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(Circulation Research. 1997;81:742-752.)
© 1997 American Heart Association, Inc.

Articles

Direct Inhibition of Expressed Cardiac L-Type Ca²⁺ Channels by S-Nitrosothiol Nitric Oxide Donors

Hai Hu Nipavan Chiamvimonvat Toshio Yamagishi Eduardo Marban

From the Section of Molecular and Cellular Cardiology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Eduardo Marban, MD, PhD, 844 Ross Bldg, The Johns Hopkins University School of Medicine, Baltimore, MD 21205. E-mail marban{at}welchlink.welch.jhu.edu

	Abstract

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Abstract NO donors have complex effects on Ca²⁺ currents in native cardiac cells, with reports of direct stimulation and indirect cGMP-mediated inhibition or stimulation. To investigate the molecular basis of these effects, we tested the effects of one class of NO donors, S-nitrosothiols (RSNOs), on expressed cardiovascular L-type Ca²⁺ channels (_1C±ß_1a±₂ or _1C±ß_2a±₂) in human embryonic kidney (HEK293) cells. The RSNO compounds we used were S-nitroso-N-acetylpenicillamine (SNAP, 5 to 10 nmol/L or 100 to 800 µmol/L), S-nitrosocysteine (SNC, 100 µmol/L or 1 mmol/L), and S-nitrosoglutathione (GSNO, 1 mmol/L). Currents were measured using whole-cell patch recordings with 2 to 10 mmol/L Ba²⁺ as the charge carrier. SNAP reduced the amplitude of barium currents (I_Ba) through all the subunit combinations, with an EC₅₀ of 360 µmol/L for _1C+ß_1a channels. SNC or GSNO also inhibited I_Ba, albeit less potently. The inhibitory effect of SNAP was not affected by methylene blue (10 to 30 µmol/L) or 8-bromo-cGMP (200 to 400 µmol/L). The effects are relatively specific for Ca²⁺ channels, as expressed cardiac or skeletal muscle Na⁺ channels, which have a similar overall architecture, were barely affected by SNAP at concentrations as high as 1 mmol/L. We conclude that in the HEK293 expression system, the S-nitrosothiol NO donors inhibit L-type Ca²⁺ channels by a mechanism independent of cGMP.

Key Words: nitric oxide • Ca²⁺ channel • Na⁺ channel • cysteine • oxidation

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nitric oxide is an important messenger that regulates various physiological processes in the cardiovascular system. As the major endothelium-derived relaxing factor, it causes vasorelaxation, inhibits platelet aggregation, and reduces blood pressure.^1,2 NO modulates cardiac contractility both in vitro and in vivo.^3,4 The functions of NO in the cardiovascular system appear to be mediated at least in part by modulation of Ca²⁺ channels. In cardiomyocytes, NO usually has no effect on basal I_Ca^5-8 but readily exerts inhibitory^6,8 or biphasic effects on cAMP-stimulated I_Ca.^5,7 In frog ventricular myocytes, the NO donor SIN-1 induced a pronounced stimulation of I_Ca at low concentrations (0.1 to 10 nmol/L), whereas at higher concentrations (100 nmol/L to 1 mmol/L), SIN-1 reduced I_Ca.⁵ The stimulatory effects were explained by the following mechanism: NO activates guanylyl cyclase, resulting in accumulation of intracellular cGMP,^5,7 which in turn suppresses cGMP-inhibited phosphodiesterases and thus elevates cAMP-stimulated I_Ca.^5,9 The inhibition of the current is due to the activation of cGMP-stimulated phosphodiesterases^5,10 or cGMP-dependent protein kinases.^7,11,12 Although in many preparations NO exerted no effect on basal I_Ca, it has been reported that NO stimulates (human atrial myocytes¹³) or dually modulates (ferret ventricular myocytes¹⁴) basal I_Ca. Besides cGMP-dependent indirect mechanisms similar to those described above for stimulation and inhibition of basal I_Ca, Campbell et al¹⁴ postulated an additional direct activation of the Ca²⁺ channel by S-nitrosation.

Given these observations in native cardiac myocytes, we became interested in elucidating the effects of NO on defined molecular components of the Ca²⁺ channel itself. Because of their biological relevance, we chose NO donors from the RSNO class to study. Biological systems use RSNOs in order to overcome the diffusional constraints that would otherwise restrict the redox signaling specificity of NO.¹⁵ We specifically sought to determine whether RSNOs modulate expressed Ca²⁺ channels and, if so, whether it is a direct modulation or an indirect modulation mediated by cGMP.

We expressed various subunit combinations of cardiac and skeletal muscle L-type Ca²⁺ channels (_1C±ß_1a±₂ or _1C±ß_2a±₂) in HEK293 cells and studied the effects of RSNO NO donors on the channels. To probe the mechanism of the modulation of I_Ba, the effects of NO donors were compared and contrasted with the effects of various cysteine-oxidizing reagents. Expressed human cardiac and rat skeletal muscle Na⁺ channels were studied to check the specificity of the NO donor effect. Preliminary results have been published in abstract form.¹⁶

	Materials and Methods

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Functional Expression of Rabbit Cardiac Ca²⁺ Channel _1C Subunits in HEK293 Cells
The transient transfection method of L-type Ca²⁺ channels in HEK293 cells was modified from previous publications.¹⁷ Briefly, HEK293 cells were maintained in DMEM with glucose and L-glutamine, supplemented with 10% fetal calf serum (GIBCO-BRL) and 1% penicillin and streptomycin (GIBCO-BRL). Cells were plated on 35-mm Petri dishes at a density of 0.2 million cells/dish at 1 day before transfection and maintained in a 37°C incubator. Cells were then transfected by calcium phosphate precipitation¹⁸ (Calcium Phosphate Transfection System, GIBCO-BRL) with 2 to 3 µg/dish plasmid DNA encoding Ca²⁺ channel subunits (see below), 0.5 µg/dish simian virus 40 T antigen, and 0.2 µg/dish mitochondrially targeted green fluorescent protein.¹⁹ The calcium phosphate–DNA mixture was left on cells for 5 to 6 hours before washing with PBS and adding fresh media. The admixture of green fluorescent protein cDNA enabled us to identify transfected cells visually by fluorescent excitation.¹⁹

Cells were transfected with plasmid DNA encoding the rabbit cardiac Ca²⁺ channel _1C subunit²⁰ alone or in combination with rabbit skeletal muscle ß_1a subunit²¹ or rat cardiac ß_2a subunit²² with or without rabbit skeletal muscle ₂ subunit.²³ All subunit DNAs were subcloned in the mammalian expression vector pGW1H (British Biotechnology Ltd) and transfected at equimolar ratios. We chose _1C+ß_1a channels as the main object of study to facilitate comparison with previous studies performed using this combination in a variety of expression systems, including Xenopus oocytes, mouse L cells, CHO cells, and HEK293 cells.^17,24-29 To address more physiologically relevant channels, we also characterized the effects of NO donors on _1C+ß_2a channels with or without ₂ subunit coexpression.

Functional Expression of Na⁺ Channels
In the Xenopus oocyte expression system, expression plasmids (pSP64T*) containing either the subunit of the human cardiac tetrodotoxin-resistant Na⁺ channel (hH1) cDNA³⁰ or the subunit of the rat skeletal muscle Na⁺ channel (µ1) cDNA³¹ were used for transcription in vitro. Stage V and VI oocytes were removed from adult female Xenopus laevis (Xenopus 1 or Nasco) and isolated by collagenase treatment (2 mg/mL, type IA, Sigma Chemical Co) in modified Barth's solution (mmol/L: NaCl 88, KCl 1, NaHCO₃ 2.4, Tris 15, CaNO₃·4H₂O 0.3, CaCl₂·6H₂O 0.41, MgSO₄·7H₂O 0.82, sodium pyruvate 5, and theophylline 0.5) supplemented with penicillin (100 U/mL), streptomycin (100 mg/mL), fungizone (250 ng/mL), and gentamicin (50 mg/mL). After digestion, oocytes were maintained in modified Barth's solution. Oocytes were then injected with 50 to 100 nL of RNA and used for electrophysiological recording 1 to 2 days after injection. The hH1 or µ1 -subunit RNA was coinjected with RNA encoding the rat brain ß₁ subunit as described.³²

The µ1 subunit (expression vector pGW1H) was also expressed in the HEK293 cells, in way similar to that described above for the Ca²⁺ channel expression.

Electrophysiology and Data Processing
In the study of Ca²⁺ channel currents, electrophysiological recordings were made 18 to 72 hours after transfection. Membrane current was recorded using the traditional whole-cell configuration,³³ with bath solutions containing (mmol/L) BaCl₂ 2, CsCl 147, HEPES 10 (pH 7.4 titrated with CsOH) or BaCl₂ 10, CsCl 125, and HEPES 10 (pH 7.4 titrated with CsOH), depending on the amplitude of the expressed currents. When performing experiments with _1C alone (which expresses small currents), we routinely used solutions containing 10 mmol/L Ba²⁺; most other experiments were performed with 2 mmol/L Ba²⁺ as the permeant ion.

Pipettes were pulled from borosilicate glass and fire-polished to resistances of 0.5 to 2 M when filled with pipette solutions containing (mmol/L) CsCl 108, MgATP 4.5, EGTA 9, and HEPES 9 (pH 7.4 titrated with CsOH). In the whole-cell configuration, the series resistance was typically 2 to 5 M. In most of the experiments, the series resistance was not compensated; this would have introduced a maximal voltage error of <2 to 5 mV, since the peak current magnitude was generally <1 nA.

A coverslip with cells was placed in a 0.3-mL perfusion chamber connected to a gravity-driven perfusion system. Flow was maintained throughout the experiment at a rate of 0.5 to 3 mL/min. Whole-cell currents were measured >10 minutes after patch rupture to allow for equilibration between intracellular and pipette solutions. Currents were elicited by depolarizing pulses to 0 mV for 25 milliseconds from a holding potential of -80 mV at intervals of 20 to 120 seconds and denoted as I_Ba. When current-voltage relationships were measured, the cell was depolarized to a family of potentials (from -50 to +60 mV) for 25 milliseconds from a holding potential of -80 mV, at intervals of 10 to 20 seconds. Currents were recorded using a patch-clamp amplifier (Axopatch 200, Axon Instruments, Inc) and sampled at 10 kHz after analog filtering at 2 to 5 kHz. To quantify ionic current amplitude, data were leak-subtracted by a P/4 protocol. Acquisition and analysis of the data were performed with custom software.

In some experiments, there was "rundown" of I_Ba, which could be fit with a first-order exponential, A₁+B₁exp(-t/₁). After addition of a drug, the time course of the rundown could change, and the altered time course of I_Ba could in general be fit with another first-order exponential, A₂+B₂exp(-t/₂). In these experiments, we used A₁ and A₂ as the projected stable currents to quantify the steady-state effect of drugs.³⁴ With this technique, the results from cells in which currents exhibited rundown were comparable to those with stable currents, so that all data for any given protocol were pooled for statistical analysis.

In the dose-response study of the effect of NO donors, the data were fit using a sigmoidal function of the following form:

where y is the normalized remaining current, x is the concentration of SNAP, x₅₀ is the half-effective concentration, and n is the Hill coefficient.

For Na⁺ currents expressed in oocytes, the two-microelectrode voltage-clamp technique was used.³⁵ Glass pipettes were filled with 3 mol/L KCl solution (resistance, 1 to 2 M). Currents were recorded in a bath solution containing (mmol/L) NaCl 96, MgCl₂ 1, and HEPES 10 (pH 7.6 titrated with NaOH). Signals were low-pass–filtered at 1 kHz by an eight-pole Bessel filter (Frequency Devices Inc) and digitized on-line at 10 to 20 kHz with 12-bit resolution onto a personal computer.

For Na⁺ currents expressed in HEK293 cells, a method similar to that in the Ca²⁺ channel study was applied. The pipette solution contained (mmol/L) NaCl 35, CsF 105, MgCl₂ 1, HEPES 10, and EGTA 10 (pH 7.2). The bath solution contained (mmol/L) NaCl 140, KCl 5, CaCl₂ 2, MgCl₂ 1, HEPES 10, and glucose 10 (pH 7.4). In all these studies, series resistance was compensated typically to 70% to 90%, and the maximal voltage error was <6 mV for the largest current studied.

All electrophysiological recordings were obtained at room temperature (22°C to 24°C).

RSNO NO Donors and Other Solutions
SNAP powder (Sigma) was dissolved in DMSO (Sigma), forming a 1 mol/L stock solution, and stored in the dark on ice until use. Stock solution of NAP (Sigma), a control for SNAP, was prepared in the same way. Stock solutions of SNC (0.1 mol/L) were synthesized by mixing equal volumes of fresh 0.2 mol/L NaNO₂ (Sigma) and 0.2 mol/L DL-cysteine (Sigma) just before use.^8,14,36 As a control for SNC, DL-cysteine (1 mol/L) was directly dissolved in bath solution. The solution of GSNO (Sigma) was also prepared by directly dissolving the powder in bath solution. During the experiments, all the NO donor solutions were prepared fresh and used within 4 minutes of preparation^36-38; typically, two to five such preparations were used in an individual experiment when the NO donor was applied for 5 to 20 minutes. Thus, the time courses of the changes in ionic currents are not limited by extracellular degradation of the RSNO compounds. Control experiments with NAP or cysteine were performed in a similar way.

Stock solutions (0.1 mol/L) of 8-bromo-cGMP (Sigma) were prepared fresh every day and stored in the dark on ice until use. During each experiment, 8-bromo-cGMP solutions were made just before use by diluting the stock solution, forming a final concentration of 200 to 400 mmol/L. MB (Sigma) stock solutions were kept frozen, thawed the day when used, and diluted to final concentrations of 10 to 30 µmol/L.

Saturating MTS (Toronto Research Chemicals) reagent solutions³⁹ of MTSEA (2.5 mmol/L), MTSES (5 mmol/L), and MTSET (1 mmol/L) were prepared fresh from stock before experiments. Stock solutions of thimerosal ([(O-carboxyphenyl)thio]-ethylmercury sodium salt, Sigma) were stored in the dark on ice until use and were diluted 1000 times in bath solution to form a 10 µmol/L test solution. Stock solutions of DTDP (Sigma) were prepared in DMSO and were also diluted 1000 times in bath solution to form a final solution of 50 µmol/L.

Statistics
Pooled data are presented as mean±SEM. Statistical comparison was evaluated by the two-tailed paired or unpaired Student's t test or by one-way ANOVA, where appropriate, with a value of P<.05 considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
RSNO NO Donor SNAP Inhibits _1C+ß_1a Channels
Fig 1 shows the effects of SNAP, an RSNO NO donor, on I_Ba through _1C+ß_1a channels. Panel C shows the time course of I_Ba (at 0 mV) and the holding current (at -80 mV). Exposure to SNAP inhibited I_Ba, with partial recovery on washout. The holding current remained basically unchanged, suggesting that the cell was otherwise stable throughout the course of the experiment. Panel A shows representative records of I_Ba before, during, and after application of SNAP (200 µmol/L) at the times indicated in panel C. The current-voltage curves before and during application of SNAP were measured at points a and c, as shown in panel B. I_Ba was inhibited at all test potentials from -20 to +40 mV.

View larger version (23K): [in this window] [in a new window]   Figure 1. NO donor SNAP inhibits IBa through 1C+ß1a channels. A, Sample traces of IBa in the control condition and in the presence and during washout of 200 µmol/L SNAP. Voltage protocol and scale bars are as indicated. B, Current-voltage relations of IBa in control () and SNAP () solutions. C, Time course of IBa at 0 mV (IBa, ) and the holding current at -80 mV (Iholding, ). The recording was interrupted in the middle to establish the current-voltage relations of IBa. I (on the ordinate) indicates current. Duration of SNAP application is as indicated. Lowercase letters indicate the times at which traces in panel A (b, c, and d) and curves in panel B (a and c) were recorded.

At concentrations of 100 µmol/L to 800 µmol/L, SNAP steadily decreased I_Ba. In many of the experiments, after the peak inhibition of I_Ba caused by exposure to SNAP, the current partially recovered but remained below control levels even after 30 minutes of washout. In 40% of experiments, no recovery was observed. This suggests that SNAP often caused persistent modification of channel activity under these experimental conditions. In any case, after inhibition of I_Ba by SNAP (400 to 800 µmol/L), application of the reducing agent DTT (5 mmol/L) for >10 minutes did not recover more I_Ba than did simple washout with SNAP-free bath solution (n=4, data not shown).

To exclude the possibility that the inhibition of I_Ba might be caused by the solvent (DMSO) or by the products of the chemical degradation of SNAP after release of NO, in five control experiments we exposed cells to 2- to 6-hour-old solutions containing 400 to 800 µmol/L SNAP for 5 minutes, which is long enough for fresh SNAP to show effects. Much weaker inhibition of I_Ba was observed. Specifically, in one experiment when a 6-hour-old 800 µmol/L SNAP solution was applied, I_Ba was reduced by only 10%. In these control experiments, subsequent exposure of the same cells to freshly prepared SNAP solutions further reduced I_Ba to an extent comparable to that in virgin cells (data not shown). These findings confirmed that it was NO or intact SNAP itself, not DMSO or the degraded products of SNAP, that inhibited I_Ba. In another series of control experiments, 1 mmol/L NAP did not alter I_Ba (-1±8%, n=4).

To test whether the reduction of I_Ba was due to accelerated rundown or to a specific effect of SNAP, we designed experiments such as that in Fig 2. Cells were washed with SNAP-free bath solution intermittently between exposures to SNAP solutions during the early declining phase of I_Ba; we reasoned that if the reduction of I_Ba was due to rundown of the current, then I_Ba during the intermittent removal of SNAP should continue to decline as in the SNAP solution. However, Fig 2 shows that removal of SNAP halted the reduction of I_Ba instantaneously, whereas subsequent reexposure to SNAP further depressed I_Ba. These data indicated that I_Ba was indeed inhibited by SNAP and that SNAP did not function by simply accelerating the rundown of I_Ba.

View larger version (15K): [in this window] [in a new window]   Figure 2. SNAP did not inhibit IBa by triggering a rundown mechanism. The experiment was designed such that the cell was washed intermittently between 800 µmol/L SNAP solutions during the early declining phase of IBa. Top, Traces of IBa recorded at a, b, c, and d as indicated in the graph below. The voltage protocol and the scale bars are as indicated. Bottom, Time sequence of IBa at a test potential of 0 mV. SNAP inhibited IBa. Subsequent wash with SNAP-free bath solution halted the reduction of IBa instantaneously, although it did not remove the inhibition in this experiment. Reexposure to SNAP continued to inhibit IBa. Applications of 800 µmol/L SNAP are indicated by horizontal bars. I (on the ordinate) indicates current.

We also tested the effect of 5 to 10 nmol/L SNAP on I_Ba, since it has been reported that low concentrations of NO donors steadily stimulated I_Ca in native cardiomyocytes.^5,13 No detectable effect (neither stimulation nor inhibition) was observed in our system (n=3, data not shown).

Next, we studied the dose-response relationship of I_Ba of the _1C+ß_1a channel to SNAP. Fig 3 shows a plot of the normalized remaining current versus SNAP concentration. The half-inhibitory concentration was 360±39 µmol/L, with a Hill coefficient of 0.89±0.13.

View larger version (11K): [in this window] [in a new window]   Figure 3. Dose-response curve of SNAP inhibition on IBa of the 1C+ß1a channel. Normalized currents that remained after SNAP applications (Iremaining) are plotted against SNAP concentrations. Data are mean±SEM, with n indicating the number of experiments. A dummy point at 1 µmol/L SNAP was added in the plot for the convenience of sigmoidal fitting of the data. The smooth curve represents the sigmoidal fitting (see "Materials and Methods"), with half effective concentration of 360±39 µmol/L and a Hill coefficient of 0.89±0.13.

SNAP Inhibition of I_Ba Is Not Absolutely Dependent on Auxiliary Subunits
To test whether SNAP inhibition of I_Ca was dependent on auxiliary subunits, we examined the effects of 800 µmol/L SNAP on channels containing different subunit combinations: _1C alone, _1C+ß_1a, _1C+ß_1a+₂, _1C+ß_2a, and _1C+ß_2a+₂; coexpression of ß subunits increased the current density, and coexpression of the ₂ subunit sped up the activation of the current during depolarizing pulses. Fig 4 summarizes the results of these experiments, with panel A showing percent inhibition of I_Ba versus subunit combinations and panel B showing pooled data for absolute current density. SNAP inhibited I_Ba through all the channel subunit combinations tested, suggesting that the _1C subunit itself is an important target for the SNAP effects. Coexpression of ß_1a or ß_2a subunits enhanced SNAP inhibition of the _1C channel, with clear-cut or marginal significance (P<.034 or .087 for ß_1a or ß_2a, respectively). The inhibitory effect of SNAP on the _1C+ß_1a channel was similar to that on the _1C+ß_2a channel, suggesting that SNAP does not distinguish differences between the skeletal muscle and cardiac ß subunits. In addition, there was no consistent effect of coexpressing the ₂ subunit with _1C+ß channels. In the _1C+ß_1a channel, ₂-subunit coexpression modestly potentiated the inhibition relative to that on the _1C channel (P<.003 for _1C and _1C+ß_1a+₂ compared with P<.034 for _1C and _1C+ß_1a). In contrast, in _1C+ß_2a channels, ₂-subunit coexpression slightly reduced the inhibition by SNAP and eliminated the modest difference relative to _1C channels (P<.837 for ₁C and _1C+ß_2a+₂ compared with P<.087 for _1C and _1C+ß_2a).

View larger version (16K): [in this window] [in a new window]   Figure 4. SNAP (800 µmol/L) inhibited IBa through channels of all subunit combinations tested, including 1C alone, 1C+ß1a, 1C+ß1a+2, 1C+ß2a, and 1C+ß2a+2. A, Percent inhibitions are plotted as mean±SEM. Pairs of groups of data that are significantly different are indicated (*P<.05, ANOVA). B, Absolute currents normalized by the cell surface area, in the control condition and when 800 µmol/L was applied. Because of differences in experimental conditions, such data from different groups are not necessarily comparable. All experiments in the 1C-alone group and two of the seven experiments in the 1C+ß1a group were performed using 10 mmol/L Ba2+. All the experiments in the 1C+ß1a+2 group were also performed using 10 mmol/L Ba2+, yet we did not conduct experiments on cells expressing large currents (>1 nA). All other experiments were performed using 2 mmol/L Ba2+. Also, in general, the cells studied later (after transfection) exhibited larger currents; in our experiments, results from first- and second-day cells after transfection were pooled together. Note that a smaller scale is used for the 1C group.

Overall, the maximal inhibition (76% on the _1C+ß_1a+₂ channel) was approximately twice that of the minimal inhibition (41% on the _1C channel). We conclude that SNAP inhibits a site(s) on the _1C subunit. The presence of auxiliary subunits is not required for I_Ba inhibition by SNAP, although such subunits can potentiate the inhibitory effect (particularly in the case of ß subunits).

RSNO Compounds SNC and GSNO Also Inhibit I_Ba
We first tested the effects of SNC, another RSNO compound, in our expression system. Fig 5A shows membrane currents through _1C+ß_1a channels before, during, and after exposure to 1 mmol/L SNC. The NO donor inhibited I_Ba in our system by 46±4% (n=3), and no recovery of I_Ba was observed during washout. Interestingly, in this and two other similar experiments, the inhibition was preceded by a small initial transient elevation of 9±1% (n=3); the experiment in Fig 5 was the one containing the most pronounced transient elevation of I_Ba. In this experiment, during the 27.5-minute application time, eight fresh preparations of 5 mL SNC solutions were applied consecutively. Thus, the transient stimulation of I_Ba could not have been due to degradation of the NO donor. Considering the possibility that the initial transient might be a genuine effect of a lower concentration of SNC before the 1 mmol/L concentration was established, in three other cells we examined the effect of 100 µmol/L SNC. However, no stimulation was observed. If anything, there was an average inhibition of 7%. Thus, the small transient elevation of current during wash-in of 1 mmol/L SNC remains unexplained but appears not to be a steady-state effect of lower SNC concentrations.

View larger version (20K): [in this window] [in a new window]   Figure 5. A, RSNO SNC of 1 mmol/L inhibited IBa of the 1C+ß1a channel after a small initial transient stimulation. Presented here is the result containing the most pronounced initial stimulation in the three experiments. At the top of panel A, current traces show the control condition, transient initial SNC stimulation, steady-state SNC inhibition (>25 minutes into SNC application), and washout on removal of SNAP, denoted by a, b, c, and d, respectively, as indicated in the graph immediately below. Voltage protocol and scale bars are as indicated. At the bottom of panel A, the time course of IBa (I on the ordinate) at a test potential of 0 mV is shown, with the application period of SNC appearing as horizontal bars. In this experiment, eight fresh preparations of 5 mL SNC solutions were used consecutively. B, Another RSNO, GSNO at 1 mmol/L, also inhibited IBa through the 1C+ß1a channels. For comparison, the effects by SNAP (800 µmol/L), NAP (1 mmol/L), SNC (1 mmol/L, steady state), and cysteine (1 mmol/L) are also plotted. *P<.05 compared with the drug-free conditions.

We also examined the effects of 1 mmol/L SNC on I_Ba through _1C+ß_1a+₂ channels. Similar inhibition was observed, but in this case there was no initial stimulation (n=3). The overall inhibition was 45±4%, compared with 46±4% for the _1C+ß_1a channels.

As a control for SNC, we examined the effect of 1 mmol/L DL-cysteine on I_Ba through the _1C+ß_1a channels. Only a barely detectable inhibition of 9±11% (n=4) was observed.

We then tested the effect of GSNO, another RSNO NO donor, on the _1C+ß_1a channels. A clear inhibition of 39±5% was observed (n=5). Under no circumstances did we observe stimulation of I_Ba, similar to SNAP. Fig 5B compares the effects of 1 mmol/L GSNO, 800 µmol/L SNAP, 1 mmol/L NAP, 1 mmol/L SNC, and 1 mmol/L cysteine on I_Ba through the _1C+ß_1a channels. All the three NO donors were clearly inhibitory, but the controls (NAP or cysteine) had no significant effect.

Inhibition of I_Ba Is Not via a cGMP-Dependent Pathway
Since all the NO-induced inhibitory effects on Ca²⁺ channels described to date have been reported to occur via a cGMP-dependent pathway, we tested whether this is also the case under our experimental conditions. We explored the effect of 8-bromo-cGMP on cells expressing _1C+ß_1a channels. Cell-permeable 8-bromo-cGMP was used to activate cGMP-dependent protein kinase, which has been reported to mediate NO inhibition of Ca²⁺ currents in native cardiac myocytes.^7,12 Recordings from such an experiment are shown in Fig 6. The top panel plots currents recorded at baseline, in the presence of 200 µmol/L 8-bromo-cGMP, in the presence of cGMP with the addition of 800 µmol/L SNAP, and during washout of SNAP. The bottom panel shows the time course of I_Ba recorded under these experimental conditions. Application of 200 µmol/L 8-bromo-cGMP alone for 10 minutes did not affect I_Ba. Nevertheless, the addition of 800 µmol/L SNAP still blocked I_Ba, as found in the absence of cGMP. Inhibition of I_Ba was partially removed on washout of SNAP, again as found in the absence of cGMP. In general, in the presence of high concentrations of 8-bromo-cGMP (200 µmol/L), SNAP still readily blocked I_Ba by 56±12% (n=3), and the peak inhibition was comparable to that of 66±6% (n=7) in the absence of 8-bromo-cGMP. The inhibition was partially reversible. Similar observations were made in another cell in which 400 µmol/L 8-bromo-cGMP was applied.

View larger version (16K): [in this window] [in a new window]   Figure 6. SNAP inhibition of IBa from 1C+ß1a channels in the presence of cGMP. Top, Recorded IBa traces at baseline, in the presence of 200 µmol/L cGMP, with the addition of 800 µmol/L SNAP, and during washout of SNAP. Voltage protocol and scale bars are as indicated. Bottom, Time course of IBa (I on the ordinate) at a test potential of 0 mV. cGMP of 200 µmol/L had no effects on IBa. In addition, application of 800 µmol/L SNAP still significantly inhibited IBa with partial recovery on washout.

Because in native cardiac cells, all the NO-induced elevation of intracellular cGMP was concluded to be due to NO activation of guanylyl cyclase, we tested the effect of cell-penetrating MB on the SNAP inhibition of I_Ba. MB is an inhibitor of the soluble guanylyl cyclase; at a concentration of 10 to 50 µmol/L, it blocked the inhibition of NO on cAMP-stimulated I_Ba in cardiomyocytes.^5,6,8 In Fig 7, panel A shows representative current records, and panel B depicts the time course of SNAP inhibition of _1C+ß_1a channels. In the presence of 10 µmol/L MB, I_Ba was still significantly depressed by 800 µmol/L SNAP, with partial recovery on washout of SNAP. In general, in the presence of 10 to 30 µmol/L MB (n=3), 800 µmol/L SNAP still reduced I_Ba by 57±8% (n=3), which is comparable to a reduction of 66±6% (n=7) in the absence of MB. In another group of experiments, applying MB alone did not affect I_Ba (n=4).

View larger version (18K): [in this window] [in a new window]   Figure 7. SNAP inhibition of IBa from 1C+ß1a channels in the presence of MB. A, Traces of IBa during the application of 10 µmol/L MB, with the addition of 800 µmol/L SNAP, and during washout of SNAP. B, Time course of IBa (I on the ordinate). In the presence of 10 µmol/L MB, 800 µmol/L SNAP still significantly inhibited IBa, with partial recovery on washout.

From the results of these experiments, we conclude that the SNAP inhibition of I_Ba in our expression system is not via a cGMP-dependent pathway. Indeed, we find no evidence for effects of cGMP on basal current through expressed cardiac channels.

Inhibition of I_Ba Might Be due to Redox Modification of Cysteine(s) Not in the Pore
Because SNAP inhibition of I_Ba does not occur via a cGMP-dependent pathway in the HEK293 expression system, we explored other possible mechanisms that may underlie the inhibition. It is known that RSNOs or the NO they release can modify thiol side chains in proteins.^38,40 Examination of the amino acid sequence of the _1C subunit revealed at least two cysteines within the putative pore region of the channel, in domain I (C389 in the Slish et al²⁰ sequence) and domain IV (C1441 in the same sequence). It is possible that S-nitrosation of these pore-lining cysteines by the NO donor inhibits I_Ba by partially occluding ion flux through the channels. To test this idea, we first examined the effect of sulfhydryl-specific modifying reagents MTSEA, MTSET, or MTSES on I_Ba through the _1C+ß_1a channels. All three reagents are hydrophilic, although MTSEA is not as hydrophilic as the latter two. These highly reactive reagents can modify free thiol side chains that are facing the aqueous pore in regions 6 Å in diameter, covalently attaching a positively charged (MTSEA and MTSET) or negatively charged (MTSES) bulky group to the side chain.^35,39 MTSEA or MTSET modification may result in a decrease of the current by steric hindrance and/or by electrostatic repulsion. MTSES, on the other hand, may increase channel conductance in cation-selective channels, depending on the balance of the steric blocking effect versus the electrostatic attraction of permeant ions secondary to the introduced negative charge.³⁵

We briefly (for 10 to 15 seconds) exposed cells to saturating concentrations of MTSEA (2.5 mmol/L), MTSET (1 mmol/L), or MTSES (5 mmol/L), because prolonged exposures often result in deterioration of the seal. Fig 8 summarizes the effects of these reagents on the current-voltage relations of I_Ba. Although highly hydrophilic MTSET (panel B) and MTSES (panel C) did not appreciably affect the current, MTSEA, which is not as hydrophilic, did modestly inhibit the current (panel A). Such inhibition was not reversible by the reducing agent, DTT (n=3, data not shown). At test potentials of 0 or 10 mV, MTSEA produced an inhibition of 30%, compared with 66% by 800 µmol/L SNAP, 46% by 1 mmol/L SNC, and 39% by 1 mmol/L GSNO. This 30% inhibition of the Ca²⁺ channel current, however, is considerably smaller than that typically induced by MTSEA modification of naturally occurring or engineered cysteines in the pore of Na⁺ channels,^35,41 which generally exceeds 50%. Thus, our experiments with MTS reagents do not support the idea that RSNOs inhibit I_Ba by S-nitrosation of cysteines in the hydrophilic pathway of the channel.

View larger version (17K): [in this window] [in a new window]   Figure 8. Effects of thiol-specific modifiers on IBa through 1C+ß1a channels. A, Normalized current-voltage relationships of IBa at control () and after a brief 10- to 15-second exposure to MTSEA (2.5 mmol/L, ). MTSEA caused a minor reduction in the current. Current traces before and after drug application and the voltage protocols are shown in the inset. B, Current-voltage relations at control and after a brief (10- to 15-second) exposure to MTSET (1 mmol/L). MTSET did not produce an appreciable effect on the current. C, Similar to MTSET, MTSES (5 mmol/L) did not produce an appreciable effect on the current. D, Time course of the inhibition by 10 µmol/L thimerosal on IBa (I on ordinate) and its complete reversal by 5 mmol/L DTT. The sequential recording was interrupted at times a, b, and c to measure the current-voltage relations. E, Current-voltage relations measured in the control, thimerosal, and DTT solutions at points indicated in panel E. F, Graph showing the effects of brief exposures to the three MTS reagents and steady-state effects of thimerosal (10 µmol/L) and DTDP (50 µmol/L). All were on peak IBa at 0 mV. Note that MTSET and MTSES are highly hydrophilic, MTSEA is not as hydrophilic, thimerosal is less hydrophilic compared with the MTS reagents, and DTDP is hydrophobic. Data are mean±SEM, with n representing the number of experiments.

We further tested the effects of two other thiol-modifying reagents, thimerosal (10 µmol/L) and DTDP (50 µmol/L), which are much less hydrophilic than are the MTS reagents. These reagents can oxidize free thiol groups and attach a neutral side chain to the cysteine residue via a disulfide bond. Thimerosal and DTDP have been found to inhibit I_Ca through smooth muscle _1C-b-alone channels expressed in CHO cells, an effect that was fully reversible by DTT.⁴² In our HEK293 expression system, similar effects were observed. In Fig 8, panel D shows the time course of thimerosal inhibition and its complete reversal by DTT, and panel E is a plot of the current-voltage curves for control, thimerosal, and DTT at the times indicated in panel D. Overall, thimerosal at 10 µmol/L inhibited I_Ba by 66±6% (n=7), and 5 mmol/L DTT recovered the current by 95±4% (n=6). The inhibition by 50 µmol/L DTDP was more potent, reaching 91±4% (n=6), as shown in panel F, which was also readily reversed by DTT. These findings indicate that cardiac Ca²⁺ channels expressed in HEK293 cells exhibit a redox modulation similar to that described previously for the smooth muscle splice variant _1C-b in CHO cells. Also plotted in panel F for comparison are the averaged inhibitions by MTS reagents at saturating concentrations and thimerosal at 10 µmol/L. The hydrophobic reagents tend to exert more pronounced inhibitory effects than the most hydrophilic ones (MTSET and MTSES).

SNAP Does Not Inhibit Heterologously Expressed Na⁺ Channels
To determine whether the inhibition of NO donors is specific to the expressed Ca²⁺ channels, we tested the effect of 1 mmol/L SNAP on µ1 (rat skeletal muscle) Na⁺ channels expressed in HEK293 cells. In Fig 9, panel A shows representative records of the current in control and in SNAP solutions, and panel B shows the average current-voltage curves from five cells before and during exposure to SNAP. Even at this high concentration, SNAP did not affect the current. Clearly, Na⁺ channels show a much lower sensitivity to RSNO NO donors than do Ca²⁺ channels.

View larger version (17K): [in this window] [in a new window]   Figure 9. Effect of SNAP on Na+ currents. A, Whole-cell Na+ current recorded from HEK293 cells expressing the µ1 Na+ channels in control (left) and in 1 mmol/L SNAP (right). The pulse protocols are shown above the current traces. B, Current-voltage curves of the µ1 Na+ channels expressed in HEK293 cells in control () and 1 mmol SNAP solutions (). C, Whole-cell Na+ current recorded from Xenopus oocytes expressing hH1 Na+ channels in control (left) and in 600 µmol/L SNAP (right) solutions. The pulse protocols are shown above the current traces. D, Peak Na+ current remaining after different concentrations of SNAP (Iremaining) obtained from a group of oocytes expressing hH1 () or µ1 Na+ channels ().

We also studied the effect of SNAP on hH1 (human heart) and µ1 Na⁺ channels expressed in Xenopus oocytes. In Fig 9, panel C shows representative current traces obtained from an oocyte expressing hH1 Na⁺ channels in the control condition (left) and after treatment with 600 µmol/L SNAP (right). The dose-response data shown in panel D indicate that at 200 µmol/L SNAP, the currents were not appreciably affected and that when up to 1 mmol/L SNAP was used, only 20% reduction in whole-cell current was seen. This inhibition by SNAP was slow in onset and nonsaturating (data not shown), hinting at a nonspecific effect. Whatever effect there may be is quite small. The results in Fig 9 also indicate that in terms of responsiveness to SNAP, there is no appreciable difference between the cardiac and skeletal muscle isoforms of the Na⁺ channel. In contrast to expressed Ca²⁺ channels, which show a half-blocking concentration for SNAP of 360 µmol/L, Na⁺ channels have a half-effective concentration for SNAP far in excess of 1 mmol/L, as judged from the dose-response data for hH1 and µ1 Na⁺ currents shown in panel D.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In our HEK293 cell expression system, all three RSNOs we tested suppressed the currents through expressed L-type Ca²⁺ channels in the steady state. The suppression occurs at concentrations of RSNO compounds of 1 mmol/L. At nanomolar concentrations, SNAP showed no detectable effects on the channel currents. Under no circumstances did we observe a stimulatory effect of SNAP or GSNO, although SNC at a concentration of 1 mmol/L caused a small transient stimulation of I_Ba through _1C+ß_1a channels.

Inhibition of I_Ba by RSNO Compounds Is Independent of Subunit Combinations
SNAP inhibits currents through Ca²⁺ channels composed of all the subunit combinations we tested. The maximal inhibition was about twice the minimal inhibition. These facts suggest that the _1C subunit, which is the pore-forming subunit minimally required for Ca²⁺ channel expression, is the primary target of SNAP inhibition. Although coexpression of either ß subunit tended to potentiate the SNAP inhibition, additional coexpression of the ₂ subunit did not show a clear result. When ₂ is coexpressed, it is believed to be an extracellular subunit that is tethered to the Ca²⁺ channel _1C- complex by an extracellular disulfide bond.⁴³ Our finding that there is no clear role for the ₂ subunit in the SNAP inhibition of I_Ba suggests that the thiol site in the _1C- complex for the disulfide cross-link with ₂ is not the target of SNAP inhibition in the multisubunit complex.

NO Donor Inhibition of I_Ba Is Independent of the cGMP-Dependent Pathway
Biochemical studies have shown that HEK293 cells express the enzymes required for cGMP-dependent NO modulation of ion channels. Bischof et al⁴⁴ reported that the cGMP concentration in HEK293 cells was reduced by 50% to 75% by exposure to the guanylyl cyclase inhibitor LY83583 or to the NO synthase inhibitor N-nitro-L-arginine; furthermore, expression of neuronal NO synthase in HEK293 cells increased cGMP concentration by 20-fold. These results demonstrate that the NO/guanylyl cyclase/cGMP pathway does exist in HEK293 cells. In another study, Dinerman et al⁴⁵ confirmed the presence of the full NO/guanylyl cyclase/cGMP pathway and further demonstrated that cGMP stimulated endogenous cGMP-dependent protein kinases in these cells. Characterization of the cascade was completed by Piriev et al,⁴⁶ who demonstrated the existence of cGMP-dependent phosphodiesterases in HEK293 cells transfected with a control expression vector using calcium phosphate precipitation. Thus, guanylyl cyclase, cGMP-dependent protein kinases, and cGMP-dependent phosphodiesterases, which are critical to NO modulation via a cGMP-dependent pathway, do exist in HEK293 cells. In the present study, the inhibition of I_Ba by SNAP was not affected by concomitant application of 8-bromo-cGMP or MB; the fact that the critical enzymes of cGMP metabolism are present in HEK293 cells indicates that such inhibition was independent of the cGMP pathway.

Coexpression of either ß subunit tended to potentiate the SNAP inhibition of Ca²⁺ channels. This fact would be in line with the idea that the basal current is appreciably phosphorylated, since it has been reported that _1C is a poor substrate of protein kinase A^47,48 and that both ß₁ and ß₂ subunits have multiple sites for phosphorylation.⁴⁹ Thus, it is possible that expressed _1C+ß_1a/ß_2a channels are more phosphorylated than are _1C channels in the basal condition and that dephosphorylation by NO donors would result in a larger reduction of I_Ba in _1C+ß_1a/ß_2a subunits. If this were true, the preferential suppression of phosphorylated channels would need to be via a cGMP-independent mechanism. Our data would be equally consistent with the idea that the potentiating effect of ß subunits on SNAP-induced inhibition is entirely unrelated to changes in phosphorylation.

Inhibition of I_Ba by RSNOs May Occur via a Redox Mechanism
Our findings of SNAP inhibition of I_Ba by a cGMP-independent pathway raise the question of what alternative mechanisms might produce the inhibition. Besides phosphorylation/dephosphorylation, a possible mechanism is direct inhibition of the Ca²⁺ channel by redox chemical reactions. Direct modulation by NO has been described in the Ca²⁺-activated K⁺ channel of rabbit aortic smooth muscle.⁵⁰ As in the present study, such modulation was only partially removed on washout of NO donors, indicating possible covalent modification of the channels. In addition, redox reaction on thiol groups by NO donors has been proposed to be responsible for the downregulation of N-methyl-D-aspartate receptor activity⁴⁰ and for the stimulatory (but not the inhibitory) effect of NO donors on Ca²⁺ channels in ferret ventricular cells.¹⁴

For rabbit smooth muscle _1C-b-alone channels expressed in CHO cells, redox modifications have been shown by Chiamvimonvat et al.⁴² We have reproduced their findings that thimerosal or DTDP inhibited I_Ba and that such inhibitions were fully reversible by DTT, indicating that this redox property of the _1C subunit was generalizable between splice variants and remained intact after coexpression of the ß_1a subunit.

Where within the Ca²⁺ channel molecule are the likely sites for redox modulation? The data summarized in Fig 8F indicate that for redox modulation of the expressed Ca²⁺ channels, the more hydrophobic the modifying reagents, the more potent the inhibition. Thus, there seem to be one or more hydrophobically accessible redox sites in the Ca²⁺ channel. NO molecules would likely have access to the same site(s), given that they are small molecules and move freely through cellular membranes. Nevertheless, even if the targets for NO donors are the same as those for thiol-specific modifying reagents, the chemical reactions may not be identical. The RSNOs we used can modify thiol side chains in three ways. They may undergo a transnitrosation reaction with thiols in the channel to form S-nitrosothiols (R_x'SNOs, where R_x' refers to cysteine residue x in the channel protein), they may form mixed disulfide bonds with cysteines (RSSR_x'), or if cysteines x and y are close to each other, RSNOs may facilitate the formation of a disulfide bond between the cysteines^38,40 (R_x'SSR_y'). Furthermore, NO released by RSNOs may modify channel thiol side chains in similar ways, with the exception of forming mixed disulfide bonds. The thiol-specific modifying reagents we used, on the other hand, can form only mixed disulfide bonds with cysteines, attaching a bulky group to the side chain. These differences in chemical reactions may contribute to the observed differences between the DTT-induced reversibility of modifications by the NO donors (no effect) and by the thiol-specific modifying reagents (full reversal of the thimerosal and DTDP effects by DTT). In principle, it might be possible to determine directly whether or not expressed Ca²⁺ channels had undergone S-nitrosation using the Saville reaction or other methods.^51,52 We have not attempted such biochemical measurements because of the relatively low amounts of expressed channel proteins in the KEK293 cells.⁵³ Finally, it is worth noting that RSNOs release not only NO but also NO⁺ and NO^- (Reference 37³⁷ ); some of the results reported here may be attributable to these other NO species.

Physiological and Pathological Implications
The concentrations of NO donors used for most experiments in the present study were relatively high compared with those that induce vasodilation, although they were comparable to those used in previous native cardiac myocyte studies.¹⁴ High concentrations of SNAP did not significantly inhibit Na⁺ channels, suggesting that the inhibition of Ca²⁺ channels by SNAP is specific. The concentrations of NO released by SNAP depend on the composition of solutions and other factors that are difficult to standardize in general. The physiological concentrations of NO, on the other hand, are in the submicromolar range.⁵⁴ Excessive amounts of NO could be toxic to the heart. Submillimolar concentrations of SNAP have been reported to reduce the contractility of both cardiomyocytes and cardiac muscles.^55,56 NO has also been shown to have negative inotropic effects on isolated ventricular myocytes.⁵⁷ Inhibition of Ca²⁺ channels by NO donors would certainly help to explain the observed negative inotropic effects.

	Selected Abbreviations and Acronyms

 

CHO = Chinese hamster ovarian DMSO = dimethyl sulfoxide DTDP = 2,2'-dithiodipyridine DTT = 1,4-dithiothreitol GSNO = S-nitrosoglutathione HEK293 = human embryonic kidney IBa = Ba2+ current through Ca2+ channels ICa = L-type Ca2+ current MB = methylene blue MTS = methanethiosulfonate MTSEA = methanethiosulfonate-ethylammonium MTSES = methanethiosulfonate-ethylsulfonate MTSET = methanethiosulfonate-ethyltrimethylammonium NAP = N-acetylpenicillamine RSNO = S-nitrosothiol SIN-1 = 3-morpholinosydnonimine hydrochloride SNAP = S-nitroso-N-acetylpenicillamine SNC = S-nitrosocysteine

	Acknowledgments

 
This study was supported by National Institutes of Health grants (R01 HL-44065 to Dr Marban and T32 HL-07227 to Dr Hu). We thank Drs Timothy J. Kamp, Jane Lalli, Ebenezer N. Yamoah, and Charles Lowenstein for helpful discussions and Maria Janecki for technical assistance.

	Footnotes

 
Previously published in abstract form (Biophys J. 1997;72:A111).

Received August 4, 1997; accepted August 26, 1997.

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