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CARDIOVASCULAR

Saxitoxin Blocks L-Type I_Ca

Cardiology Division, University of Utah Health Sciences Center, Salt Lake City, Utah (Z.S., M.S., F.L., W.H.B.); and Department of Physiology, Tokai University, Isahara, Japan (H.I.)

Received July 3, 2003; accepted October 9, 2003.

	Abstract

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Saxitoxin (STX) and tetrodotoxin (TTX) are frequently used to selectively block sodium channels. In this study, we provide evidence that commercial STX also inhibits L-type Ca²⁺ currents (I_Ca,L) in adult mouse ventricular myocytes (VMs) and tsA-201 cells that were transiently cotransfected with three calcium channel subunits. We measured inhibition of sodium currents (I_Na) in mouse VMs, of I_Ca,L in mouse VM and tsA-201 cells, and intracellular calcium concentration ([Ca²⁺]_i) transients in single mouse VMs. STX or TTX was abruptly applied before the test voltage pulse using a rapid solution switcher device. STX (10 µM; Calbiochem) and TTX (60 µM; Sigma-Aldrich) completely blocked I_Na in mouse VMs. However, STX at 10 µM also reduced I_Ca,L in mouse VM by 39% (P < 0.0001; n = 14), whereas TTX at 60 µM had no effect on I_Ca,L. STX (10 µM; Calbiochem) reduced the amplitude of the [Ca²⁺]_i transients in mouse VMs by 36% (P < 0.0001; n = 10). In contrast, TTX (60 µM; Sigma-Aldrich) only reduced the amplitude of the [Ca²⁺]_i transients by 9% (P = 0.003; n = 5). STX (10 µM) obtained from Sigma-Aldrich showed a similar inhibitory effect on I_Ca,L (33%) (P < 0.0001; n = 5) in mouse VMs. STX (Calbiochem) inhibited the calcium currents of tsA-201 cells in a dose-dependent manner. This inhibition was voltage-independent. The current-voltage relationship of calcium currents in tsA-201 cells was not altered by STX. These results indicate that STX partially blocks L-type Ca²⁺ channels and thus provide further evidence that its effects are not specific for Na⁺ channels.

	Materials and Methods

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Dissociation of Adult Mouse Ventricular Myocytes. Single mouse ventricular myocytes were isolated as described previously (Yao et al., 1998). After retrograde perfusion with modified Tyrode's solution (Ca²⁺-free) for 5 min, the heart was digested for 7 to 12 min with 0.9 mg/ml collagenase D (Roche Diagnostics, Indianapolis, IN) in modified Tyrode's solution containing 25 µM CaCl₂. The modified Tyrode's solution (pH 7.4) contained the following: 126 mM NaCl, 4.4 mM KCl, 1.0 mM MgCl₂, 18 mM NaHCO₃, 11 mM glucose, 4 mM HEPES, 30 mM butanedione monoxime, and 0.13 U/ml insulin, and was gassed with 5% CO₂, 95% O₂. The digested left ventricle was cut into small pieces in modified Tyrode's solution containing 100 µM Ca²⁺. These pieces were gently agitated to release single myocytes and then incubated in the same solution with 2% albumin at 30°C for 20 min. The cell suspension was centrifuged at 300 rpm for 3 min, and the pellet of cells was resuspended in modified Tyrode's solution containing 200 µM Ca²⁺ and 2% albumin and allowed to settle for another 20 min at 30°C. Cells were then suspended in culture media composed of 5% fetal bovine serum, 47.5% minimal essential medium (Invitrogen, Carlsbad, MD), 47.5% modified Tyrode's solution, 10 mM pyruvic acid, 4.0 HEPES, and 6.1 mM glucose and finally maintained in a 5% CO₂ atmosphere at 30°C until use. All experiments in this study were performed at 23–25°C.

Transient Expression of L-Type Calcium Channels in tsA-201 Cells. We maintained tsA-201 cells (large T antigen-transformed human embryonic kidney cells) at 37°C with Dulbecco's modified Eagle's medium (Invitrogen) plus 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin in a 5% CO₂ atmosphere (Sheets et al., 1996). TsA-201 cells were fused as described previously (Sheets et al., 1996). Fused tsA-201 cells were transiently cotransfected by calcium phosphate-mediated transfection with 5 µg each of three calcium channel subunits (rabbit _1C, rat _2a and ₂/) per 10-cm plate (Perez-Reyes et al., 1992; Wei et al., 2000). Rabbit _1C was subcloned into pRc/CMV (Invitrogen), rat _2a into p91023(B), and rat ₂/ into pZEM229 (ZymoGenetics, Seattle, WA). Because tsA-201 cells normally have no detectable Ca²⁺ currents, the presence of L-type Ca²⁺ currents provided confirmation of transfection.

For electrophysiological study, the fused tsA-210 cells (2–3 days after transfection) were harvested from the culture dish by trypsinization, washed once with Dulbecco's modified Eagle's medium plus 10% fetal bovine serum, and maintained in this medium at room temperature for later use. Only cells that showed a current amplitude >300 pA were used for drug studies.

Electrophysiology. The setup for voltage clamp has been described in detail previously (Su et al., 1998). Cells were voltage clamped at room temperature with single suction pipettes that were made from borosilicate glass tubing (Corning 7052, 1.65 mm o.d., 1.2 mm i.d.; A-M System Inc., Everett, WA) and had initial resistances of 1.0 to 2.5 M when filled with pipette solution containing 15 mM NaCl, 100 mM CsCl, 30 mM tetraethylammonium chloride, 5 mM MgATP, 10 mM EGTA, 10 mM HEPES, and 5.5 mM dextrose (pH 7.1 adjusted with CsOH). To measure [Ca²⁺]_i transients in voltage-clamped myocytes (Su et al., 2001), EGTA was omitted from pipette solution. Inhibition of both sodium currents (I_Na) in mouse VMs and L-type calcium currents (I_Ca,L) in mouse VMs and tsA-201 cells was measured by a whole-cell voltage-clamp technique using an Axopatch 200A amplifier (Axon Instruments, Union City, CA). Series resistance compensation (70–80%) was used in all experiments. Currents were filtered at 2 to 5 kHz and digitized at 5 to 10 kHz. Cell capacitance was also measured to calculate the current density.

Mouse VM were perfused with an external solution containing 126 mM NaCl, 1.0 mM MgCl₂, 1.08 mM CaCl₂, 4.4 mM CsCl, 11 mM dextrose, and 24 mM HEPES (pH 7.4 adjusted with NaOH). TsA-201 cells were superfused with an external solution containing 126 mM NaCl, 5 mM CaCl₂, 4.4 mM CsCl, 11 mM dextrose, and 24 mM HEPES (pH 7.4 adjusted with NaOH).

Measurement of [Ca²⁺]_i. The [Ca²⁺]_i was measured as described previously (Yao et al., 1998, Su et al., 2001). Myocytes were loaded with fluo-3 by exposure to 1 µM fluo-3 AM (Molecular Probes, Eugene, OR) at 30°C for 30 min. Fluo 3-loaded myocytes were placed in a chamber mounted on an inverted microscope. Once myocytes had settled to the bottom, they were superfused with a HEPES-buffered solution containing 126 mM NaCl, 4.4 mM KCl, 1.0 mM MgCl₂, 1.08 mM CaCl₂, 11 mM dextrose, 0.5 mM probenecid, and 24 mM HEPES (pH 7.4 adjusted with NaOH to give a final external Na⁺ concentration of 140 mM).

Myocytes were held at -80 mV at room temperature and clamped to 0 mV for 200 ms to trigger [Ca²⁺]_i transients. Eight conditioning pulses (200 ms, -80 to 0 mV, 0.25 Hz) were applied before test pulse to provide a steady-state loading of SR with Ca²⁺.

Fluo-3-loaded myocytes were illuminated by 485-nm excitation light via an epifluorescence attachment (510-nm dichroic mirror; Omega Optical, Brattleboro, VT) and a 40x Fluor oil objective lens. The resulting fluorescence signals at 530 nm (DF30; Omega) were detected with a photomultiplier (SFX-2; Solamere Technology Group, Salt Lake City, UT). Fluo-3 fluorescence was transformed to [Ca²⁺]_i by a "pseudoratio method" (Cheng et al., 1993): [Ca²⁺]_i = K_d(F/F₀)/(K_d/[Ca²⁺]_irest + 1 - (F/F₀)), where K_d is the dissociation constant for fluo-3 (493 nM at 25°C), F the fluorescence intensity, F₀ the intensity at rest, and [Ca²⁺]_irest the [Ca²⁺]_i at rest and assumed to be 80 nM under our experimental conditions (Yao et al., 1998).

Rapid Application of Drugs. To observe the effects of STX (B19491 [GenBank] , Calbiochem, San Diego, CA; 39H1139, Sigma-Aldrich, St. Louis, MO) and TTX (99H0832; Sigma-Aldrich) on [Ca²⁺]_i transients, sodium currents and/or L-type calcium currents, we rapidly applied those drugs to clamped single cells. Rapid applications of STX and TTX were accomplished with a fast solution switcher device (Yao et al., 1997), which minimizes the amounts of test solution required and eliminates uncertainty regarding the precise time of exposure.

Measurement of Cation Content. Cation content in the commercially prepared STX solution was determined by inductively coupled plasma (ICP) emission spectroscopy (D'souza and Holz, 1999) by use of an ICPS-7500 instrument (Shimadzu, Kyoto, Japan). All glassware that was used was soaked in concentrated nitric acid and rinsed in chelated neuro pure water to remove any metal contaminants.

Data Analysis. All recordings were digitized online with a Digi-Data 1200 interface (Axon Instruments) and stored on disk. The digitized data were analyzed with pCLAMP6 (Axon Instruments) and ORIGIN (OriginLab Corp., Northampton, MA). Results were expressed as means ± S.E.M., and statistical differences were determined by unpaired or paired t tests. The n indicates the number of myocytes or cells studied. One to three myocytes were studied from each heart dissociation. Differences were considered statistically significant at P < 0.05.

	Results

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Effects of STX and TTX on [Ca²⁺]_i Transients in Mouse Ventricular Myocytes. To induce a stable [Ca²⁺]_i transient magnitude, a prepulse protocol (eight pulses at 0.25 Hz, -80 to 0 mV, for 200 ms) was applied before each test pulse. The left panel of Fig. 1A shows the last of a series of eight conditioning voltage-clamp pulses (trace a) and its corresponding membrane currents (trace b) as well as the triggered [Ca²⁺]_i transient (trace d). The test pulse was initiated 4 s after the start of the last conditioning pulse. To determine the effects of STX or TTX on [Ca²⁺]_i transients, the clamped myocyte was superfused with STX (10 µM) or TTX (60 µM) for 2 s immediately before and during the test pulse by using a rapid solution switcher (trace c in Fig. 1A). In our experiments, K⁺ currents were blocked by replacing intracellular and extracellular K⁺ with Cs⁺ and including tetraethylammonium chloride in the pipette solution. Therefore, the inward currents in a 200-ms test pulse (from -80 to 0 mV) in the presence of STX or TTX should be largely ascribed to calcium currents. Figure 1A illustrates that the peak inward currents (I_Na plus I_Ca) activated during the 200-ms pulse from -80 to 0 mV were dramatically reduced by rapid application of STX (Calbiochem) at 10 µM. The [Ca²⁺]_i transient amplitude was also significantly decreased (36%) by STX. In contrast, previous studies in our laboratory (Fig. 1B) showed that the [Ca²⁺]_i transient was only slightly decreased (9%) by rapid application of TTX (Sigma-Aldrich) at 60 µM. We also found that the mean of the peak inward currents during the test pulse (-80 to 0 mV) in the presence of STX (Calbiochem) were smaller than those measured in the presence of TTX (Sigma-Aldrich) (624 ± 88 pA, n = 10) versus 947 ± 110 pA, n = 7; P < 0.05). These results suggested that STX may inhibit L-type calcium channel currents in mouse ventricular myocytes. We therefore carried out additional experiments to examine the effects of the commercial STX preparations on L-type calcium channel currents.

View larger version (30K): [in this window] [in a new window]   Fig. 1. A, influence of STX (Calbiochem) on [Ca2+]i transients in mouse ventricular myocytes. Left, trace a shows the last two of a series of eight voltage-clamp protocols in which the cell was held at -80 mV and depolarized to 0 mV for 200 ms; trace b, membrane currents (INa and ICa,L) in which K+ currents were blocked by tetraethylammonium chloride and Cs; trace c, rapid application of STX (10 µM) was accomplished by a double-barreled solution switcher; trace d, [Ca2+]i transients. Average values of the peak [Ca2+]i are presented as mean ± S.E.M. of 10 cells from four hearts and shown in the bar graph on the right. B, similar recordings showing the effects of TTX in an identical protocol as in Fig. 1A [B reprinted from Su et al. (2001), with permission of publisher].

Effects of STX and TTX on I_Ca,L in Mouse Ventricular Myocytes. As shown in Fig. 2a, mouse ventricular myocytes were held at -80 mV, depolarized to -40 mV for 200 ms to inactivate I_Na, and then depolarized to 0 mV to activate I_Ca,L. Application of STX (10 µM; Calbiochem) to the same cell seemed to completely block I_Na and inhibited I_Ca,L by 39% (P < 0.0001; n = 14) (Fig. 2b). Both I_Na and I_Ca,L recovered upon washout of STX (Fig. 2c). STX (10 µM) purchased from Sigma-Aldrich also showed a similar inhibitory effect on I_Ca,L in mouse ventricular myocytes (current densities were reduced by 33%, n = 5, P < 0.001; data not shown). This inhibitory effect of STX from Sigma-Aldrich was also reversible upon washout. In contrast, TTX (60 µM; Sigma Aldrich) blocked I_Na, but did not influence I_Ca,L (Fig. 3). These results provide another line of evidence for the inhibitory effect of commercial STX preparations on L-type calcium channel currents in mouse ventricular myocytes, and are consistent with STX-induced reduction in the amplitude of [Ca²⁺]_i transients.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Effects of STX from Calbiochem on membrane currents (INa and ICa,L) in mouse ventricular myocytes. a, control run of the voltage protocol in which the cell was held at -80 mV and pulsed to -40 mV to activate INa, and then to 0 mV to activate ICa,L. Potassium currents were blocked by replacing the extracellular and intracellular K+ with Cs+. B, same voltage protocol as in a was applied to the same cell in the presence of STX (10 µM). Note the INa was completely blocked and the ICa,L was partially inhibited. c, recovery of both INa and ICa,L upon washout of STX. D, an overlapped plot of L-type Ca2+ current tracings in the absence and presence of STX.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Effects of TTX (Sigma-Aldrich) on membrane currents (INa and ICa,L) in mouse ventricular myocytes. Left, control run of the voltage protocol as described in Fig. 2. Right, same voltage protocol was applied to the same cell in the presence of TTX (60 µM). TTX blocked INa, but did not influence ICa,L. Right inset, an overlapped plot of L-type calcium currents in the absence and presence of TTX. Similar results were observed in eight cells from three hearts.

To exclude the possibility that the STX-induced reduction of the inward current during a voltage pulse (0 mV) was not due, in part, to block of residual I_Na resulting from incomplete inactivation of Na⁺ channels during the prepulse to -40 mV, the effect of STX on I_Ca,L was observed in the absence of extracellular Na⁺. The same voltage protocol as described in Fig. 2a was used in this experiment. The I_Ca,L was recorded before and after STX application.

STX (10 µM; Calbiochem) showed the same inhibitory effect on I_Ca,L (decreased by 39%; n = 5) when extracellular Na⁺ was replaced by tetramethylammonium chloride. We also examined the effect of acetic acid (vehicle solvent in the commercial STX preparation) on the inhibitory effect of STX on I_Ca,L. Acetic acid at 1 mM, which was the concentration of acetic acid in the working solution of STX (10 µM), did not reduce the amplitude of I_Ca,L (data not shown).

Effects of STX on I_Ca,L in tsA-201 Cells. To confirm further that residual I_Na did not appreciably contaminate measurements of I_Ca,L, we examined the effect of STX on calcium currents mediated by L-type calcium channels expressed in fused tsA-201 cells. TsA-201 cells were transiently cotransfected with three calcium channel subunits (see Materials and Methods). As shown in Fig. 4, STX inhibited calcium currents in a dose-dependent manner. Note the absence of significant inward current due to I_Na when stepping from -80 mv to -40 mV. Current traces (recorded from the same cell) in Fig. 4, A to C, exhibited a very similar amplitude of control currents, indicating that the effects of STX on calcium currents of L-type calcium channels expressed in tsA-201 cells were completely reversible, consistent with recovery of L-type calcium channel currents after washout of STX in mouse ventricular myocytes. As shown in Fig. 4D, the maximal effect of STX on the L-type Ca current in tsA-201 cells was a 49% block, and the "K_d" (concentration required for one-half of this effect) for this effect was 0.3 µM. The current-voltage relationship (I-V curve) of calcium currents in tsA-201 cells was not altered by STX (Fig. 5C), indicating that the voltage-dependence of the activation of L-type calcium channel is not influenced by STX. This inhibition was also voltage-independent (Fig. 5D).

View larger version (21K): [in this window] [in a new window]   Fig. 4. Dose-dependent effects of STX (Calbiochem) on L-type calcium channel currents in tsA-201 cells. The same voltage protocol as used in mouse VM (Fig. 2) was used to examine the effects of STX on calcium currents mediated by L-type calcium channels expressed in tsA-201 cells. A, B, and C are representative traces showing the effects (in the same cell) of STX at 0.3, 1, and 3 µM, respectively. D, dose-dependent effect of STX on L-type calcium currents on a log scale. The data were fit using SAS and a nonlinear least-squares analysis of the equation "fractional block = maximum block x (dose/(Kd + dose))." The Kd is 0.3 µM and the maximum block 0.49. Results are expressed as means ± S.E.M., n = 5.

View larger version (27K): [in this window] [in a new window]   Fig. 5. Voltage-independent effects of STX on L-type calcium channels expressed in tsA-201 cells. L-type calcium channel currents were activated by depolarizing the cell membrane from -30 to 70 mV at a 10-mV increment. A, representative traces showing the control currents (in the absence of STX). B, representative traces showing the currents in the same cell in the presence of 3 µM STX. Pooled data (means ± S.E.M., n = 5) is shown in C and indicates that the I-V relation is not altered by STX. D, ratio of ICa,L in STX to control demonstrating lack of a voltage-dependent reduction by STX of peak ICa,L.

Content of Metal Ions in the Commercial STX Solution. Our experimental results described above shows evidence for the inhibitory effect of STX (Calbiochem or Sigma-Aldrich) on L-type calcium channels. It is important to exclude the possibility that a metal ion contaminant of the commercially prepared STX solution is responsible for this inhibitory effect. We therefore measured the content of metal ions in the commercial STX solution (Calbiochem) by ICP emission analysis. Concentrations of metal ions (Ni²⁺, Co²⁺, Mn²⁺, Cd²⁺, and La³⁺) in the STX preparation from Calbiochem were all under detection limits (400 nM) of ICP emission analysis. These results, as well as the rapid reversibility of the effects of STX on I_Ca,L, suggest that metal ions are not responsible for the inhibitory effect of the commercial STX on L-type calcium channels.

	Discussion

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It is well recognized that STX can bind to and block Na⁺ channels in cardiac myocytes as well as in neuronal cells, although the K_d values for these effects differ (Guo et al., 1987; Hille, 2001). In the present study, we have provided evidence demonstrating that STX also partially blocks L-type calcium channels. First, STX reduced the amplitude of [Ca²⁺]_i transients in mouse ventricular myocytes more markedly than did TTX. The apparent slowing of the decline of the [Ca²⁺]_i transient after STX probably is due to the reduction in its amplitude (Bers and Berlin, 1995) rather than to an effect of STX on sarcoplasmic reticulum Ca²⁺-ATPase. Second, STX from two different commercial suppliers significantly inhibited calcium currents of native L-type calcium channel in mouse ventricular myocytes. Third, the inhibitory effect of the STX on I_Ca,L is not due to the solvent, acetic acid, nor to divalent cation contamination by Ni²⁺,Co²⁺,Mn²⁺,or Cd²⁺or the trivalent cation La³⁺ in the STX preparation. Fourth, STX inhibited calcium currents mediated by L-type calcium channels expressed in tsA-201 cells, which have little or no fast Na⁺ currents (Sheets et al., 1996). The I-V relationship of calcium currents in tsA-201 cells was not altered by STX, indicating that there was no surface charge effect on I-V relationships, and there is no voltage dependence of the block of L-type calcium channel by STX. The lack of a shift in the I-V relationship further suggests that the block of I_Ca,L is not caused by metal ion contamination in the commercial STX preparation, because a shift in the I-V relationship is expected if metal ions are responsible for the block of L-type calcium channels (Hobai et al., 2000; Hille, 2001).

The inhibitory effect of STX on I_Ca,L was rapid in onset, particularly at a high concentration of STX (10 µM). Block of I_Ca,L was also rapidly reversible, with currents recovered within 40 s after washout. These basic features of STX-mediated blockade of I_Ca,L suggest that STX acts on the calcium channel at a site that has access from the external surface and may have some similarities to the binding site for STX in the external vestibule of the Na⁺ channel ion permeation pathway (Lipkind and Fozzard, 1994).

The maximal effect of STX on the L-type Ca channel was a 49% inhibition, and the "K_d" for this effect was 300 nM (Fig. 4D). The concentration of STX at which a half-maximal effect of STX on the L-type Ca channel was seen is similar to that reported by Jones and Marks (1989) in bullfrog neuronal N-type Ca²⁺ channels (400 nM). These concentrations are much higher than for the effect of STX on neuronal Na⁺ channels (K_d < 20 nM), but somewhat less than for STX inhibition of the cardiac Na channel (5000 nM), which is known to be STX resistant (Hille, 2001). Interestingly, Jones and Marks (1989) also found that STX produced an incomplete block of bullfrog neuronal Ca²⁺ channels (66% at 1 µM). Also, a mutated µ-conotoxin has been reported to only partially reduce the skeletal muscle Na⁺ current due to a partial block at the single channel level (French et al., 1996). The incomplete block of STX of L-type Ca²⁺ channel currents may be due also to only a partial inhibition by STX of single L-type Ca²⁺ channels. A channel blocker acting at a single site would be expected to completely inhibit the channel at a sufficiently high concentration. The fact that this is not observed with STX inhibition of Ca²⁺ channels may indicate that the mechanism is not a simple pore block. A recent report by Wang et al. (2003) has shown that STX can decrease hERG K⁺ currents by stabilizing closed channel states, manifest by a shift in the voltage dependence of channel opening to more depolarized membrane potentials. However, in our experiments STX had no effect on the voltage dependence of activation of the L-type Ca²⁺ channel expressed in tsA-201 cells. Our data cannot rule out the possibility that STX decreased I_Ca,L by producing a negative (leftward) shift of the steady-state inactivation curve, resulting in inactivation of I_Ca,L at our holding potential of -40 mV. However, such an effect is unlikely because it would require that STX cause a marked shift in the steady-state inactivation curve while at the same time not producing block of the channel pore. Furthermore, in Na⁺ channels STX has been demonstrated to shift gating charge by only -4.9 mV (Heggeness and Starkus, 1986).

It has been shown that the binding interaction between the Na⁺ channel and STX is different from that between the Na⁺ channel and TTX (Kirsch et al., 1994; Penzotti et al., 1998). Based on the different effect of STX and TTX on L-type calcium channel, it is plausible that L-type calcium channels may bear a binding site for STX, but not for TTX. A different channel-mediated current, the TTX-blockable calcium current (I_Ca,(TTX)), has been reported in human atrial (Lemaire et al., 1995) and ventricular (Gaughan et al., 1999) myocytes, in guinea pig (Cole et al., 1997) and rat (Aggarwal et al., 1997) ventricular cells, and in neural preparations (Meves and Vogel, 1973; Akaike and Takahashi, 1992). The identity of the channel that conducts I_Ca(TTX) is still controversial; however, it is possible that I_Ca(TTX) is mediated by a subtype of a sodium channel and not a calcium channel (Aggarwal et al., 1997). In any event, our studies in tsA201 cells expressing I_Ca,L eliminate the I_Ca(TTX) as a contributor to our results.

In our study, we also noted minor but significant variations in the sensitivity to STX depending upon the tissue studied. STX at 10 µM reduced L-type calcium currents by 39 ± 3% (n = 14) in mouse ventricular myocytes, versus 53 ± 4% (n = 6) in transfected tsA-201 cells (P < 0.05) and 58 ± 6% (n = 4) in rabbit myocytes (P < 0.01). Consequently, the degree of block of the L-type Ca channel produced by STX in cardiac myocytes from different species may vary.

	Acknowledgements

 
We are indebted to Drs. Edward Perez-Reyes and David T. Yue for providing cDNAs of L-type calcium channel subunits, Wenqing Yu for performing the transient expression of L-type calcium channel in tsA-201 cells, and Pam Larson for assistance in preparing the manuscript.

	Footnotes

 
This work was supported in part by National Institutes of Health Grants HL44630 and HL52338.

DOI: 10.1124/jpet.103.056564.

ABBREVIATIONS: STX, saxitoxin; TTX, tetrodotoxin; [Ca²⁺]_i, intracellular calcium concentration; VM, ventricular myocyte; ICP, inductively coupled plasma; I-V, current-voltage relationship.

¹ Current address: Abbott Laboratories, Chicago, IL 60064.

Address correspondence to: Dr. William H. Barry, Division of Cardiology, University of Utah Health Sciences Center, 50 North Medical Dr., Salt Lake City, UT 84132. E-mail: whbarry{at}med.utah.edu

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