. The increasing awareness of the biological importance of endogenous gases (firstly NO, but more recently CO and H2S – these are now labeled as “gasotransmitters”) has led to a wealth of studies indicating that they are important in diverse physiological functions, and can exert important influences on disease progression. These gases modulate a number of intracellular signaling pathways, and ion channels were among the first type of target proteins recognized as mediating some of their effects. More recently, we have shown that T-type channels are sensitive to gasotransmitters: CO blocks all 3 isoforms of T-type Ca2C channels with similar affinity, although the underlying mechanisms vary significantly. Such modulation appears to contribute to the inhibitory effects of this gas on cell proliferation. By contrast, H2S is selective in its effects: at low (presumed physiological) levels it selectively inhibits Cav3.2, while Cav3.1 and 3.3 are unaffected. Similarly to redox modulation, the effect of H2S depended on the presence of the metal binding site. Thus, mutation of H191 to glutamine (H191Q) abolished H2S sensitivity. Furthermore, the analogous

T-Type Ca2+ Channel Regulation by CO: A Mechanism for Control of Cell Proliferation

T-type Ca(2+) channels regulate proliferation in a number of tissue types, including vascular smooth muscle and various cancers. In such tissues, up-regulation of the inducible enzyme heme oxygenase-1 (HO-1) is often observed, and hypoxia is a key factor in its induction. HO-1 degrades heme to generate carbon monoxide (CO) along with Fe(2+) and biliverdin. Since CO is increasingly recognized as a regulator of ion channels (Peers et al. 2015), we have explored the possibility that it may regulate proliferation via modulation of T-type Ca(2+) channels.Whole-cell patch-clamp recordings revealed that CO (applied as the dissolved gas or via CORM donors) inhibited all 3 isoforms of T-type Ca(2+) channels (Cav3.1-3.3) when expressed in HEK293 cells with similar IC(50) values, and induction of HO-1 expression also suppressed T-type currents (Boycott et al. 2013). CO/HO-1 induction also suppressed the elevated basal [Ca(2+) ](i) in cells expressing these channels and reduced their proliferative rate to levels seen in non-transfected control cells (Duckles et al. 2015).Proliferation of vascular smooth muscle cells (both A7r5 and human saphenous vein cells) was also suppressed either by T-type Ca(2+) channel inhibitors (mibefradil and NNC 55-0396), HO-1 induction or application of CO. Effects of these blockers and CO were non additive. Although L-type Ca(2+) channels were also sensitive to CO (Scragg et al. 2008), they did not influence proliferation. Our data suggest that HO-1 acts to control proliferation via CO modulation of T-type Ca(2+) channels.