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

Abstract

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 Ca 2؉ currents and the recombinant ␣ 1C subunit of the human cardiac L-type Ca 2؉ 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 Ca 2؉ 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 channel's cytoplasmic C-terminal tail, resulting in channel inhibition.CO is an established and important signaling molecule in both the heart and vasculature as well as other tissues (1, 2). Cardiac atrial and ventricular myocytes express heme oxygenases HO-1 4 and HO-2, which generate CO along with biliverdin and free Fe 2ϩ by heme catabolism, and HO-1 levels can be increased by various stress factors (3), including myocardial infarction (4). CO limits the cellular damage of ischemia/reperfusion injury in the heart (5). Indeed, greater cardiac damage is seen following ischemia/reperfusion injury in HO-1 knock-out mice (6). Conversely, HO-1 overexpression in the heart reduces infarct size and other markers of damage following ischemia/ reperfusion injury (7). CO also improves cardiac blood supply through coronary vessel dilation (8, 9) and reduces cardiac contractility (9). However, the mechanisms underlying this cardioprotective effect of CO are not understood.In the vasculature, CO also exerts numerous beneficial effects. Its ability to dilate blood vessels is long established (9 -11) and endothelium-independent (12) and not due to development of hypoxia through displacement of O 2 (see Ref. 13). CO has clear, protective effects in various vascular diseases, such as systemic and pulmonary hypertension, development of atherosclerosis, and neointimal hyperplasia due to proliferation of vascular smooth muscle cells following vascular injury (all reviewed in Refs. 2, 13, and 14). Import...

The carbon monoxide donor, CORM-2, is an antagonist of ATP-gated, human P2X4 receptors

“…3d) and found that complex I inhibition was without effect on P2X4 currents or their modulation by CORM-2. Similarly, inhibition of the complex III Q o site, which would be expected to decrease cytosolic ROS [38] and has been shown to block the CORM-2 inhibition of L-type calcium channels [29], had no effect on P2X4 currents or their inhibition by CORM-2. Results obtained with the complex III inhibitor, antimycin A, were intriguing.…”

“…This is the proposed mechanism by which CO and CORM-2 exert an inhibitory effect on L-type calcium channels [29]. Incubation of cells with 100 μM KCN, which would also inhibit complex IV, did not mimic the inhibitory effect of CORM-2, and we found that the inclusion of reducing (DTT) or oxidising (H 2 O 2 ) agents in the patch pipette had no effect on the ability of CORM-2 to inhibit P2X4 currents (Fig.…”

“…Cells were maintained in Dulbecco's modified Eagle's medium/Ham's F12 (1:1) supplemented with 10% foetal calf serum, 2 mM L-glutamine, 100 U mL − 1 penicillin G, 100 μg mL − 1 streptomycin, 250 ng mL −1 amphotericin B and 150 μg mL −1 Geneticin (all purchased from Invitrogen, Paisley, Strathclyde, UK). Cells lacking mitochondria (ρ 0 cells) were generated as previously described by us and others [29,[33][34][35] by growth in media containing ethidium bromide (50 ng mL −1 ), sodium pyruvate (1 mM) and uridine (50 μg mL −1 ) for 2 months prior to experimentation. Cells were incubated at 37°C in a humidified incubator gassed with 5% CO 2 /95% air.…”

“…Previous studies have implicated the generation of ROS from mitochondria in the action of CORM-2 on L-type voltage-gated calcium channels [29]. CO inhibits complex IV, which would lead to an augmentation of superoxide anion generation from complex III.…”

“…Recent experiments have also shown CO to play important roles in cardiovascular function [26] and O 2 sensing [27,28] via an activation of large-conductance, calcium-activated potassium channel (BK Ca ). Other ion channels have also been shown to be modulated by CO such as L-type calcium channels (Ca v 1.2) [29], TREK-1 [30] and ENaC channels [31]. Recently, we observed that short applications (10 s) of CO or a CO donor molecule were able to potentiate ATP-evoked currents at rat P2X2 receptors, whilst causing a small but significant decrease in P2X4 currents [32].…”