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. Author manuscript; available in PMC: 2012 Jan 21.
Published in final edited form as: FEBS Lett. 2010 Dec 13;585(2):328–334. doi: 10.1016/j.febslet.2010.12.008

Biphasic Effect of Nitric Oxide on the Cardiac Voltage-dependent Anion Channel

Qunli Cheng a, Filip Sedlic a,b, Danijel Pravdic a, Zeljko J Bosnjak a,b, Wai-Meng Kwok a,c
PMCID: PMC3035949  NIHMSID: NIHMS258424  PMID: 21156174

Abstract

Nitric oxide (NO˙) effects on the cardiac mitochondrial voltage-dependent anion channel (VDAC) are unknown. The effects of exogenous NO˙ on VDAC purified from rat hearts were investigated in this study. When incorporated into lipid bilayers, VDAC was inhibited directly by an NO˙ donor, PAPA NONOate, in a concentration-dependent biphasic manner. This was prevented by an NO˙ scavenger, PTIO. The effect paralleled that of NO˙ in delaying the opening of the mitochondrial permeability transition (PT) pore. These biphasic effects on the cardiac VDAC and the PT pore reveal a tandem impact of NO˙ on the two mitochondrial entities.

Keywords: Voltage-dependent Anion Channel (VDAC), Nitric Oxide, Heart, Cardioprotection, Mitochondria

1. Introduction

Nitric oxide (NO˙) has been shown to play a complex role in cardioprotection against ischemia-reperfusion injury.[1] Exogenous NO˙ have been shown to mediate cardioprotection in a dose-dependent manner, where protection is abrogated at high levels of NO˙.[2] The cardioprotection afforded by NO˙ is partially attributed to its direct and indirect effects on preserving mitochondrial function. NO˙ prevents the opening of the mitochondrial permeability transition (PT) pore, thus averting a key end-stage event that leads to apoptotic and necrotic cell death.[3,4] However, the exact nature of the interaction of NO˙ with the PT pore is unknown. This is in large part due to the lack of the molecular identity of the PT pore complex.[5] NO˙ also targets the complexes of the electron transport chain and can activate the mitochondrial ATP-sensitive potassium channel.[6,7] Thus, how these multiple actions of NO˙ on the mitochondria work in concert to ultimately lead to cardioprotection is yet to be elucidated.

Other mitochondrial proteins may also be targeted by NO˙. The voltage-dependent anion channel (VDAC) is the major conduit that allows for the diffusional exchange of nucleotides and metabolites across the outer mitochondrial membrane.[8] Although recent studies have provided evidence that excluded VDAC as a component of the PT pore as previously thought, VDAC plays a critical role in the maintenance of mitochondrial function, and ultimately to preservation of cellular function.[8] Yet, its sensitivity to NO˙ has not been established. Specifically, its effect on the cardiac VDAC is unknown. The main objective of this study was to determine the direct effect of exogenous NO˙ on the cardiac VDAC. The VDAC protein was purified from rat hearts and incorporated into planar lipid bilayer to monitor its activity at the single channel level. In addition, the effects of NO˙ on the opening of the cardiac mitochondrial PT pore were also investigated and compared to those on VDAC to determine whether NO˙ had parallel effects on these two mitochondrial entities.

2. Methods

2.1. Purification of cardiac VDAC

Upon approval by the Institutional Animal Care and Use Committee (IACUC), mitochondria were isolated from hearts of male Wistar rats (250–300 g) as previously described.[7] VDAC was purified from the isolated mitochondria following a previously reported procedure with modifications.[9] Mitochondria were incubated for 30 min at 4 °C with agitation in a solution containing 10 mM Tris, 0.15 mM PMSF, 0.04 mM leupeptin hydrochloride, and 3% Triton X-100 with pH 7.0. Following centrifugation at 44,000 g for 30 min at 4 °C, the supernatant was loaded onto a dry hydroxyapatite/celite (2:1 wt/wt) column (0.1 g/mg protein) and eluted with a buffer containing 10 mM Tris, and 3% Triton X-100 with pH 6.8. Fractions of the VDAC-containing eluent were collected, diluted three-fold with 10 mM Tris, pH 7.4, and loaded onto a reactive red-agarose column which was pre-equilibrated with 10 mM Tris, pH 7.4, and 0.4% Nonidet P-40. VDAC was eluted with the same buffer but with the addition of 0.3 M NaCl. Following separation by SDS-PAGE (4–20%: Bio-Rad Laboratories, Hercules, CA) the bands were silver stained (Silver Stain Kit, Sigma-Aldrich, St. Louis, MO). Western blot analysis for VDAC followed standard procedure with a polyclonal VDAC antibody (EMD Chemical, Gibbstown, NJ).

2.2. Reconstitution of VDAC into planar lipid bilayers

Phospholipids were prepared by mixing phosphatidylethanolamine, phosphatidylserine and phosphatidylcholine (Avanti Polar Lipids, Alabaster, AL) in a ratio of 5:4:1 (v/v), dried under nitrogen, and resuspended in n-decane for a final concentration of 25 mg/ml. The cis/trans chambers contained symmetrical solutions of 10 mM HEPES, 500 mM NaCl and 1 mM CaCl2 with pH 7.4. The cis chamber was held at virtual ground and the trans chamber was held at the command voltages. The VDAC protein and mediators were added into the cis chamber. Currents were digitized at 5 kHz and low pass filtered at 1 kHz using a voltage clamp amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, CA) via a digitizer (DigiData 1332, Molecular Devices), and recorded in 2-min segments. The pClamp software (version 9, Molecular Devices) was used for data acquisition and analysis. Additional analysis was conducted using Origin 7.0 (OriginLab, Northampton, MA). PAPA NONOate (PPN) was made fresh in 10 mM NaOH and diluted to the desired concentrations in the recording buffer. The levels of NO˙ generated by the different concentrations of PPN were determined using an NO˙-sensitive electrode connected to a free radical analyzer (APOLLO 4000, World Precision Instruments, Sarasota, FL). An NO˙ scavenger, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; EMD Biosciences, San Diego, CA) was prepared in cold deionized water. The final concentration of PTIO in buffer was 25 µM. Koenig’s polyanion, a VDAC antagonist was kindly provided by Dr. Marco Colombini (University of Maryland).

2.3. Measurement of mitochondrial PT pore opening

Opening of the PT pore was measured as described previously.[10,11] Following approval by the IACUC, cardiomyocytes were enzymatically isolated from the rat hearts as previously described[12] and loaded with the fluorescent indicator tetramethylrhodamine ethyl ester (TMRE, 100 nM; Invitrogen, Carlsbad, CA) for 25 min at room temperature. After dye loading, selected regions of myocyte (50×50 µm) were consecutively illuminated with a HeNe laser at 543 nm, thereby generating oxidative stress that caused PT pore opening. The emitted fluorescence was collected at 590 nm. Cells were imaged using a confocal microscope (Eclipse TE2000-U; Nikon, Tokyo, Japan) with a 60x/1.4 oil-immersion objective. The obtained images were analyzed with MetaMorph 6.2 software (Universal Imaging, West Chester, PA). The PT pore opening time was determined as the time when TMRE fluorescence intensity decreased by 50% between the initial and residual values after PT pore opening. This correlated with the mean PT pore opening time of approximately 50% of mitochondria in the recorded region.[10] To test the effect NO˙ on PT pore opening, cells were loaded for 20 min with NO˙ donors, either PPN or S-nitroso-N-acetylpenicillamine (SNAP; Tocris Bioscience, Ellisville, MO). PPN was prepared as described above and SNAP was prepared in dimethyl sulfoxide and diluted to the desired concentrations. All the experiments were conducted at room temperature.

2.4 Statistical analysis

Data are shown as mean ± SEM. One-way ANOVA followed by Scheffe’s test was used to compare the data among groups. P < 0.05 was considered significantly different.

3. Results

3.1. Purification and identification of VDAC

The enrichment of the VDAC protein from isolated heart mitochondria is depicted in Fig. 1A. The silver staining gel shows the mitochondria and VDAC-containing fractions. Western blot analysis confirmed the identity of the VDAC (molecular weight of 31 kDa) in the VDAC-containing fractions. To further confirm the identity of the purified protein, VDAC was incorporated into a planar lipid bilayer. A key feature of VDAC is its voltage-dependence whereby the greatest conductance is observed at membrane voltages near 0 mV. This is demonstrated in Fig. 1B where VDAC activity was recorded using a voltage ramp protocol from −80 to + 80 mV with a 0 mV holding potential. As shown, the peak conductance of 1.8 nS occurred at voltages between −40 and +60 mV. The normalized conductance at the various membrane potentials are summarized in Fig. 1C depicting the voltage-dependent characteristic of VDAC. Furthermore, the reconstituted VDAC was inhibited by Koenig’s polyanion, its putative inhibitor (Fig. 1D). In the example shown, the application of the polyanion resulted in a decrease in the channel’s chord conductance from 1.7 nS to 700 pS.

Figure 1.

Figure 1

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Purification and functional identification of VDAC. VDAC was purified from mitochondria isolated from rat heart. Panel A: VDAC bands are shown on a silver-staining gel and Western blot. Std=kD standards; Mito=mitochondrial sample; columns 1,2=VDAC-containing fractions eluted from an active red-agarose column. Panel B: Channel current recorded during a linear voltage-ramp protocol from −80 to +80 mV from a holding potential of 0 mV is shown. Panel C: Normalized conductances were obtained at the various membrane potentials. Conductance was determined from the peak current amplitude recorded at the indicated membrane potentials (n=6 recordings). Panel D: Sample VDAC traces and the corresponding all-points amplitude histograms are shown in Control and in the presence of 2 µM Koenig’s polyanion, a VDAC antagonist. Recordings were obtained at a membrane potential of −10 mV. The dashed lines indicate zero current levels.

3.2. Effect of NO˙ on VDAC

The effect of NO˙ on the reconstituted VDAC was investigated using PPN. This compound was chosen due to its stability at room temperature. Each electrophysiological recording was completed within 30 minutes. A representative channel activity is depicted in Fig 2A. As shown, at a concentration of 25 µM, PPN inhibited VDAC activity and resulted in lower conductance states. The cumulative amplitude histogram from n=10 separate recordings revealed multiple low conductance states in the presence of PPN. At a higher concentration of 50 μM, the effects of PPN on VDAC appeared to be less than those of 25 μM, as shown in Fig 2B. In particular, fewer low conductance states were revealed. The result toward lower conductances in the presence of PPN is also demonstrated in Fig. 2C where VDAC activity was recorded in response to a voltage ramp protocol from three separate recordings. In the absence of PPN, the channel exhibited the characteristic voltage-dependence with the high conductance open state of 1.8 nS predominant at voltages between −60 and +60 mV. Upon the addition of 25 μM PPN, the predominant conductance through the voltage range of −80 to +80 mV was a low conductance closed state of 0.6 nS. To rule out time-dependent effects, a time course protocol was implemented to monitor VDAC activity over a 30-minute time period under control conditions, in the absence of PPN. As shown in Fig. 3, the cumulative amplitude histogram (n=5) at three different time points demonstrated the stability of the VDAC recordings. Thus, the effects of PPN were not due to channel “run-down”.

Figure 2.

Figure 2

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Effect of exogenous NO˙ on VDAC. Panel A: Sample VDAC current traces are shown in Control and in the presence of 25 μM PAPA NONOate (PPN). Recordings were obtained at a membrane potential of −10 mV. The dashed lines indicate zero current levels. The amplitude histogram is cumulative from n=10 separate recordings. Panel B: Sample VDAC recording is shown in the presence of 50 μM PPN. The amplitude histogram is cumulative from n=5 separate recordings. Panel C: VDAC current traces were recorded in Control and in the presence of 25 μM PPN in response to a voltage ramp protocol from −80 to +80 mV. The holding potential was set at 0 mV. Recordings from n=3/group are superimposed. In the presence of PPN, the dominant VDAC state was the low conductance “closed” state.

Figure 3.

Figure 3

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Stability of VDAC activity in planar bilayers. VDAC recordings were monitored over a 30-minute period to rule out time-dependent changes. The sequential amplitude histograms are cumulative from n=5 separate recordings. At the onset of each time point depicted, VDAC activity was acquired during a 2-minute recording duration.

The summary of the effects of NO˙ on VDAC is shown in Fig. 4A. Due to multiple conductance states, VDAC activity was determined as the mean current during multiple recording sets of 2-min durations. The effects of NO˙ on VDAC were determined after steady-state conditions were reached. The mean time to steady-state inhibition of VDAC by NO˙ was 16±2 minutes (n=9). NO˙ was found to have concentration-dependent and biphasic inhibitory effects on VDAC. At 25 μM where the peak inhibition was observed, PPN inhibited VDAC by approximately 40%. The inhibitory effects were attenuated at higher concentrations. The corresponding levels of NO˙ generated by PPN were also determined. NO˙ levels generated by PPN were stable under our room temperature conditions for at least 30 minutes (Fig. 4B). Based on our measurements, the biphasic inhibitory effect occurred within the range of 120 – 900 nM of NO˙. The PPN of 25 μM, where the peak inhibition of VDAC occurred, corresponded to an NO˙ level of approximately 350 nM. This was within the physiological range of NO˙ levels that have been demonstrated to be anti-apoptotic.[3]

Figure 4.

Figure 4

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Concentration-dependent effects of NO˙ on VDAC. Panel A: The concentration-response curve for various concentrations of PPN on the VDAC mean current amplitude is shown (n=5–10/group). Panel B: The steady-state levels of NO˙ generated by the various concentrations of PPN are shown. NO˙ levels were measured using the APOLLO 4000 free radical analyzer (World Precision Instruments; n=3/group). Panel C: Summary of the effects of 25 μM PPN in the absence and presence of PTIO is shown. Vehicle was composed of the base solution of 10 mM Na-OH diluted 1:200 to mimic the solution the PPN was prepared in. *denotes significantly different from the Vehicle and PTIO+PPN groups.

To further confirm the effect of PPN, experiments were conducted to test whether an NO˙ scavenger would prevent the inhibition on VDAC. PTIO was added simultaneously with PPN to the recording buffer solution. In the presence of PTIO, PPN at 25 μM was unable to inhibit VDAC activity (Fig. 4C). Consequently, this supported the observation that the inhibitory effect of PPN on VDAC was due to the generated NO˙.

3.3. Effect of NO˙ on the cardiac mitochondrial PT pore

NO˙ has been reported to inhibit the opening of the PT pore of mitochondria isolated from liver in a concentration-dependent manner.[3] Low levels of NO˙ inhibited PT pore opening, while higher levels accelerated its opening. Therefore, we investigated whether there was a similar effect of NO˙ on the PT pore opening in isolated cardiomyocytes and whether this paralleled the effects of NO˙ on VDAC observed above. Changes in mitochondrial membrane potential (ΔΨm) as a function of TMRE fluorescence were used as an indicator of PT pore opening. Two commonly used NO˙ donors, SNAP and PPN, were used for these experiments, and the experimental protocol is shown in Fig. 5A. As depicted in a representative recording in Fig. 5B, SNAP triggered a concentration-dependent delay in the opening of the cardiac PT pore. At the lowest concentration of 0.1 mM, SNAP triggered a delay in the PT pore opening compared to the control condition in the absence of the NO˙ donor. This was not evident at the higher SNAP concentrations. The results are summarized in Fig. 5C. Experiments with PPN showed a similar trend, with a delay in PT pore opening at a concentration of 25 µM and no significant delay at 100 µM. Based on these results, NO˙ triggered a significant delay in the opening of the cardiac mitochondrial PT pore only at the low concentrations of NO˙ donors. These effects of NO˙ paralleled those on VDAC.

Figure 5.

Figure 5

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Effect of exogenous NO˙ on the cardiac PT pore. Panel A: Schematic of the experimental protocol used for measuring PT pore opening. TMRE denotes the loading time of the fluorescent dye and Tyrode denotes the incubation of the cardiomyocytes in this solution. The arrow indicates the time at which the PT pore opening measurements were initiated. Panel B: Changes in TMRE fluorescence as a function of time in the presence of different concentrations of SNAP are shown. Opening of the PT pore was correlated with the abrupt decrease in the fluorescent signal. Panel C: Summary of the effects of SNAP on time to PT pore opening is shown. Panel D: Summary of PT pore opening times recorded in cardiomyocytes in control and following pretreatment with 25 and 100 µM PPN. *denotes significantly different from Control (n=7–11/group in Panel C and 8/group in Panel D).

4. Discussion

The results from this study provided functional evidence of the novel ability of exogenous NO˙ to inhibit the cardiac VDAC in a concentration-dependent biphasic manner. This effect was direct and independent of guanylate cyclase since no cytosolic factors were present. The range of NO˙ levels where the inhibition of VDAC was observed was within the physiological range associated with the anti-apoptotic effects of NO˙. The inhibition occurred within a limited range of NO˙ levels, 120 – 900 nM, suggesting a rather steep sensitivity of VDAC to NO˙. A similar concentration-dependent biphasic effect of NO˙ was also observed in delaying mitochondrial PT pore opening. The NO˙-induced delay in PT pore opening paralleled the effect of NO˙ in triggering the transition of VDAC to the “closed” state, implicative of a synergistic effect. Furthermore, at higher concentrations, the effects of NO˙ on both VDAC and the PT pore were abolished.

Recent studies have brought into question the role of the VDAC as being part of the PT pore.[5] Genetic knock-out studies showed that PT pore opening can occur in the absence of VDAC.[13] Nevertheless, VDAC is essential for mitochondrial function and cell survival as it is the main gateway for the exchange of ATP, ADP and metabolites across the outer mitochondrial membrane.[8] Previous studies have demonstrated that closure of VDAC by hexokinase prevented opening of the PT pore, and hence apoptosis.[14] However, at the time of that study, VDAC was considered to be part of the PT pore complex. Based on the current view that VDAC is not part of the PT pore complex, our study raises an intriguing possibility that low levels of NO˙ afford cardioprotection by its dual effects on VDAC and the PT pore. While opening of the PT pore, the end-stage mitochondrial event leading to cell death, is delayed by NO˙, it is conceivable, although speculative at this time, the effect of NO˙ on VDAC may play a role in preserving mitochondrial function, and, consequently, contributing as an upstream modulator in delaying the PT pore’s opening. Furthermore, our observation that high concentrations of NO˙ diminished both the inhibitory effects on VDAC activity and delay in the PT pore opening support the duality of NO˙ on cardiac function whereby low concentrations are cardioprotective, while high concentrations are cardiotoxic. Thus, our results show that the tandem effects of NO˙ on both VDAC and the PT pore can potentially function synergistically.

The physiological consequence of the multiple low conductance states in the presence of PPN observed in the present study is yet to be determined. In fact, whether the “open” high conductance or the “closed” low conductance states of VDAC contributes to cardioprotection is currently unresolved. A complex biophysical property of VDAC is its ability to conduct negatively charged nucleotides and metabolites at the high conductance “open” state and cations such as calcium at the low conductance “closed” state.[15] Phosphorylation of VDAC induces the low conductance “closed” state, and discrepancies abound as to whether phosphorylation or dephosphorylation of VDAC is cardioprotective. For example, VDAC has been identified as one of the phosphorylation targets of PKC-ε translocation to the OMM triggered by preconditioning.[16] Yet, a significant reduction in the ischemia-induced phosphorylation of VDAC was found to underlie the cardioprotective mechanism of an inhibitor of p38 mitogen-activated protein kinase.[17] Consequently, these studies suggest that VDAC is highly modulated and that this modulation is a crucial component of cardioprotection. The biphasic effect of NO˙ on VDAC likely contributes to this modulation.

VDAC has been reported to be a binding partner of eNOS, and this interaction was shown to increase eNOS activity.[18] In our study, because purified VDAC was incorporated into an artificial bilayer, the confounding impact of eNOS was absent. On the other hand, the close interaction of eNOS with VDAC coupled with the direct effect of NO˙ on VDAC suggests a highly regulated and efficient modulation of the channel protein. However, the mechanism of the biphasic NO˙ effect on VDAC has yet to be determined. Based on the sequence and structure of VDAC, there are two cysteine residues that may potentially be sites for S-nitrosylation.[19] Modification by S-nitrosylation is associated with protein regulation and has been shown to be involved in apoptosis.[20,21] The correlation between S-nitrosylation and the biphasic inhibition of VDAC requires further studies. Given the complex biophysical characteristic of VDAC, other modulators of the channel may also impact its regulation by NO˙.

We have previously reported on the activating effect of NO˙ the mitochondrial ATP-sensitive potassium channel.[7] Thus, NO˙ may be an important modulator of mitochondrial ion channels. However, what the cumulative effects of NO˙ on these ion channels in modulating mitochondrial function are, and the exact roles of these channels have in contributing to cardioprotection are yet to be determined.

5. Conclusions

In summary, our results showed that NO˙ directly inhibits the cardiac mitochondrial VDAC in a biphasic manner. This inhibition paralleled the delay in the opening of the cardiac PT pore by NO˙. This tandem, synergistic effect on both the VDAC and PT pore may potentially contribute to the cardioprotective effects of low levels of NO˙.

Research Highlight.

  • Nitric oxide (NO˙) inhibited the cardiac voltage-dependent anion channel (VDAC) in a concentration-dependent biphasic manner.

  • NO˙ also delayed the opening of the mitochondrial permeability transition (PT) pore in a similar biphasic manner within the same concentration range.

  • These effects on the cardiac VDAC and PT pore reveal a tandem impact of NO˙ on the two mitochondrial entities.

  • The cardioprotective effects of low concentrations of NO˙ may be due to a synergistic effect on the VDAC and PT pore.

Acknowledgements

The authors thank Dr. Martin Bienengraeber at the Medical College of Wisconsin for helpful scientific discussions. Supported by P01 GM066730 to Z.J.B.

List of abbreviations

NO˙

nitric oxide

VDAC

voltage-dependent anion channel

PPN

PAPA NONOate

PTIO

2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide

SNAP

S-nitroso-N-acetylpenicillamine

PT pore

mitochondrial permeability transition pore

Footnotes

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