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(Circulation Research. 2000;86:628.)
© 2000 American Heart Association, Inc.

Cellular Biology

T-Type and Tetrodotoxin-Sensitive Ca²⁺ Currents Coexist in Guinea Pig Ventricular Myocytes and Are Both Blocked by Mibefradil

Jürgen F. Heubach, Arndt Köhler, Erich Wettwer, Ursula Ravens

From the Institut für Pharmakologie und Toxikologie, Universitätsklinikum Carl Gustav Carus, Technische Universität Dresden, Dresden, Germany.

Correspondence to Prof Dr Ursula Ravens, Institut für Pharmakologie und Toxikologie, Universitätsklinikum der TU Dresden, Karl-Marx-Str. 3, D-01109 Dresden, Germany. E-mail ravens{at}rcs.urz.tu-dresden.de

	Abstract

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Abstract—Under Na⁺-free conditions, low-voltage–activated Ca²⁺ currents in cardiomyocytes from various species have been described either as Ni²⁺-sensitive T-type Ca²⁺ current (I_Ca(T)) or as tetrodotoxin (TTX)-sensitive Ca²⁺ current (I_Ca(TTX)). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which I_Ca(T) and I_Ca(TTX) can be separated and studied in the same cell. Rat and guinea pig ventricular myocytes were investigated with the whole-cell voltage-clamp technique in Na⁺-free solutions. Whereas rat myocytes lack I_Ca(T) and exhibit I_Ca(TTX) only, guinea pig myocytes possess both of these low-voltage–activated Ca²⁺ currents, which are separated pharmacologically by superfusion with TTX or Ni²⁺. I_Ca(T) and I_Ca(TTX) were of similar amplitude but significantly differed in their electrophysiological properties: I_Ca(TTX) activated at more negative potentials than did I_Ca(T), the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster. I_Ca(TTX) but not I_Ca(T) increased after membrane rupture ("run-up"). Isolation of I_Ca(TTX) by application of the bivalent cation Ni²⁺ is critical because of possible shifts in voltage dependence. Therefore, we investigated whether the T-type Ca²⁺ channel blocker mibefradil (10 µmol/L) is a suitable tool for the study of I_Ca(TTX). However, mibefradil not only blocked I_Ca(T) by 85±2% but also decreased I_Ca(TTX) by 48±8%. We conclude that under Na⁺-free conditions I_Ca(T) and I_Ca(TTX) coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of I_Ca(T) will have to consider the TTX-sensitive current component to avoid possible interference.

Key Words: T-type Ca²⁺ currents • tetrodotoxin-sensitive Ca²⁺ currents • guinea pig ventricular myocytes • mibefradil

	Introduction

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Low-voltage–activated inward currents of myocardial cells are critical for the generation and conduction of physiological excitation. Well-characterized currents include the T-type Ca²⁺ current (I_Ca(T))^{1 2} and the tetrodotoxin (TTX)-sensitive Na⁺ current (I_Na).³ I_Ca(T) has been reported to contribute to pacemaking in sinoatrial cells⁴ in conjunction with the hyperpolarization-activated current I_f.^{5 6} Very recently, 3 T-type Ca²⁺ channel–forming 1 subunits have been cloned and expressed, ie, 1G, 1H, and 1I.^{7 8 9} Significant mRNA levels of the former 2 were detected in cardiac tissue.^{7 8} The expression of T-type Ca²⁺ channels in myocytes from the working myocardium declines with maturation¹⁰ but is reactivated in certain models of disease.^{11 12 13}

I_Na, on the other hand, determines excitation of the working myocardium. Under physiological conditions, this current is much larger in amplitude than I_Ca(T).¹⁴ In the past, ion selectivity of the Na⁺ channel has been considered an exclusive channel property linked to distinct amino acid sequences within the pore-forming loop of the channel protein,¹⁵ but recent reports suggest that cardiac Na⁺ channels may in fact allow Ca²⁺ to pass when the channels become modified by phosphorylation or by the presence of cardiotonic steroids like ouabain or digoxin ("slip-mode conductance"¹⁶ ).

Recently, another low-voltage–activated inward current was observed under Na⁺-free experimental conditions in human atrial cells¹⁷ as well as in rat¹⁸ and guinea pig¹⁹ ventricular myocytes. This current was found to be carried by Ca²⁺ passing through a subpopulation of distinct TTX-sensitive Na⁺ channels and was therefore referred to as TTX-sensitive Ca²⁺ current (I_Ca(TTX)). The relation between I_Ca(TTX) and the slip-mode conductance of Na⁺ channels is still a matter of debate.²⁰

Rat ventricular myocytes lack a macroscopic I_Ca(T),²¹ and I_Ca(TTX) is the only Ca²⁺ current that is activated in the low potential range.¹⁸ With ventricular myocytes from guinea pig heart, results from various studies are more controversial. Both I_Ca(T) and I_Ca(TTX) have been demonstrated individually in separate studies; however, to the best of our knowledge, evidence for coexistence of the 2 currents in the same myocytes has never been reported. One reason may be that when the focus was on I_Ca(T), TTX was often applied in addition to Na⁺-free superfusion solutions to ensure complete elimination of I_Na. Remarkably, in the only study so far involving I_Ca(TTX) in guinea pig ventricular myocytes, I_Ca(T) has not been detected.¹⁹

In the present study, we analyzed the low-voltage–activated Ca²⁺ current (LVACC) of guinea pig ventricular myocytes for a possible coexistence of I_Ca(T) and I_Ca(TTX). Identification of I_Ca(TTX) was supported by a comparison with rat myocytes, in which I_Ca(TTX) is the only LVACC.

	Materials and Methods

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Cell Isolation
All studies complied with the German Home Office Regulations Governing the Care and Use of Laboratory Animals. Ventricular myocytes were isolated by standard enzymatic dissociation of hearts from adult male Wistar rats (360±22 g, n=11) and Dunkin-Hartley guinea pigs (305±11 g, n=34), respectively. For rat hearts, 300 U/mL collagenase (Worthington type 1, 254 U/mg) was used, and for guinea pig hearts, an additional 0.034 mg/mL pronase E (Merck, 4000 proteolytic units per milligram) was used. The preparation buffer contained 150 mmol/L NaCl. Single myocytes were harvested into Na⁺-free storage buffer and washed 3 times before use.

Current Measurements
Whole-cell voltage-clamp details have been reported previously.²² Average values for membrane capacitance measured with 5-millisecond depolarizing ramp pulses (from -55 to -50 mV) were 195±6 pF (rat, n=29) and 138±4 pF (guinea pig, n=75; mean±SEM). Series resistance was routinely compensated by 50% to 70%. Currents were low-pass–filtered at 2 kHz.

In general, experiments were performed at 36±1°C with the following Na⁺-free superfusion solution (mmol/L): tetraethylammonium chloride 120, CsCl 10, HEPES 10, CaCl₂ 2, MgCl₂ 1, and glucose 10, pH 7.4 (adjusted with CsOH). Tail currents (Figure 6) were recorded at 21±1°C and with 10 mmol/L [Ca²⁺]_o. The pipette solution (pH 7.2) included (mmol/L) cesium methanesulfonate 90, CsCl 20, HEPES 10, Mg-ATP 4, Tris-GTP 0.4, EGTA 10, and CaCl₂ 3, with calculated free Ca²⁺ and Mg²⁺ concentrations of 60 nmol/L and 450 µmol/L, respectively.²³ After experimentation, all membrane potentials were corrected for the calculated liquid junction potential of -15 mV (JPCalc version 2.2 by Barry²⁴ ).

View larger version (15K): [in this window] [in a new window]   Figure 6. Deactivation of Ca2+ current tails in guinea pig myocytes. A, Clamp protocol. B, Original current traces. C, Difference current obtained by subtraction of traces in panel B. Tail currents were recorded at -60 mV after brief current activation for 4 to 8 milliseconds from Vh -115 mV (•). Capacitive transients during the step to -60 mV were measured after a 200-millisecond Ca2+ current inactivating step to 0 mV () and subtracted from the current obtained with the activating protocol. Time constants of current deactivation were deduced from monoexponential curve fits to the tail currents (difference currents). All tail currents were recorded at 21±1°C and 10 mmol/L [Ca2+]o. Under these conditions, voltage dependence was shifted by 10 mV toward more positive potentials, compared with the conditions at 36°C and 2 mmol/L [Ca2+]o. Separation of individual current components was as follows: for ICa(L), Ni2+ (100 µmol/L) and TTX (30 µmol/L); for ICa(T), TTX (30 µmol/L) and nifedipine (5 µmol/L); and for ICa(TTX), Ni2+ (100 µmol/L) and nifedipine (5 µmol/L).

Current amplitude was determined as the difference between peak inward current and current at the end of the depolarizing step. LVACCs were separated from high-voltage–activated L-type Ca²⁺ current (I_Ca(L)) by 2 protocols: (1) with test steps (3-second interval) from -115 to -50 mV, ie, negative to activation threshold for I_Ca(L), and (2) as difference current between the traces from the holding potential, which was -115 mV (both LVACC and I_Ca(L) available), and from -65 mV (only I_Ca(L) current available). The latter method was used for determining maximum LVACC amplitude and the respective potential (V_peak).

Chemicals
Mibefradil (ASTA Medica AWD) and TTX citrate (Tocris) were dissolved in H₂O as stock solutions (10 mmol/L). Nifedipine (RBI) was dissolved in dimethyl sulfoxide (10 mmol/L). Aliquots were stored at -20°C until use. All chemicals were purchased from commercial suppliers and were of analytical grade.

Data Analysis
Absolute current amplitudes (in pA) were corrected for cell size and expressed in pA/pF. pClamp software (Clampfit) or Prism (Graphpad Software) was used for appropriate curve fitting to experimental data.

Significance of differences between means (±SEM) of 2 groups was analyzed by use of the unpaired Student t test or Mann-Whitney test. Deactivation kinetics of tail currents were compared by ANOVA followed by the Bonferroni multiple comparisons test. Differences were considered significant at P<0.05.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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LVACCs of Rat and Guinea Pig Ventricular Myocytes
Current-voltage relations for rat and guinea pig ventricular myocytes clearly indicated LVACC (Figure 1). In rat cells, the LVACC peaked at -53.0±1.1 mV and had a maximum amplitude of -1.07±0.18 pA/pF (n=11). The LVACC of guinea pig myocytes was of similar amplitude (-1.18±0.12 pA/pF, n=11) but peaked at a more positive potential (-43.2±1.0 mV, P<0.0001). Typical current tracings for LVACC and I_Ca(L) are shown in Figure 1 (bottom) for rat and guinea pig myocytes.

View larger version (18K): [in this window] [in a new window]   Figure 1. Current-voltage (I-Vm) relations and original Ca2+ current traces of representative ventricular myocytes from rat heart (A) and guinea pig heart (B). Top, I-Vm relations recorded from 2 holding potentials (-115 mV [•] and -65 mV []) to allow separation of LVACCs and ICa(L). Bottom, Original current recordings at steps to -55 mV (LVACC maximum in rat myocyte), -45 mV (LVACC maximum in guinea pig myocyte), and -5 mV (range of ICa(L) maximum). Capacitive transients at the beginning of the test pulse were cut. Arrowheads indicate zero current.

In rat myocytes, current-voltage relations exhibited clear separation of LVACC and I_Ca(L). LVACC was completely blocked by 30 µmol/L TTX and therefore identified as I_Ca(TTX) (Figure 2D). In guinea pig myocytes, however, LVACC partially overlapped I_Ca(L) because of activation over a broader potential range (Figure 2A), suggesting that LVACC might consist of >1 component. The contribution of I_Ca(T) was tested with Ni²⁺, which clearly decreased the LVACC at potentials positive to -50 mV (Figure 2B). Ni²⁺-sensitive current peaked at -40.9±0.6 mV and had a maximum amplitude of -0.88±0.06 pA/pF (n=11), whereas Ni²⁺-resistant current was similar in amplitude (-0.72±0.07 pA/pF) but peaked at a more negative potential (-49.1±0.6 mV, P<0.0001). The current-voltage relation of Ni²⁺-resistant LVACC remarkably resembled that of I_Ca(TTX) found in rat myocytes (Figure 2D). Indeed, Ni²⁺-resistant current was blocked by TTX and hence identified as I_Ca(TTX) (Figure 2C). Concentration-response curves (Figure 2E) indicated that the entire LVACC in rat myocytes was sensitive to TTX (IC₅₀ 1.7 µmol/L, Hill coefficient [n_H] -0.96). In guinea pig cells, TTX (IC₅₀ 1.2 µmol/L, n_H -1.3) and Ni²⁺ (IC₅₀ 16 µmol/L, n_H -2.1) each blocked 50% of the total current.

View larger version (28K): [in this window] [in a new window]   Figure 2. Block of LVACCs by Ni2+ and TTX. In panels A through C, I-Vm relations of a representative guinea pig myocyte are shown. A, Control (same data are indicated by lines in panels B and C). Vh indicates holding potential. B, In the presence of Ni2+. C, With Ni2+ and TTX (10 µmol/L). D, I-Vm relations in a representative rat myocyte in the presence and absence of TTX (30 µmol/L). E, Concentration-response curves for block of LVACCs. In guinea pig myocytes (test potential -50 mV), Ni2+ and TTX each blocked 50% of the current, whereas in rat myocytes (test potential -55 mV), the entire LVACC was sensitive to TTX. There were 3 to 6 values per concentration (see text for IC50 and nH values).

Because both LVACC components of guinea pig myocytes could be activated by clamp steps from -115 to -50 mV without interference from I_Ca(L), the time courses of the effects of TTX and Ni²⁺ were studied at this potential (Figure 3). The amplitude of LVACC increased substantially during the initial few minutes after membrane rupture. Therefore, at least 6 minutes of equilibration was always allowed before starting further experimental protocols. Successive application of TTX or Ni²⁺ blocked 50% of the current (Figure 3A). Total LVACC and the residual currents I_Ca(T) and I_Ca(TTX) (indicated as a, b, and c, respectively, in Figure 3A) had similar kinetics of inactivation. The time constants from monoexponential curve fitting were 6.7±0.2 milliseconds (n=45), 6.6±0.3 milliseconds (n=20), and 6.3±0.3 milliseconds (n=22), respectively. Cumulative application of TTX and Ni²⁺ almost completely blocked the total LVACC (Figure 3B).

View larger version (18K): [in this window] [in a new window]   Figure 3. Time course (top) and individual current traces (bottom) of LVACC of guinea pig ventricular myocytes, with small letters indicating time point of current traces below (arrowheads indicate zero current). A, Successive superfusion with TTX and Ni2+. B, Cumulative application of Ni2+ and TTX. C, Effects of various extracellular Na+ concentrations and removal of extracellular Ca2+ on TTX-sensitive current, ICa(T), blocked with Ni2+. Note fast washout of 1000 µmol/L Na+ and absence of current during superfusion with Ca2+-free solution (CaCl2 replaced with MgCl2).

Although the cells were superfused with nominally Na⁺-free solutions, Na⁺ contamination originating from cell isolation procedures could still be present and could provide the charge for current passing through TTX-sensitive Na⁺ channels. However, such Na⁺ should be washed out rapidly, as simulated in the experiment shown in Figure 3C, in which the current increase that was due to 1 mmol/L Na⁺ was reversed on washout within <1 minute. The remaining amplitude was completely abolished when all extracellular Ca²⁺ was replaced with Mg²⁺.

Development of LVACCs After Rupture of Membrane
LVACCs increased within the first minutes after access to the cell (Figure 3). The contribution of I_Ca(T) and I_Ca(TTX) to the total current increase in guinea pig myocytes was studied in the presence of TTX and Ni²⁺, respectively; I_Ca(TTX) of rat myocytes was recorded without blockers. Figure 4 shows that I_Ca(T) could be measured immediately after series resistance compensation and that it remained relatively constant. In contrast, I_Ca(TTX) of guinea pig and rat myocytes was often undetectable during the first 2 minutes before it started to increase ("run-up").

View larger version (17K): [in this window] [in a new window]   Figure 4. Representative examples of current amplitude after rupture of cell membrane. ICa(T) and ICa(TTX) from guinea pig ventricular myocytes were measured in the presence of TTX (30 µmol/L) and Ni2+ (40 µmol/L), respectively; ICa(TTX) in rat myocytes was recorded without any blockers. Test potentials were -55 mV in guinea pig cells and -50 mV in rat cells.

Steady-State Inactivation of I_Ca(T) and I_Ca(TTX)
Steady-state inactivation curves for I_Ca(T) and I_Ca(TTX) were obtained by using a conventional protocol (see inset in Figure 5B), in which I_Ca(T) and I_Ca(TTX) were dissected pharmacologically by adding TTX and Ni²⁺, respectively. In this set of experiments, I_Ca(T) had a higher amplitude than I_Ca(TTX) (Figure 5A) and therefore dominated steady-state inactivation of the total LVACC. In the absence of any blocker (ie, I_Ca(T)+I_Ca(TTX)), the potential for half-maximum steady-state inactivation (V_0.5) and the slope factor were -69.0±1.0 mV and -3.9±0.3 mV (n=9), respectively. However, after separation of the 2 current components and normalizing to unity (Figure 5B), it became evident that I_Ca(TTX) inactivated at more negative potentials than did I_Ca(T) (-73.9±0.8 versus -69.5±0.4 mV, respectively; P<0.001; n=8). The slope factors were not significantly different (-3.9±1.1 mV for I_Ca(TTX) and -3.2±0.3 mV for I_Ca(T)).

View larger version (27K): [in this window] [in a new window]   Figure 5. Steady-state inactivation and recovery from inactivation of LVACCs of guinea pig ventricular myocytes. ICa(T) and ICa(TTX) were measured in TTX (30 µmol/L) and Ni2+ (40 µmol/L), respectively. Top, Steady-state inactivation of total LVACC and its 2 components. For voltage protocol, see inset in panel B. Absolute (A) and normalized (B) data were fit with Boltzmann functions. Bottom, Recovery from inactivation. For voltage protocol, see inset in panel C. For typical current traces, see inset in panel D. Arrowheads indicate zero current. Absolute (C) and normalized (D) data were fit with double exponential functions (see Table).

Recovery From Inactivation
The kinetics of recovery from inactivation are characteristic properties that may also help to distinguish between current components (Figures 5C and 5D). As shown by the original recordings (insets in Figure 5D), I_Ca(TTX) recovered faster from inactivation than did I_Ca(T). For a more detailed analysis, the currents of individual cells were fitted with double exponential functions, which described the data with the highest accuracy for all 3 conditions (total LVACC, I_Ca(T), and I_Ca(TTX)). The curve fits provided time constants _f and _s and amplitudes A_f and A_s for fast and slowly recovering current fractions, respectively. The values are summarized in the Table. The time constant _f for the fast recovering current fraction of I_Ca(TTX) was significantly smaller than _f of I_Ca(T). The time constants _f and _s of total LVACCs had intermediate values.

View this table: [in this window] [in a new window]   Table 1. Recovery of LVACCs From Inactivation

Deactivation Kinetics of I_Ca(L), I_Ca(T), and I_Ca(TTX)
Deactivation of I_Ca(L) occurs significantly faster than deactivation of I_Ca(T).²⁵ Therefore, we investigated whether this current characteristic is also suitable to distinguish between I_Ca(T) and I_Ca(TTX). Tail currents of all 3 Ca²⁺ currents were recorded at -60 mV (Figure 6). To slow the pace of deactivation for more accurate recordings, these experiments were performed at 21±1°C. The time constant for I_Ca(T) deactivation (4.6±0.5 milliseconds, n=7; P<0.001) was significantly larger than the time constants for I_Ca(L) (1.3±0.2 milliseconds, n=8) and for I_Ca(TTX) (0.7±0.1 milliseconds, n=8). There was no significant difference between the latter values.

Mibefradil Block of I_Ca(T) and I_Ca(TTX)
Separation of the 2 LVACCs by application of Ni²⁺ might be critical because of the possible shifts in voltage dependence of the remaining currents. Therefore, we aimed to suppress I_Ca(T) with the T-type Ca²⁺ channel blocker mibefradil. However, mibefradil may be used as a tool to separate the 2 LVACCs only if it is highly selective for I_Ca(T) and does not affect I_Ca(TTX). This was investigated by applying mibefradil (10 µmol/L) in the presence of TTX or Ni²⁺. Mibefradil reversibly reduced not only I_Ca(T) (Figure 7A) but also I_Ca(TTX) (Figure 7B). At test steps from -115 to -50 mV, the mean block of I_Ca(T) by mibefradil was 85±2% (n=6), whereas the mean block of I_Ca(TTX) was 48±8% (n=5). Mibefradil blocked I_Ca(TTX) over the whole potential range for activation, as illustrated by the current-voltage relations in the absence and presence of the drug (Figure 7C).

View larger version (25K): [in this window] [in a new window]   Figure 7. A and B, Effects of mibefradil on peak ICa(T) (A) and ICa(TTX) (B) in guinea pig ventricular myocytes. LVACC components were dissected with TTX and Ni2+ as indicated. C, I-Vm relations of LVACC and part of ICa(L) under control conditions (left), in the presence of Ni2+ (middle), and with Ni2+ and mibefradil (right, n=4). Vh values were -115 mV (filled symbols) and -65 mV (open symbols). The dashed lines represent control data at Vh -115 mV. Mibefradil blocked ICa(TTX) over the whole potential range for activation (right).

	Discussion

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Characterization of the Two LVACCs
In guinea pig ventricular myocytes, 2 LVACCs can be distinguished under nominally Na⁺-free conditions on the basis of their sensitivity to the blockers Ni²⁺ and TTX as well as by their distinct electrophysiological properties. The Ni²⁺-sensitive current shares many characteristics with I_Ca(T) previously identified under similar conditions.²⁶ When studying I_Ca(T) of guinea pig ventricular myocytes, almost all investigators eliminated I_Na by using Na⁺-free superfusion solutions.^{2 21 25 26 27} Most investigators even added Na⁺ channel blockers like TTX to the external solutions to block any residual current due to Na⁺ contamination or Ca²⁺ permeation.²⁷ The presence of TTX certainly excluded the characterization of I_Ca(TTX) described here.

The I_Ca(TTX) characterized in the present study corresponds well to the I_Ca(TTX) described previously in human atrial¹⁷ and rat ventricular ¹⁸ myocytes. An I_Ca(TTX) was also detected in 1 study involving guinea pig ventricular myocytes; the IC₅₀ value of 2.4 µmol/L resembled the value reported in the present study, although for reasons unknown, I_Ca(T) was not found.¹⁹ The lack of I_Ca(T) could have been caused by significant differences in cell isolation procedures, which are often critical for detection of a specific ionic current.²⁸ In contrast to that previous work,¹⁹ we found the coexistence of I_Ca(TTX) and I_Ca(T) under comparable (21°C, 10 mmol/L [Ca²⁺]_o; Figure 6) and more physiological (36°C, 2 mmol/L [Ca²⁺]_o) conditions.

We consider Ca²⁺ to be the charge carrier for TTX-sensitive current, because amplitude was abolished on removal of extracellular Ca²⁺. Certainly, Na⁺ contamination originating from cell isolation procedures could have a large impact because of the enormous size of regular Na⁺ current amplitude in relation to the currents investigated in the present study. However, Figure 3C shows that 1 mmol/L Na⁺, which simulates Na⁺ contamination, could be washed out rapidly. Nevertheless, traces of Na⁺ have to be assumed in our nominally Na⁺-free solutions. This Na⁺ contamination could affect current amplitude in 2 ways, depending on the true, yet unsettled, nature of the I_Ca(TTX)-conducting channel: (1) I_Ca(TTX) might be conducted by classic Na⁺ channels that reversibly alter their properties under nominally Na⁺-free conditions.¹⁹ This hypothesis was based on the finding that Na⁺ concentrations in the micromolar range reduced I_Ca(TTX) by a competitive interaction between Ca²⁺ and Na⁺ at the channel pore, leading to block of Ca²⁺ conduction and switch from Ca²⁺ to Na⁺ permeation as the Na⁺ concentration is increased.¹⁹ (2) I_Ca(TTX) is conducted by a distinct TTX-sensitive channel that is different from the classic Na⁺ channel.¹⁸ In this case, Na⁺ contamination should not decrease I_Ca(TTX) but add an additional current (ie, I_Na) with increasing Na⁺ concentration. In support of the latter hypothesis, we did not observe I_Ca(TTX) reduction after the addition of low Na⁺ concentrations (Figure 3C), although this was not studied in detail.

Separation of I_Ca(T) and I_Ca(TTX)
Superfusion of the myocytes with TTX isolates I_Ca(T), whereas Ni²⁺ preserves I_Ca(TTX). V_peak and V_0.5 of I_Ca(TTX) were more negative than V_peak and V_0.5 of I_Ca(T). These differences may be taken as evidence for 2 independent currents, but only if the respective separating procedure by itself does not affect the voltage dependence of residual currents. In the case of I_Ca(T), there is general agreement that current characteristics are not modified by TTX,^{1 27} whereas in the case of I_Ca(TTX), it cannot be excluded that Ni²⁺ influences current properties. Application of 50 µmol/L Ni²⁺ was found to alter the inactivation of current elicited at -25 mV. However, at this potential, I_Ca(TTX) is contaminated by I_Ca(L) (see Figure 1B),¹⁹ which limits interpretation. In rat ventricular myocytes, the same Ni²⁺ concentration affected neither current amplitude nor the current-voltage relation of I_Ca(TTX).¹⁸ Theoretically, Ni²⁺ would shift activation and steady-state inactivation curves toward more positive potentials by screening off fixed membrane charges because of its nature as a bivalent cation.²⁹ Therefore, the more negative values for V_peak and V_0.5 of I_Ca(TTX) cannot be caused by Ni²⁺, which is expected to produce a shift into the opposite direction. If anything, the presence of Ni²⁺ would lead to an underestimation of the true differences between the 2 LVACCs.

I_Ca(T) and I_Ca(TTX) differ not only in their voltage dependence but also in current development after breaking the membrane (Figure 4). The reason for run-up of I_Ca(TTX) is presently unclear; its time course suggests some relation to cell dialysis. In addition, the kinetics of recovery from inactivation and current deactivation are significantly different between I_Ca(TTX) and I_Ca(T). As shown previously,²⁵ tail currents of I_Ca(T) decayed with a relatively slow time constant.

Selectivity of Mibefradil
Only few blockers of Ca²⁺ channels are sufficiently selective for a distinct Ca²⁺ channel subtype in order to be exploited as pharmacological tools. The nondihydropyridine mibefradil was introduced as a T-type Ca²⁺ channel blocker³⁰ because of its selectivity for T-type over L-type Ca²⁺ channels. Because separation of I_Ca(TTX) by application of Ni²⁺ is critical for the reasons discussed, we aimed to block I_Ca(T) by mibefradil. We found that mibefradil not only blocked I_Ca(T) but also decreased I_Ca(TTX). Thus, the compound is not a suitable tool for the study of I_Ca(TTX) in guinea pig ventricular myocytes. Block of I_Ca(TTX) provides another example of the limited selectivity of mibefradil, which also impairs currents other than I_Ca(T) at moderately higher concentrations.

Relation Between I_Ca(TTX) and Slip-Mode Conductance
A detailed study of I_Ca(TTX) suggested that the current is conducted by Na⁺ channels that are functionally distinct from those conducting the classic I_Na.¹⁸ Another study involving rat ventricular myocytes suggested that classic Na⁺ channels become "promiscuous" by phosphorylation or by the presence of cardiotonic steroids (slip-mode conductance).¹⁶ Under these conditions, the modified Na⁺ channels conduct I_Ca(TTX), which triggers Ca²⁺-induced Ca²⁺ release, which in turn leads to intracellular Ca²⁺ transients. At present, the relation between I_Ca(TTX) and slip-mode conductance is the subject of controversy.²⁰

In our view, the Ca²⁺ current due to slip-mode conductance and I_Ca(TTX) are not identical. One reason is that activation of protein kinase A or the presence of cardiotonic steroids was not a prerequisite for the detection of I_Ca(TTX) in the present or any previous study.^{17 18 19 20} Certainly, any basal activity of protein kinase A could modify a small fraction of Na⁺ channels to conduct I_Ca(TTX) even in the absence of protein kinase A activators. However, the IC₅₀ values for the TTX block of I_Ca(TTX) of 1.2 and 1.7 µmol/L in guinea pig and rat myocytes, respectively, reported in the present study, argue against this possibility for the following reasons: All experiments involving slip-mode conductance were performed in the presence of extracellular Na⁺. Thus, the Ca²⁺ current through modified Na⁺ channels was always measured in the simultaneous presence of I_Na and was therefore not assessed directly. Instead, the intracellular Ca²⁺ transient assumed to be triggered by this Ca²⁺ current was used as an indicator. These transients were blocked by TTX with an IC₅₀ value of 0.1 µmol/L. In contrast, the IC₅₀ value for the block of the current conducted by all Na⁺ channels (modified and unmodified) was 1 µmol/L.¹⁶ The authors concluded that modified Na⁺ channels are more sensitive to TTX than unmodified Na⁺ channels. In the present study, I_Ca(TTX) was measured under Na⁺-free conditions, ie, without interference of I_Na. Provided that I_Ca(TTX) was conducted by these modified Na⁺ channels, an IC₅₀ in the range of 0.1 µmol/L would have been expected. However, the 12-fold and 17-fold higher values measured in the present study argue against the conductance of I_Ca(TTX) by modified Na⁺ channels. We conclude that I_Ca(TTX) and Ca²⁺ current via slip-mode conductance are 2 separate currents.

In summary, the present study demonstrates that I_Ca(T) and I_Ca(TTX) coexist in guinea pig ventricular myocytes under Na⁺-free conditions. Both LVACCs are sensitive to mibefradil. Future physiological and pharmacological investigations of I_Ca(T) will have to consider the TTX-sensitive Ca²⁺ current to avoid possible interference.

	Acknowledgments

 
The excellent technical assistance of Manja Schöne and Margarete Siess is gratefully acknowledged.

Received June 29, 1999; accepted December 22, 1999.

	References

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