Delineating an extracellular redox-sensitive module in T-type Ca2+ channels.

T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing a high-affinity metal-binding site formed by the extracellular IS1-IS2 and IS3-IS4 loops of domain I and a cluster of extracellular cysteines in the IS1-IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide, as well as a reactive oxygen species-producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1-IS2 loop of Cav3.2 reduced its sensitivity to MTSES and SP. We hypothesize that oxidative modification of IS1-IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2 so that it becomes sensitive to ambient zinc.

Intracellular zinc activates KCNQ channels by reducing their dependence on phosphatidylinositol 4,5-bisphosphate.

M-type (Kv7, KCNQ) potassium channels are proteins that control the excitability of neurons and muscle cells. Many physiological and pathological mechanisms of excitation operate via the suppression of M channel activity or expression. Conversely, pharmacological augmentation of M channel activity is a recognized strategy for the treatment of hyperexcitability disorders such as pain and epilepsy. However, physiological mechanisms resulting in M channel potentiation are rare. Here we report that intracellular free zinc directly and reversibly augments the activity of recombinant and native M channels. This effect is mechanistically distinct from the known redox-dependent KCNQ channel potentiation. Interestingly, the effect of zinc cannot be attributed to a single histidine- or cysteine-containing zinc-binding site within KCNQ channels. Instead, zinc dramatically reduces KCNQ channel dependence on its obligatory physiological activator, phosphatidylinositol 4,5-bisphosphate (PIP2). We hypothesize that zinc facilitates interactions of the lipid-facing interface of a KCNQ protein with the inner leaflet of the plasma membrane in a way similar to that promoted by PIP2 Because zinc is increasingly recognized as a ubiquitous intracellular second messenger, this discovery might represent a hitherto unknown native pathway of M channel modulation and provide a fresh strategy for the design of M channel activators for therapeutic purposes.

Coupling of Smoothened to inhibitory G proteins reduces voltage-gated K+ currents in cardiomyocytes and prolongs cardiac action potential duration.

SMO (Smoothened), the central transducer of Hedgehog signaling, is coupled to heterotrimeric Gi proteins in many cell types, including cardiomyocytes. In this study, we report that activation of SMO with SHH (Sonic Hedgehog) or a small agonist, purmorphamine, rapidly causes a prolongation of the action potential duration that is sensitive to a SMO inhibitor. In contrast, neither of the SMO agonists prolonged the action potential in cardiomyocytes from transgenic GiCT/TTA mice, in which Gi signaling is impaired, suggesting that the effect of SMO is mediated by Gi proteins. Investigation of the mechanism underlying the change in action potential kinetics revealed that activation of SMO selectively reduces outward voltage-gated K+ repolarizing (Kv) currents in isolated cardiomyocytes and that it induces a down-regulation of membrane levels of Kv4.3 in cardiomyocytes and intact hearts from WT but not from GiCT/TTA mice. Moreover, perfusion of intact hearts with Shh or purmorphamine increased the ventricular repolarization time (QT interval) and induced ventricular arrhythmias. Our data constitute the first report that acute, noncanonical Hh signaling mediated by Gi proteins regulates K+ currents density in cardiomyocytes and sensitizes the heart to the development of ventricular arrhythmias.

A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide-induced proarrhythmic early afterdepolarizations.

Exposure to CO causes early afterdepolarization arrhythmias. Previous studies in rats have indicated that arrhythmias arose as a result of augmentation of the late Na+ current. The purpose of the present study was to examine the basis for CO-induced arrhythmias in guinea pig myocytes in which action potentials (APs) more closely resemble those of human myocytes. Whole-cell current- and voltage-clamp recordings were made from isolated guinea pig myocytes as well as from human embryonic kidney 293 (HEK293) cells that express wild-type or a C723S mutant form of ether-a-go-go-related gene (ERG; Kv11.1). We also monitored the formation of peroxynitrite (ONOO-) in HEK293 cells fluorimetrically. CO-applied as the CO-releasing molecule, CORM-2-prolonged the APs and induced early afterdepolarizations in guinea pig myocytes. In HEK293 cells, CO inhibited wild-type, but not C723S mutant, Kv11.1 K+ currents. Inhibition was prevented by an antioxidant, mitochondrial inhibitors, or inhibition of NO formation. CO also raised ONOO- levels, an effect that was reversed by the ONOO- scavenger, FeTPPS [5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato-iron(III)], which also prevented the CO inhibition of Kv11.1 currents and abolished the effects of CO on Kv11.1 tail currents and APs in guinea pig myocytes. Our data suggest that CO induces arrhythmias in guinea pig cardiac myocytes via the ONOO--mediated inhibition of Kv11.1 K+ channels.-Al-Owais, M. M., Hettiarachchi, N. T., Kirton, H. M., Hardy, M. E., Boyle, J. P., Scragg, J. L., Steele, D. S., Peers, C. A key role for peroxynitrite-mediated inhibition of cardiac ERG (Kv11.1) K+ channels in carbon monoxide-induced proarrhythmic early afterdepolarizations.

Astrocytic transporters in Alzheimer's disease.

Astrocytes play a fundamental role in maintaining the health and function of the central nervous system. Increasing evidence indicates that astrocytes undergo both cellular and molecular changes at an early stage in neurological diseases, including Alzheimer's disease (AD). These changes may reflect a change from a neuroprotective to a neurotoxic phenotype. Given the lack of current disease-modifying therapies for AD, astrocytes have become an interesting and viable target for therapeutic intervention. The astrocyte transport system covers a diverse array of proteins involved in metabolic support, neurotransmission and synaptic architecture. Therefore, specific targeting of individual transporter families has the potential to suppress neurodegeneration, a characteristic hallmark of AD. A small number of the 400 transporter superfamilies are expressed in astrocytes, with evidence highlighting a fraction of these are implicated in AD. Here, we review the current evidence for six astrocytic transporter subfamilies involved in AD, as reported in both animal and human studies. This review confirms that astrocytes are indeed a viable target, highlights the complexities of studying astrocytes and provides future directives to exploit the potential of astrocytes in tackling AD.

Modulation of Potassium Channels Inhibits Bunyavirus Infection.

Bunyaviruses are considered to be emerging pathogens facilitated by the segmented nature of their genome that allows reassortment between different species to generate novel viruses with altered pathogenicity. Bunyaviruses are transmitted via a diverse range of arthropod vectors, as well as rodents, and have established a global disease range with massive importance in healthcare, animal welfare, and economics. There are no vaccines or anti-viral therapies available to treat human bunyavirus infections and so development of new anti-viral strategies is urgently required. Bunyamwera virus (BUNV; genus Orthobunyavirus) is the model bunyavirus, sharing aspects of its molecular and cellular biology with all Bunyaviridae family members. Here, we show for the first time that BUNV activates and requires cellular potassium (K(+)) channels to infect cells. Time of addition assays using K(+) channel modulating agents demonstrated that K(+) channel function is critical to events shortly after virus entry but prior to viral RNA synthesis/replication. A similar K(+) channel dependence was identified for other bunyaviruses namely Schmallenberg virus (Orthobunyavirus) as well as the more distantly related Hazara virus (Nairovirus). Using a rational pharmacological screening regimen, two-pore domain K(+) channels (K2P) were identified as the K(+) channel family mediating BUNV K(+) channel dependence. As several K2P channel modulators are currently in clinical use, our work suggests they may represent a new and safe drug class for the treatment of potentially lethal bunyavirus disease.

Oxygen-dependent hydroxylation by FIH regulates the TRPV3 ion channel.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels.

Regulation of the T-type Ca(2+) channel Cav3.2 by hydrogen sulfide: emerging controversies concerning the role of H2 S 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 H2 S is a modulator of low voltage-activated T-type Ca(2+) channels, and discriminates between the different subtypes of T-type Ca(2+) channel in that it selectively modulates Cav3.2, whilst Cav3.1 and Cav3.3 are unaffected. At high concentrations, H2 S augments Cav3.2 currents, an observation which has led to the suggestion that H2 S exerts its pro-nociceptive effects via this channel, since Cav3.2 plays a central role in sensory nerve excitability. However, at more physiological concentrations, H2 S 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 H2 S 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 H2 S modulation of T-type Ca(2+) 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 Ca(2+) channel modulation by H2 S might be exploited as a novel approach to pain management.

Resistance of Dynamin-related Protein 1 Oligomers to Disassembly Impairs Mitophagy, Resulting in Myocardial Inflammation and Heart Failure.

We have reported previously that a missense mutation in the mitochondrial fission gene Dynamin-related protein 1 (Drp1) underlies the Python mouse model of monogenic dilated cardiomyopathy. The aim of this study was to investigate the consequences of the C452F mutation on Drp1 protein function and to define the cellular sequelae leading to heart failure in the Python monogenic dilated cardiomyopathy model. We found that the C452F mutation increased Drp1 GTPase activity. The mutation also conferred resistance to oligomer disassembly by guanine nucleotides and high ionic strength solutions. In a mouse embryonic fibroblast model, Drp1 C452F cells exhibited abnormal mitochondrial morphology and defective mitophagy. Mitochondria in C452F mouse embryonic fibroblasts were depolarized and had reduced calcium uptake with impaired ATP production by oxidative phosphorylation. In the Python heart, we found a corresponding progressive decline in oxidative phosphorylation with age and activation of sterile inflammation. As a corollary, enhancing autophagy by exposure to a prolonged low-protein diet improved cardiac function in Python mice. In conclusion, failure of Drp1 disassembly impairs mitophagy, leading to a downstream cascade of mitochondrial depolarization, aberrant calcium handling, impaired ATP synthesis, and activation of sterile myocardial inflammation, resulting in heart failure.

Hepatitis C virus attenuates mitochondrial lipid ß-oxidation by downregulating mitochondrial trifunctional-protein expression.

UNLABELLED: The course of hepatitis C virus (HCV) infection and disease progression involves alterations in lipid metabolism, leading to symptoms such as hypocholesterolemia and steatosis. Steatosis can be induced by multiple mechanisms, including increases in lipid biosynthesis and uptake, impaired lipoprotein secretion, and/or attenuation of lipid ß-oxidation. However, little is known about the effects of HCV on lipid ß-oxidation. A previous proteomics study revealed that HCV interacted with both the α- and ß-subunits of the mitochondrial trifunctional protein (MTP), an enzyme complex which catalyzes the last 3 steps of mitochondrial lipid ß-oxidation for cellular energy production. Here we show that in HCV-infected Huh7.5 cells, lipid ß-oxidation was significantly attenuated. Consistently with this, MTP protein and mRNA levels were suppressed by HCV infection. A loss-of-function study showed that MTP depletion rendered cells less responsive to alpha interferon (IFN-α) treatment by impairing IFN-stimulated gene expression. These aspects of host-virus interaction explain how HCV alters host energy homeostasis and how it may also contribute to the establishment of persistent infection in the liver. IMPORTANCE: HCV infection triggers metabolic alterations, which lead to significant disease outcomes, such as fatty liver (steatosis). This study revealed that HCV impairs mitochondrial lipid ß-oxidation, which results in low lipid combustion. On the other hand, the HCV-induced defects in metabolic status played an important role in the control of the type I interferon system. Under the conditions of impaired lipid ß-oxidation, host cells were less responsive to the ability of exogenously added IFN-α to suppress HCV replication. This suggests that interference with lipid ß-oxidation may assist the virus in the establishment of a long-term, persistent infection. Further understanding of this aspect of virus-host interaction may lead to improvements in the current standard therapy.

Inhibition of the cardiac Na⁺ channel Nav1.5 by carbon monoxide.

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 N(ω)-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.

Gasotransmitter regulation of ion channels: a key step in O2 sensing by the carotid body.

Carotid bodies detect hypoxia in arterial blood, translating this stimulus into physiological responses via the CNS. It is long established that ion channels are critical to this process. More recent evidence indicates that gasotransmitters exert powerful influences on O2 sensing by the carotid body. Here, we review current understanding of hypoxia-dependent production of gasotransmitters, how they regulate ion channels in the carotid body, and how this impacts carotid body function.

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 Ca(2+) channels in HEK293 cells raised basal [Ca(2+)]i and increased proliferation as compared with non-transfected cells. Proliferation and [Ca(2+)]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) Ca(2+) 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 Ca(2+) channels. This signalling pathway provides a novel means by which proliferation of VSMCs (and other cells) may be regulated therapeutically.

GABAB receptors inhibit low-voltage activated and high-voltage activated Ca(2+) channels in sensory neurons via distinct mechanisms.

Growing evidence suggests that mammalian peripheral somatosensory neurons express functional receptors for gamma-aminobutyric acid, GABAA and GABAB. Moreover, local release of GABA by pain-sensing (nociceptive) nerve fibres has also been suggested. Yet, the functional significance of GABA receptor triggering in nociceptive neurons is not fully understood. Here we used patch-clamp recordings from small-diameter cultured DRG neurons to investigate effects of GABAB receptor agonist baclofen on voltage-gated Ca(2+) currents. We found that baclofen inhibited both low-voltage activated (LVA, T-type) and high-voltage activated (HVA) Ca(2+) currents in a proportion of DRG neurons by 22% and 32% respectively; both effects were sensitive to Gi/o inhibitor pertussis toxin. Inhibitory effect of baclofen on both current types was about twice less efficacious as compared to that of the µ-opioid receptor agonist DAMGO. Surprisingly, only HVA but not LVA current modulation by baclofen was partially prevented by G protein inhibitor GDP-ß-S. In contrast, only LVA but not HVA current modulation was reversed by the application of a reducing agent dithiothreitol (DTT). Inhibition of T-type Ca(2+) current by baclofen and the recovery of such inhibition by DTT were successfully reconstituted in the expression system. Our data suggest that inhibition of LVA current in DRG neurons by baclofen is partially mediated by an unconventional signaling pathway that involves a redox mechanism. These findings reinforce the idea of targeting peripheral GABA receptors for pain relief.

H2S does not regulate proliferation via T-type Ca2+ channels.

T-type Ca(2+) channels (Cav3.1, 3.2 and 3.3) strongly influence proliferation of various cell types, including vascular smooth muscle cells (VSMCs) and certain cancers. We have recently shown that the gasotransmitter carbon monoxide (CO) inhibits T-type Ca(2+) channels and, in so doing, attenuates proliferation of VSMC. We have also shown that the T-type Ca(2+) channel Cav3.2 is selectively inhibited by hydrogen sulfide (H2S) whilst the other channel isoforms (Cav3.1 and Cav3.3) are unaffected. Here, we explored whether inhibition of Cav3.2 by H2S could account for the anti-proliferative effects of this gasotransmitter. H2S suppressed proliferation in HEK293 cells expressing Cav3.2, as predicted by our previous observations. However, H2S was similarly effective in suppressing proliferation in wild type (non-transfected) HEK293 cells and those expressing the H2S insensitive channel, Cav3.1. Further studies demonstrated that T-type Ca(2+) channels in the smooth muscle cell line A7r5 and in human coronary VSMCs strongly influenced proliferation. In both cell types, H2S caused a concentration-dependent inhibition of proliferation, yet by far the dominant T-type Ca(2+) channel isoform was the H2S-insensitive channel, Cav3.1. Our data indicate that inhibition of T-type Ca(2+) channel-mediated proliferation by H2S is independent of the channels' sensitivity to H2S.

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 Ca(2+) 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 Zn(2+) to this channel. Chelation of Zn(2+) 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 Zn(2+) 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 Ca(2+) channel Cav3.2, and suggest that such modulation cannot account for the pronociceptive effects of this gasotransmitter.

Heme Oxygenase-1 Influences Apoptosis via CO-mediated Inhibition of K+ Channels.

Hypoxic/ischemic episodes can trigger oxidative stress-mediated loss of central neurons via apoptosis, and low pO2 is also a feature of the tumor microenvironment, where cancer cells are particularly resistant to apoptosis. In the CNS, ischemic insult increases expression of the CO-generating enzyme heme oxygenase-1 (HO-1), which is commonly constitutively active in cancer cells. It has been proposed that apoptosis can be regulated by the trafficking and activity of K(+) channels, particularly Kv2.1. We have explored the idea that HO-1 may influence apoptosis via regulation of Kv2.1. Overexpression of Kv2.1 in HEK293 cells increased their vulnerability to oxidant-induced apoptosis. CO (applied as the donor CORM-2) protected cells against apoptosis and inhibited Kv2.1 channels. Similarly in hippocampal neurones, CO selectively inhibited Kv2.1 and protected neurones against oxidant-induced apoptosis. In medulloblastoma sections we identified constitutive expression of HO-1 and Kv2.1, and in the medulloblastoma-derived cell line DAOY, hypoxic HO-1 induction or exposure to CO protected cells against apoptosis, and also selectively inhibited Kv2.1 channels expressed in these cells. These studies are consistent with a central role for Kv2.1 in apoptosis in both central neurones and cancer cells. They also suggest that HO-1 expression can strongly influence apoptosis via CO-mediated regulation of Kv2.1 activity.

GAL-021 and GAL-160 are Efficacious in Rat Models of Obstructive and Central Sleep Apnea and Inhibit BKCa in Isolated Rat Carotid Body Glomus Cells.

GAL-021 and GAL-160 are alkylamino triazine analogues, which stimulate ventilation in rodents, non-human primates and (for GAL-021) in humans. To probe the site and mechanism of action of GAL-021 and GAL-160 we utilized spirometry in urethane anesthetized rats subjected to acute bilateral carotid sinus nerve transection (CSNTX) or sham surgery. In addition, using patch clamp electrophysiology we evaluated ionic currents in carotid body glomus cells isolated from neonatal rats. Acute CSNTX markedly attenuated and in some instances abolished the ventilatory stimulant effects of GAL-021 and GAL-160 (0.3 mg/kg IV), suggesting the carotid body is a/the major locus of action. Electrophysiology studies, in isolated Type I cells, established that GAL-021 (30 µM) and GAL-160 (30 µM) inhibited the BK(Ca) current without affecting the delayed rectifier K(+), leak K(+) or inward Ca(2+) currents. At a higher concentration of GAL-160 (100 µM), inhibition of the delayed rectifier K(+) current and leak K(+) current were observed. These data are consistent with the concept that GAL-021 and GAL-160 influence breathing control by acting as peripheral chemoreceptor modulators predominantly by inhibiting BK(Ca) mediated currents in glomus cells of the carotid body.

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.

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.