Introduction

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Large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels are found in many tissues and may participate in a variety of cellular processes. These channels are gated open by both binding of intracellular Ca²⁺ and by membrane depolarization (Tseng-Crank et al., 1994; Wei et al., 1994; Kaczorowski et al., 1996). Because of the large unitary conductance and dense distribution, the activity of these channels is partly responsible for spike repolarization and the early afterhyperpolarization that follows each action potential (Sah and McLachlan, 1991; Kaczorowski et al., 1996). Previous studies also showed that presynaptic Ca²⁺ signals and transmitter release from nerve terminals can be regulated by the activity of BK_Ca channels (Robitaille and Charlton, 1992). Cloning and expression of both  and  subunits of this channel will allow us to examine structural components that control these gating processes (Kaczorowski et al., 1996; Toro et al., 1998). To determine the structural components of channel gating and to study the role that these channels may play in different cellular processes, small organic modulators for the gating of these channels will be useful tools.

Ruthenium red, an inorganic polycationic dye, is known to antagonize capsaicin-induced response (Staszewska-Woolley and Woolley, 1991; Wang and Hakanson, 1993; Lo et al., 1997), to inhibit Ca²⁺ release from the mitochondria (Broekemeier et al., 1994; Gunter et al., 1998; Wood and Gillespie, 1998) or from endoplasmic reticulum (Cheek et al., 1991; Sah and McLachlan, 1991; Otun et al., 1996), and to induce neurodegeneration (Velasco and Tapia, 1997). This inorganic polycationic dye has been reported to block voltage-dependent Ca²⁺ current in various cell types, including chromaffin cells (Gomis et al., 1994), mouse sensory neurons (Duchen, 1992), and synaptosomes and neuromuscular preparations (Hamilton and Lundy, 1995; Tapia and Velasco, 1997). However, to date, the effect of ruthenium red on K⁺ currents still remains controversial. For instance, ruthenium red can inhibit Ca²⁺-activated K⁺ channels in neurons (Sah and McLachlan, 1991; Wann and Richards, 1994) and smooth myocytes (Duridanova et al., 1996; Hirano et al., 1998). Ruthenium red was also found to have no effect on voltage-dependent K⁺ current in chromaffin cells (Gomis et al., 1994) or bladder smooth myocytes (Hirano et al., 1998). In contrast, ruthenium red is recently reported to enhance both voltage-dependent and Ca²⁺-activated K⁺ currents in mouse motor nerve terminals (Lin and Lin-Shiau, 1996).

In the present study, we sought to examine the effect of ruthenium red on Ca²⁺-activated K⁺ currents in rat pituitary GH₃ cells and to determine whether ruthenium red affects the activity of BK_Ca channels. These cells exhibit a wide variety of ionic conductances, including at least two types of Ca²⁺-activated K⁺ conductance (Ritchie, 1987). These results show a ruthenium red-induced inhibition of BK_Ca channels in these cells.

	Materials and Methods

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Cell Culture. The clonal strain GH₃ cell line, originally derived from a rat anterior pituitary adenoma, was obtained from American Type Culture Collection [(CLL-82.1), Manassas, VA]. GH₃ cells were grown in monolayer culture in 50-ml plastic culture flasks in a humidified environment of 5% CO₂/95% air at 37°C. Cells were maintained at a density of 10⁶/ml in 5 ml of Ham's F-12 nutrient media (Life Technologies, Grand Island, NY) supplemented with 15% heat-inactivated horse serum (v/v), 2.5% fetal calf serum (v/v), and 2 mM L-glutamine (Life Technologies). Cells were subcultured once a week, and a new stock line was generated from frozen cells (frozen in 10% glycerol in medium plus serum) every 3 months. The experiments were performed after 5 or 6 days of subcultivation (60-80% confluence).

Electrophysiological Measurements. Immediately before each experiment, GH₃ cells were dissociated, and an aliquot of the cell suspension was transferred to a recording chamber positioned on the stage of an inverted phase-contrast microscope (Diaphot-200; Nikon, Tokyo, Japan). The microscope was coupled to a video camera system with magnification up to 1500× to continually monitor cell size during the experiments. Cells were bathed at room temperature (20-25°C) in normal Tyrode's solution containing 1.8 mM CaCl₂. The patch pipettes were prepared from Kimax capillary tubes (Vineland, NJ) using a vertical two-step electrode puller (PB-7; Narishige, Tokyo, Japan), and the tips were fire-polished with a microforge (MF-83; Narishige). The resistance of the patch pipette was 3 to 5 M when it was immersed in normal Tyrode's solution. Voltage pulses were digitally generated by a programmable stimulator (SMP-311; Biologic, Claix, France). Ionic currents were recorded with glass pipettes in whole-cell or inside-out configuration of patch-clamp technique, using an RK-400 patch amplifier (Biologic) (Hamill et al., 1981; Wu et al., 1998b). All potentials were corrected for liquid junction potential that developed at the tip of the pipette when the composition of pipette solution was different from that of bath. Tested drugs were applied by perfusion or added to the bath to obtain the final concentrations indicated.

Data Recording and Analysis. The signals, consisting of voltage and current tracings, were monitored with a digital-storage oscilloscope (model 1602; Gould, Valley View, OH) and recorded on-line using a digital audio tape recorder (model 1204, Biologic). After the experiments, the stored data were then fed back and digitized at 5 to 10 kHz with a Digidata 1200 analog-to-digital device (Axon Instruments, Foster City, CA) interfaced to a Pentium-grade computer and pClamp 6.03 software package (Axon Instruments). Voltage-activated currents recorded during whole-cell experiments are stored digitally without leakage correction and analyzed using Clampfit subroutine (Axon Instruments) to establish a current-voltage relationship for ionic currents.

Drugs and Solutions. Tetraethylammonium chloride, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), cyclosporin A, neuraminidase, and tetrodotoxin were purchased from Sigma Chemical (St. Louis, MO). Ruthenium red (trans-tetradecaamine di-µ-oxotriruthenium⁶⁺ hexachloride) was obtained from Research Biochemicals (Natick, MA). All other chemicals were commercially available and of reagent grade. The composition of normal Tyrode's solution was as follows: 136.5 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl₂, 0.53 mM MgCl₂, 5.5 mM glucose, and 5.5 mM HEPES-NaOH buffer (pH 7.4). To record K⁺ currents, the patch pipette was filled with solution: 140 mM KCl, 1 mM MgCl₂, 3 mM Na₂ATP, 0.1 mM Na₂GTP, 0.1 mM EGTA, and 5 mM HEPES-KOH buffer 5 (pH 7.2). To record Ca²⁺ current, KCl inside the pipette solution was replaced with equimolar CsCl, and pH was adjusted to 7.2 with CsOH. In single-channel recording, high K⁺ bathing solution contained 145 mM KCl, 0.53 mM MgCl₂, and 5 mM HEPES-KOH 5 (pH 7.4). The pipette solution contained 145 mM KCl, 2 mM MgCl₂, and 5 mM HEPES-KOH (pH 7.2).

	Results

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Effect of Ruthenium Red on Ca²⁺-Activated K⁺ Current in GH₃ Cells. The whole-cell configuration of the patch-clamp technique was used to investigate the effect of ruthenium red on macroscopic ionic currents in these cells. In these experiments, the cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl₂. To inactivate other voltage-dependent K⁺ currents, each cell was held at 0 mV. As shown in Fig. 1, when the cell was held at 0 mV, voltage pulses from 30 to +70 mV with 20-mV increments elicited a family of large, noisy, outward currents. The amplitudes of these currents were increased with greater depolarization. These outward currents are identified as Ca²⁺-activated K⁺ currents [I_K(Ca)] (Olesen et al., 1994). Within 1 min of exposing the cells to ruthenium red (5 and 50 µM), the amplitude of outward currents was significantly decreased throughout the entire voltage-clamp step. For instance, when the voltage step from 0 to +70 mV was evoked, ruthenium red (50 µM) significantly decreased the current amplitude from 1609 ± 108 to 538 ± 48 pA (n = 7). This inhibitory effect was readily reversed after the removal of ruthenium red (Fig. 1). The averaged current-voltage (I-V) relations for these currents in the absence and presence of ruthenium red (5 and 50 µM) were shown in Fig. 1B. Figure 1C shows the relationships between the concentration of ruthenium red and the percentage of inhibition of I_K(Ca). Ruthenium red (5-200 µM) inhibited the amplitude of I_K(Ca) in a concentration-dependent manner. The half-maximal concentration required for the inhibitory effect of ruthenium red on I_K(Ca) was 15 µM, and 200 µM ruthenium red almost completely suppressed the amplitude of I_K(Ca). These results demonstrate that ruthenium red significantly reduces the action on I_K(Ca) in these cells.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Inhibitory effect of ruthenium red on IK(Ca) in GH3 cells. A, superimposed current traces in control, during the exposure to ruthenium red (5 and 50 µM) and after washout of ruthenium red. The cells, bathed in normal Tyrode's solution containing 1.8 mM CaCl2, were held at 0 mV, and voltage pulses from 30 to +70 mV in 20-mV increments were then applied. The leftmost side in A indicates the voltage protocol. Arrows denote the 0-current level. B, the averaged current-voltage relations of outward currents measured at the end of voltage pulses in control (), during exposure to 5 µM (), and 50 µM ruthenium red (), and washout of ruthenium red () (mean ± S.E.; n = 6-10 for each point). C, concentration-dependent inhibition of IK(Ca) by ruthenium red. The relation between the percentage of inhibition of IK(Ca) and the concentration of ruthenium red was illustrated. Each cell was depolarized from 0 to +50 mV with a duration of 300 ms. Various concentrations of ruthenium red (5-200 µM) were applied. The amplitude of IK(Ca) during the application of ruthenium red was compared to the control value, i.e., in the absence of ruthenium red (mean ± S.E.; n = 6-10 for each point). The smooth line represents the best fit to the Hill equation). The values of IC50 and maximally inhibited percentage of IK(Ca) in the presence of ruthenium red were 15 µM and 98%, respectively. The Hill coefficient was 1.2.

Comparison between the Effect of Ruthenium Red and Those of CCCP, Cyclosporin A, 4-Aminopyridine, and Tetraethylammonium Chloride. Effects of CCCP, cyclosporin A, 4-aminopyridine, tetraethylammonium chloride, and ruthenium red on I_K(Ca) in GH₃ cells were examined and compared. CCCP is an uncoupler of oxidative phosphorylation (Hehl et al., 1996), whereas cyclosporin A is considered to be a specific inhibitor of mitochondrial permeability transition (Shinohara et al., 1998). As depicted in Fig. 2, CCCP (10 µM), cyclosporin A (200 nM), or 4-aminopyridine (1 mM) did not produce a significant reduction of the amplitude of I_K(Ca). However, tetraethylammonium chloride (10 mM) and ruthenium red (50 µM) suppressed I_K(Ca) significantly. Thus, ruthenium red-induced inhibition of I_K(Ca) in GH₃ cells appears to be unrelated to its inhibition of mitochondrial function. However, at this point, it is still not easily excluded that the reduction of I_K(Ca) by ruthenium red may be due to a decrease in intracellular Ca²⁺ concentration.

View larger version (25K): [in this window] [in a new window]   Fig. 2.   Comparison of the effect of ruthenium red and CCCP, cyclosporin A, 4-aminopyridine, and tetraethylammonium chloride on IK(Ca). The cells were held at 0 mV, and voltage pulses to +50 mV (300 ms in duration) were applied. The amplitude of IK(Ca) in the control was considered to be 1.0, and the relative amplitude of IK(Ca) after application of each agent was then plotted. The parentheses denote the number of cells examined. Mean ± S.E. CCCP (10 µM); Cyc, cyclosporin A (200 nM); 4-AP, 4-aminopyridine (1 mM); TEA, tetraethylammonium chloride (10 mM); Ruth, ruthenium red (50 µM). *, significantly different from controls.

Inhibitory Effect of Ruthenium Red on the Activity of Large-Conductance Ca²⁺-Activated K⁺ (BK_Ca) Channels in Inside-Out Patches. To characterize the effect of ruthenium red on ionic currents, we also examined the effect of ruthenium red on BK_Ca channels. In an inside-out configuration, when ruthenium red (50 µM) was applied to the bath containing 1 µM Ca²⁺, the channel activity was profoundly reduced. A representative recording of BK_Ca channels after the application of ruthenium red is shown in Fig. 3. The opening probability of BK_Ca channels in control at the level of +80 mV was found to be 0.75 ± 0.06 (n = 12). One minute after the application of ruthenium red into the bath, the opening probability was significantly decreased to 0.08 ± 0.01 (n = 12). The further addition of 10 µM Ca²⁺ into the bath rapidly increased the channel activity to 0.55 ± 0.06 (n = 5). However, when ruthenium red (50 µM) was included in the pipette solution, the channel activity was unaffected (data not shown). Figure 4A illustrates current-voltage relations of BK_Ca channels in the absence and presence of ruthenium red (50 µM). Fitting current amplitudes with a linear regression revealed a single-channel conductance of BK_Ca channels in control of 151 ± 6 pS (n = 14). This value was not significantly different from that (150 ± 5 pS, n = 10) measured in the presence of ruthenium red (50 µM). These results clearly indicate that ruthenium red causes no change in the single-channel conductance but inhibits the opening probability of BK_Ca channels in GH₃ cells.

View larger version (33K): [in this window] [in a new window]   Fig. 3.   Effect of ruthenium red on BKCa channels in GH3 cells. The single-channel experiments in an excised inside-out membrane patch were conducted with symmetrical K+ concentration (145 mM). The bath solution contained 1 µM Ca2+. The membrane potential was held at +100 mV. A, the original current trace showing the change in the activity of BKCa channels after addition of ruthenium red. Channel openings are shown as upward deflection. The lower parts in A show the current traces obtained in expanded time scale. Note that the channel shows only a few, brief openings in the presence of ruthenium red (50 µM) and the further addition of 10 µM Ca2+ into the bath rapidly increases the channel activity. B, open probability (NPo) for the activity of BKCa channels shown in A plotted against time of recording. Bin width is 0.3 sec. Horizontal bars shown in the panel indicate the application of ruthenium red (50 µM) and Ca2+ (10 µM).

View larger version (16K): [in this window] [in a new window]   Fig. 4.   Effect of ruthenium red on the current-voltage relation of BKCa channels in GH3 cells. The cells were bathed in symmetrical K+ solution (145 mM), and the experiments were conducted under inside-out configuration. A, examples of BKCa channels in the absence (left) and presence (right) of ruthenium red (50 µM) measured from cells at various membrane potentials. Ruthenium red was applied to the bathing solution. The arrows and numbers at the beginning of each current trace mark the 0 current and voltage applied to the patch pipette, respectively. Upward deflections are the opening events of the channel. Currents were filtered at 1 kHz. B, the current-voltage relations of BKCa channels in the absence () and presence () of ruthenium red (50 µM). Of note, the single-channel conductance in the absence and presence of ruthenium red is nearly identical. C, the relationship between opening probability of BKCa channels and membrane potential in the absence () and presence () of 5 µM ruthenium red. The smooth lines represent the best fit to the Boltzmann equation.

Effect of Ruthenium Red on the Activation Curve of BK_Ca Channels. Figure 4C shows the activation curve of BK_Ca channels in the absence and presence of ruthenium red. The relationships between membrane potentials and opening probability of BK_Ca channels with or without the application of ruthenium red (5 and 50 µM) were plotted and filled by the Boltzmann equation using a nonlinear regression analysis
where N = the number of channels in patches, n = the maximal NP_o level, V = the membrane potential in mV, a = the membrane potential for half-maximal activation, and b = the slope factor of the activation curve. The values for a, b, and n are 61 ± 4 mV, 4.8 ± 2.5 mV, and 0.92 ± 0.04 (n = 6) in control; and 75 ± 3 mV, 5.2 ± 2.4 mV, and 0.52 ± 0.01 (n = 5) in the presence of ruthenium red (5 µM). Thus, ruthenium red not only decreased the maximal opening probability of BK_Ca channels but also shifted the activation curve to more positive membrane potentials. However, the slope (i.e., b value) of the voltage dependence of channel activation was unchanged by the presence of 5 µM ruthenium red.

Effect of Ruthenium Red on Kinetic Behavior of BK_Ca Channels in GH₃ Cells. The effect of ruthenium red on open and closed time of BK_Ca channels was examined and analyzed during recordings from patches showing only single-channel openings. As shown in Fig. 5, in control cells (i.e., in the absence of ruthenium red), the open-time histogram of BK_Ca channels at +40 mV can be fitted by a single-exponential curve with a mean open time of 3.3 ± 0.4 ms (n = 6), whereas the closed-time histogram was fitted by a two-exponential curve with a mean closed time of 2.9 ± 0.3 and 38.1 ± 1.4 msec (n = 6). The presence of ruthenium red decreased the lifetime of the open state to 1.6 ± 0.2 ms (n = 5) and increased the mean closed time to 7.3 ± 0.2 and 72.2 ± 8.2 msec (n = 5). Thus, it is clear that the inhibitory effect of ruthenium red on BK_Ca channel activity is due to both a decrease in open time and an increase in closed time.

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Effect of ruthenium red on mean open (left) and closed time (right) of BKCa channel in GH3 cells. Under symmetrical K+ condition, the cells were held at +40 mV in inside-out configuration. Open-time histogram in control was fitted by a single exponential function with a mean open time of 3.3 ms, whereas closed-time histogram in control was fitted by a sum of two-exponential function with a mean closed time of 2.9 and 38.1 ms, respectively. The open time (upper) and closed-time (lower) histograms after the addition of ruthenium red (10 µM) into the bath are shown in the lower part. The mean open time was decreased to 1.6 ms, whereas the mean closed time was increased to 7.3 and 72.2 ms. Of note, the abscissa and ordinate show the logarithm of the open or closed time (msec) and the square root of the number of events (n1/2), respectively. The solid curve indicates single- or two-exponential fitting using the least-squares method. In the control, data were obtained from a measurement of 984 channel openings with a total record time of 30 s. In the presence of ruthenium red, data were measured from 895 channel openings with a total record time of 2 min. Bin width for the open- and closed-time histograms is 0.5 and 3 ms, respectively.

Lack of Effect of Ruthenium Red on Voltage-Dependent K⁺ Outward Current (I_K) in GH₃ Cells. To determine whether ruthenium red also affects the amplitude of I_K in these cells, the experiments were conducted in cells bathed in Ca²⁺-free Tyrode's solution containing 1 µM tetrodotoxin and 0.5 mM CdCl₂. When the cell was held at 60 mV and various potentials ranging from 70 to +70 mV were applied, no significant difference in current amplitudes at each voltage step tested between the absence and presence of ruthenium red (50 µM) can be demonstrated (Fig. 6). Similar results were obtained in seven different cells. Thus, these results indicate that ruthenium red did not affect the amplitude of I_K in GH₃ cells.

View larger version (20K): [in this window] [in a new window]   Fig. 6.   Lack of effect of ruthenium red on voltage-dependent K+ outward currents in GH3 cells. The cells, bathed in Ca2+-free Tyrode's solution containing tetrodotoxin (1 µM) and CdCl2 (0.5 mM), were held at the level of 60 mV, and voltage pulses from 70 to +70 mV in 20-mV increments were applied. Superimposed current traces shown in the upper part are control, and those in lower part were recorded 2 min after addition of ruthenium red (50 µM). Voltage protocols are shown in uppermost part. Arrows indicate 0 current level.

Inhibitory Effect of Ruthenium Red on Voltage-Dependent L-Type Ca²⁺ Current (I_Ca,L) in GH₃ Cells. The effect of ruthenium red on I_Ca,L was also examined. The experiments were conducted with the Cs⁺-containing pipette solution. As shown in Fig. 7A, the cell was held at 50 mV, and the depolarizing pulses (300 ms in duration) to 0 mV were delivered at 0.1 Hz. Ruthenium red can inhibit the amplitude of I_Ca,L in a concentration-dependent manner. When the cells were depolarized from 50 to 0 mV, the amplitude of I_Ca,L was significantly decreased by ruthenium red (10 µM) to 52 ± 5 pA from a control value of 227 ± 12 pA (n = 8). In addition, the current-voltage relation of I_Ca,L was unaffected by ruthenium red (Fig. 7B). These results indicate that ruthenium red also suppressed the amplitude of I_Ca,L.

View larger version (11K): [in this window] [in a new window]   Fig. 7.   Effect of ruthenium red on ICa,L in GH3 cells. The patch pipette was filled with Cs+-containing solution, and cells were bathed in normal Tyrode's solution containing 1.8 mM CaCl2, 1 µM tetrodotoxin, and 10 mM tetraethylammonium chloride. A, superimposed current traces when the cell was depolarized from 50 to 0 mV with a duration of 300 ms. a, control. b, c, and d were obtained 1 min after the application of 3, 10, and 30 µM ruthenium red, respectively. Arrow indicates the 0 current level. B, the averaged current-voltage relations for ICa,L in the absence () and presence () of ruthenium red (10 µM) (mean ± S.E.; n = 8 for each point).

	Discussion

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The major findings of this study were as follows: 1) in rat pituitary GH₃ cells, ruthenium red, an organometallic dye, suppressed I_K(Ca); 2) ruthenium red decreases the activity of BK_Ca channels but does not change single-channel conductance; 3) ruthenium red shifts the activation curve of BK_Ca channels to more positive potentials; 4) ruthenium red-induced block in BK_Ca channels can be reversed by an increase in intracellular Ca²⁺ concentration; and 5) ruthenium red inhibits voltage-dependent I_Ca,L. These findings suggest that ruthenium red-induced decrease in the activity of BK_Ca channels and that inhibition of I_Ca,L may contribute to its modulatory effects on neuronal or neuroendocrine function.

Previous reports have demonstrated that ruthenium red can affect Ca²⁺ release from mitochondria (Broekemeier et al., 1994; Gunter et al., 1998; Wood and Gillespie, 1998). However, the present study shows that in GH₃ cells, neither CCCP (10 µM), an uncoupler of oxidative phosphorylation (Hehl et al., 1996), nor cyclosporin A, an inhibitor of mitochondrial permeability transition pore (Shinohara et al., 1998), can produce any effect on the amplitude of I_K(Ca). Additionally, our results from single-channel experiments with an excised membrane patch led to the interpretation that the inhibitory effect of ruthenium red on BK_Ca channels is mainly due to the result of direct binding to the inner surface of channel. Thus, these results make it unlikely that the effect of ruthenium red on mitochondrial function in these cells is directly responsible for the reduced activity of BK_Ca channels, although the two agents used to differentiate among potential sites of action of ruthenium red are not specific.

The IC₅₀ value for ruthenium red-induced inhibition of BK_Ca channels in GH₃ cells was 15 µM. This value is higher than that which produced the inhibition of BK_Ca channels in bladder smooth myocytes, with an IC₅₀ value of 5 µM (Hirano et al., 1998), but similar to those required to inhibit mitochondrial Ca²⁺-induced Ca²⁺ release (Broekemeier et al., 1994; Gunter et al., 1998; Wood and Gillespie, 1998), to block vanilloid receptor (Staszewska-Woolley and Woolley, 1991; Lo et al., 1997), and to inhibit Ca²⁺ release from endoplasmic reticulum (Cheek et al., 1991; Sah and McLachlan, 1991; Otun et al., 1996). Therefore, BK_Ca channels are likely to be a relevant "target" for the action of ruthenium red, although it remains to be determined whether this effect occurs in other types of cells.

Ruthenium red had no effect on single-channel conductance of BK_Ca channels in GH₃ cells (Fig. 4). Thus, the reduction in the conductance of whole-cell current shown in Fig. 1 must be due to a decrease in channel-open probability. In addition, ruthenium red also caused a shift in the midpoint for voltage-dependent opening. Thus, it is clear that ruthenium red can suppress the activity of BK_Ca channels in a voltage-dependent fashion.

Previous reports showed that ruthenium red can interact with the negative charges of the sialic acid moieties, thus disturbing synaptic transmission in rat hippocampal slices (Wieraszko, 1986). The reflex effects caused by capsaicin can be reversed by treatment with ruthenium red (Wang and Hakanson, 1993; Lo et al., 1997) or neuraminidase, an enzyme that cleaves sialic acid residues from glycosides and sialoglycoproteins (Staszewska-Woolley and Woolley, 1991). However, in our study, the inhibition of I_K(Ca) caused by ruthenium red was not affected by pretreatment of cells with neuraminidase (1 U/ml) (data not shown). Thus, it is unlikely that the blockade of BK_Ca channels by ruthenium red is due to its binding to sialic acid residues. In fact, a recent study showing that ruthenium red-induced neurotoxicity cannot be prevented by treatment with neuraminidase (Velasco and Tapia, 1997) appears to be compatible with our findings. Furthermore, the inhibitory effects of ruthenium red on BK_Ca channels cannot be due to its antagonistic action on vanilloid receptors because GH₃ cells do not express these receptors (Wu et al., 1998a).

The present results appear to be inconsistent with a previous report in which it was found that ruthenium red can increase both I_K and BK_Ca at mouse motor nerve terminals (Lin and Lin-Shiau, 1996). However, several studies, including the present results, showed that ruthenium red can suppress the activity of BK_Ca channels in hippocampal neurons (Wann and Richards, 1994) and smooth myocytes (Duridanova et al., 1996; Hirano et al., 1998). Alternatively, the activity of BK_Ca channels was reported to affect presynaptic Ca²⁺ signals and transmitter release from nerve terminals (Robitaille and Charlton, 1992). This discrepancy is currently unexplained, but it may be related to different channel isoforms in various tissues as reported (Tseng-Crank et al., 1994). However, our work showed that ruthenium red did not affect the amplitude of voltage-dependent K⁺ current, but did inhibit voltage-dependent L-type Ca²⁺ current. The inhibitory effect of ruthenium red on Ca²⁺ current is consistent with previous reports observed in chromaffin cells (Gomis et al., 1994), mouse sensory neurons (Duchen, 1992), and synaptosomes and neuromuscular preparations (Hamilton and Lundy, 1995; Tapia and Velasco, 1997).

One interesting finding is the antagonism by an increase in internal Ca²⁺ of ruthenium red-mediated inhibition of BK_Ca channels. This property distinguishes ruthenium red from some other types of channel blockers that act as open channel or use-dependent blockers (Wu et al., 1997). For such drugs, block is increased as the channel opening is promoted. The functional antagonism between the effect of internal Ca²⁺ and ruthenium red on BK_Ca channels may be interpreted to mean that these two molecules interact to bind allosterically. This notion is based on the findings showing the rapid relief of ruthenium red block by elevated Ca²⁺ (Fig. 3), as compared with the slow decrease in the channel activity during the exposure to ruthenium red and slow recovery from the inhibition of ruthenium red during washout.

In summary, the results of our study provide substantial evidence that in addition to suppressing L-type Ca²⁺ current, ruthenium red can also suppress the activity of BK_Ca channels in GH₃ cells. Ruthenium red may prove to be a useful tool for learning about BK_Ca channels at both molecular and macroscopic levels. These compounds are small molecules that can bind to BK_Ca channels at sites that are important for channel gating and are coupled to Ca²⁺ binding sites. They may also give insights into the role of BK_Ca channels in cell and tissue function.