T-Type and Tetrodotoxin-Sensitive Ca2+ Currents Coexist in Guinea Pig Ventricular Myocytes and Are Both Blocked by Mibefradil
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 (ICa(T)) or as tetrodotoxin (TTX)-sensitive Ca2+ current (ICa(TTX)). So far, coexistence of the 2 currents within the same type of myocyte has never been reported. We describe experimental conditions under which ICa(T) and ICa(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 ICa(T) and exhibit ICa(TTX) only, guinea pig myocytes possess both of these low-voltage–activated Ca2+ currents, which are separated pharmacologically by superfusion with TTX or Ni2+. ICa(T) and ICa(TTX) were of similar amplitude but significantly differed in their electrophysiological properties: ICa(TTX) activated at more negative potentials than did ICa(T), the potential for half-maximum steady-state inactivation was more negative, and current deactivation and recovery from inactivation were faster. ICa(TTX) but not ICa(T) increased after membrane rupture (“run-up”). Isolation of ICa(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 ICa(TTX). However, mibefradil not only blocked ICa(T) by 85±2% but also decreased ICa(TTX) by 48±8%. We conclude that under Na+-free conditions ICa(T) and ICa(TTX) coexist in guinea pig ventricular myocytes and that both currents are sensitive to mibefradil. Future investigations of ICa(T) will have to consider the TTX-sensitive current component to avoid possible interference.
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 (ICa(T))1 2 and the tetrodotoxin (TTX)-sensitive Na+ current (INa).3 ICa(T) has been reported to contribute to pacemaking in sinoatrial cells4 in conjunction with the hyperpolarization-activated current If.5 6 Very recently, 3 T-type Ca2+ channel–forming α1 subunits have been cloned and expressed, ie, α1G, α1H, and α1I.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
INa, on the other hand, determines excitation of the working myocardium. Under physiological conditions, this current is much larger in amplitude than ICa(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 (ICa(TTX)). The relation between ICa(TTX) and the slip-mode conductance of Na+ channels is still a matter of debate.20
Rat ventricular myocytes lack a macroscopic ICa(T),21 and ICa(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 ICa(T) and ICa(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 ICa(T), TTX was often applied in addition to Na+-free superfusion solutions to ensure complete elimination of INa. Remarkably, in the only study so far involving ICa(TTX) in guinea pig ventricular myocytes, ICa(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 ICa(T) and ICa(TTX). Identification of ICa(TTX) was supported by a comparison with rat myocytes, in which ICa(TTX) is the only LVACC.
Materials and Methods
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.
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 ).
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 (ICa(L)) by 2 protocols: (1) with test steps (3-second interval) from −115 to −50 mV, ie, negative to activation threshold for ICa(L), and (2) as difference current between the traces from the holding potential, which was −115 mV (both LVACC and ICa(L) available), and from −65 mV (only ICa(L) current available). The latter method was used for determining maximum LVACC amplitude and the respective potential (Vpeak).
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.
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.
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 ICa(L) are shown in Figure 1⇓ (bottom) for rat and guinea pig myocytes.
In rat myocytes, current-voltage relations exhibited clear separation of LVACC and ICa(L). LVACC was completely blocked by 30 μmol/L TTX and therefore identified as ICa(TTX) (Figure 2D⇓). In guinea pig myocytes, however, LVACC partially overlapped ICa(L) because of activation over a broader potential range (Figure 2A⇓), suggesting that LVACC might consist of >1 component. The contribution of ICa(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 ICa(TTX) found in rat myocytes (Figure 2D⇓). Indeed, Ni2+-resistant current was blocked by TTX and hence identified as ICa(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 [nH] −0.96). In guinea pig cells, TTX (IC50 1.2 μmol/L, nH −1.3) and Ni2+ (IC50 16 μmol/L, nH −2.1) each blocked ≈50% of the total current.
Because both LVACC components of guinea pig myocytes could be activated by clamp steps from −115 to −50 mV without interference from ICa(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 ICa(T) and ICa(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⇓).
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 ICa(T) and ICa(TTX) to the total current increase in guinea pig myocytes was studied in the presence of TTX and Ni2+, respectively; ICa(TTX) of rat myocytes was recorded without blockers. Figure 4⇓ shows that ICa(T) could be measured immediately after series resistance compensation and that it remained relatively constant. In contrast, ICa(TTX) of guinea pig and rat myocytes was often undetectable during the first 2 minutes before it started to increase (“run-up”).
Steady-State Inactivation of ICa(T) and ICa(TTX)
Steady-state inactivation curves for ICa(T) and ICa(TTX) were obtained by using a conventional protocol (see inset in Figure 5B⇓), in which ICa(T) and ICa(TTX) were dissected pharmacologically by adding TTX and Ni2+, respectively. In this set of experiments, ICa(T) had a higher amplitude than ICa(TTX) (Figure 5A⇓) and therefore dominated steady-state inactivation of the total LVACC. In the absence of any blocker (ie, ICa(T)+ICa(TTX)), the potential for half-maximum steady-state inactivation (V0.5) 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 ICa(TTX) inactivated at more negative potentials than did ICa(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 ICa(TTX) and −3.2±0.3 mV for ICa(T)).
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⇑), ICa(TTX) recovered faster from inactivation than did ICa(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, ICa(T), and ICa(TTX)). The curve fits provided time constants τf and τs and amplitudes Af and As for fast and slowly recovering current fractions, respectively. The values are summarized in the Table⇓. The time constant τf for the fast recovering current fraction of ICa(TTX) was significantly smaller than τf of ICa(T). The time constants τf and τs of total LVACCs had intermediate values.
Deactivation Kinetics of ICa(L), ICa(T), and ICa(TTX)
Deactivation of ICa(L) occurs significantly faster than deactivation of ICa(T).25 Therefore, we investigated whether this current characteristic is also suitable to distinguish between ICa(T) and ICa(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 ICa(T) deactivation (4.6±0.5 milliseconds, n=7; P<0.001) was significantly larger than the time constants for ICa(L) (1.3±0.2 milliseconds, n=8) and for ICa(TTX) (0.7±0.1 milliseconds, n=8). There was no significant difference between the latter values.
Mibefradil Block of ICa(T) and ICa(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 ICa(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 ICa(T) and does not affect ICa(TTX). This was investigated by applying mibefradil (10 μmol/L) in the presence of TTX or Ni2+. Mibefradil reversibly reduced not only ICa(T) (Figure 7A⇓) but also ICa(TTX) (Figure 7B⇓). At test steps from −115 to −50 mV, the mean block of ICa(T) by mibefradil was 85±2% (n=6), whereas the mean block of ICa(TTX) was 48±8% (n=5). Mibefradil blocked ICa(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⇓).
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 ICa(T) previously identified under similar conditions.26 When studying ICa(T) of guinea pig ventricular myocytes, almost all investigators eliminated INa 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 ICa(TTX) described here.
The ICa(TTX) characterized in the present study corresponds well to the ICa(TTX) described previously in human atrial17 and rat ventricular 18 myocytes. An ICa(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, ICa(T) was not found.19 The lack of ICa(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 ICa(TTX) and ICa(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 ICa(TTX)-conducting channel: (1) ICa(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 ICa(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) ICa(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 ICa(TTX) but add an additional current (ie, INa) with increasing Na+ concentration. In support of the latter hypothesis, we did not observe ICa(TTX) reduction after the addition of low Na+ concentrations (Figure 3C⇑), although this was not studied in detail.
Separation of ICa(T) and ICa(TTX)
Superfusion of the myocytes with TTX isolates ICa(T), whereas Ni2+ preserves ICa(TTX). Vpeak and V0.5 of ICa(TTX) were more negative than Vpeak and V0.5 of ICa(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 ICa(T), there is general agreement that current characteristics are not modified by TTX,1 27 whereas in the case of ICa(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, ICa(TTX) is contaminated by ICa(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 ICa(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 Vpeak and V0.5 of ICa(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.
ICa(T) and ICa(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 ICa(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 ICa(TTX) and ICa(T). As shown previously,25 tail currents of ICa(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 ICa(TTX) by application of Ni2+ is critical for the reasons discussed, we aimed to block ICa(T) by mibefradil. We found that mibefradil not only blocked ICa(T) but also decreased ICa(TTX). Thus, the compound is not a suitable tool for the study of ICa(TTX) in guinea pig ventricular myocytes. Block of ICa(TTX) provides another example of the limited selectivity of mibefradil, which also impairs currents other than ICa(T) at moderately higher concentrations.
Relation Between ICa(TTX) and Slip-Mode Conductance
A detailed study of ICa(TTX) suggested that the current is conducted by Na+ channels that are functionally distinct from those conducting the classic INa.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 ICa(TTX), which triggers Ca2+-induced Ca2+ release, which in turn leads to intracellular Ca2+ transients. At present, the relation between ICa(TTX) and slip-mode conductance is the subject of controversy.20
In our view, the Ca2+ current due to slip-mode conductance and ICa(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 ICa(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 ICa(TTX) even in the absence of protein kinase A activators. However, the IC50 values for the TTX block of ICa(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 INa 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, ICa(TTX) was measured under Na+-free conditions, ie, without interference of INa. Provided that ICa(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 ICa(TTX) by modified Na+ channels. We conclude that ICa(TTX) and Ca2+ current via slip-mode conductance are 2 separate currents.
In summary, the present study demonstrates that ICa(T) and ICa(TTX) coexist in guinea pig ventricular myocytes under Na+-free conditions. Both LVACCs are sensitive to mibefradil. Future physiological and pharmacological investigations of ICa(T) will have to consider the TTX-sensitive Ca2+ current to avoid possible interference.
The excellent technical assistance of Manja Schöne and Margarete Siess is gratefully acknowledged.
- Received June 29, 1999.
- Accepted December 22, 1999.
- © 2000 American Heart Association, Inc.
Bean BP. Two kinds of calcium channels in canine atrial cells: differences in kinetics, selectivity, and pharmacology. J Gen Physiol. 1985;86:1–30.
Cribbs LL, Lee J-H, Yang J, Satin J, Zhang Y, Daud A, Barclay J, Williamson MP, Fox M, Rees M, Perez-Reyes E. Cloning and characterization of α1H from human heart, a member of the T-type Ca2+ channel gene family. Circ Res. 1998;83:103–109.
Lee JH, Daud AN, Cribbs LL, Lacerda AE, Pereverzev A, Klöckner U, Schneider T, Perez-Reyez E. Cloning and expression of a novel member of the low voltage activated T-type calcium channel family. J Neurosci. 1999;19:1912–1921.
Xu X, Best PM. Increase in T-type calcium current in atrial myocytes from adult rats with growth hormone-secreting tumors. Proc Natl Acad Sci U S A. 1990;87:4655–4659.
Nuss HB, Houser SR. T-type Ca2+ current is expressed in hypertrophic adult feline left ventricular myocytes. Circ Res. 1993;73:777–782.
Sen L, Smith TW. T-type Ca2+ channels are abnormal in genetically determined cardiomyopathic hamster hearts. Circ Res. 1994;75:149–155.
Santana LF, Gómez AM, Lederer WJ. Ca2+ flux through promiscuous cardiac Na+ channels: slip-mode conductance. Science. 1998;279:1027–1033.
Cole WC, Chartier D, Martin M, Leblanc N. Ca2+ permeation through Na+ channels in guinea pig ventricular myocytes. Am J Physiol. 1997;273:H128–H137.
Nuss HB, Marban E; Balke CW, Goldman L, Aggarwal R, Shorofsky SR. Whether ‘slip-mode conductance’ occurs. Science. 1999;284:711.
Heubach JF, Trebeβ I, Wettwer E, Himmel HM, Michel MC, Kaumann AJ, Koch WJ, Harding SE, Ravens U. L-type calcium current and contractility in ventricular myocytes from mice overexpressing the cardiac β2-adrenoceptor. Cardiovasc Res. 1998;42:173–182.
Cohen CJ, Spires S, Van Skiver D. Block of T-type Ca channels in guinea pig atrial cells by antiarrhythmic agents and Ca channel antagonists. J Gen Physiol. 1992;100:703–728.
Mitra R, Morad M. Two types of calcium channels in guinea pig ventricular myocytes. Proc Natl Acad Sci U S A. 1986;83:5340–5344.
Li G-R, Feng J, Yue L, Carrier M, Nattel S. Evidence for two components of delayed rectifier K+ current in human ventricular myocytes. Circ Res. 1996;78:689–696.