Properties of voltage-gated Ca2+ channels in rabbit ventricular myocytes expressing Ca2+ channel alpha 1E cDNA

Michihiro Tateyama1, Shuqin Zong2, Tsutomu Tanabe2, and Rikuo Ochi1

1 Department of Physiology, Juntendo University School of Medicine, Tokyo 113-8421; and 2 Department of Pharmacology and Neurobiology, Graduate School of Medicine, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8519, Japan


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Using the whole-cell patch-clamp technique, we have studied the properties of alpha 1E Ca2+ channel transfected in cardiac myocytes. We have also investigated the effect of foreign gene expression on the intrinsic L-type current (ICa,L). Expression of green fluorescent protein significantly decreased the ICa,L. By contrast, expression of alpha 1E with beta 2b and alpha 2/delta significantly increased the total Ca2+ current, and in these cells a Ca2+ antagonist, PN-200-110 (PN), only partially blocked the current. The remaining PN-resistant current was abolished by the application of a low concentration of Ni2+ and was little affected by changing the charge carrier from Ca2+ to Ba2+ or by beta -adrenergic stimulation. On the basis of its voltage range for activation, this channel was classified as a high-voltage activated channel. Thus the expression of alpha 1E did not generate T-like current in cardiac myocytes. On the other hand, expression of alpha 1E decreased ICa,L and slowed the ICa,L inactivation. This inactivation slowing was attenuated by the beta 2b coexpression, suggesting that the alpha 1E may slow the inactivation of ICa,L by scrambling with alpha 1C for intrinsic auxiliary beta .

green fluorescent protein; culture; L-type calcium channel; calcium channel beta -subunit; transfection


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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IN MAMMALIAN CARDIAC MYOCYTES, Ca2+ influx through L-type Ca2+ channels triggers excitation-contraction coupling. Functional L-type Ca2+ channel in cardiac myocytes is composed of at least four subunits: alpha 1C, alpha 2/delta , and beta , similar to the other high-voltage-activated (HVA) Ca2+ channels (11). Ion conducting pore, voltage sensor, and the binding sites for dihydropyridine (DHP) are all located on alpha 1C-subunit, while auxiliary subunits beta  and alpha 2/delta serve regulatory roles affecting the biophysical and pharmacological properties of evoked Ca2+ current (13, 15, 19). In cardiac myocytes, beta -subunit is considered to play a critical role in regulating L-type channel function, since overexpression of cardiac beta -subunit (beta 2a) enhances the L-type current amplitude and alters the inactivation kinetics (38). In addition, application of the antisense oligonucleotide against beta 2-subunit results in slowing of the decay of L-type current (35).

Recently, expression of alpha 1E-, alpha 1G-, and alpha 1H-subunit has been reported in young rat atrial myocytes (16). alpha 1E gene has been suggested to constitute part of the T-type current (3, 14, 21, 32, 33, 36), although major parts of the T-type current are thought to consist of alpha 1G (29), alpha 1H (6), and/or alpha 1I (18). In cardiac myocytes, application of antisense oligonucleotide against alpha 1E-subunit resulted in suppression of the cardiac T-type current induced by insulin-like growth factor-1 (IGF-1) (4, 30). T-type current is not detected in ventricular myocytes of normal rat and feline heart but is detected in those of hypertrophied hearts (20, 27). Moreover, properties of L-type current in hypertrophied hearts are reported to be altered, resulting in reduction of the current amplitude and slowing of the inactivation. These alterations might reflect the expression of alpha 1E gene, because alpha 1E-subunit is a candidate for T-type current and interacts with auxiliary subunits (25, 28). The latter could cause deficiency of auxiliary subunits for alpha 1C, leading to the alteration of L-type current properties. In the present study, we investigated the properties of voltage-gated Ca2+ channels in cultured adult ventricular myocytes expressing several exogenous cDNAs. In rabbit ventricular myocytes, neither alpha 1E-subunit (26) nor T-type channel (23) is expressed, so we have used these cells to investigate whether the alpha 1E channel expressed in cardiac myocytes shows T-like channel character. We have also investigated whether the expression of alpha 1E-subunit could affect the properties of intrinsic L-type current by the possible removal of accessory subunits from the alpha 1C-subunit.

We found that green fluorescent protein (GFP), which was used as a marker for successful cDNA expression, decreased L-type Ca2+ current (ICa,L) amplitude without affecting the biophysical properties of L-type channel. Expression of alpha 1E gene did not generate T-like current but slowed the decay of ICa,L as expected for the decrease of intrinsic beta -subunit associated with the L-type channel.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Plasmid construct. The 7.7-kb Hind III/Xba I fragment from pSPCBII-2 was ligated with Hind III/Xba I-cleaved pCDNA1/Amp (Invitrogen) to yield pBII-2E, encoding the alpha 1E-subunit (37). The 4.7-kb Sal I/Not I fragment from pBH17 was ligated with Sal I/Not I-cleaved pSV SPORT 1 (GIBCO BRL) to yield PSVCabeta 2b, encoding the cardiac beta  (beta 2b)-subunit (13). PKCRalpha 2, encoding the alpha 2/delta -subunit, was described previously (42). PCAGS65A, encoding a mutated form of GFP, was a gift from Dr. C. Akazawa.

Isolation and culture of ventricular myocytes. Single ventricular myocytes were isolated from the hearts of female Japanese White rabbit (1.5 kg) or male guinea pig (300 g) by collagenase treatment during Langendorff perfusion (1). Guinea pig myocytes were prepared and used for electrophysiology within 8 h after dissociation. Isolated rabbit myocytes were suspended in medium 199 without glutamate, plated onto laminin-coated glass coverslips in 35-mm culture dishes, and allowed to attach for 2 h at 37°C under an atmosphere of 5% CO2-95% air. Plated cells were washed twice and maintained in medium 199 supplemented with 5% fetal bovine serum, 0.25 µg/ml amphotericin B, 0.1 mg/ml streptomycin, and 100 U/ml penicillin. The culture medium was changed daily.

Transient transfection. After the myocytes had been cultured for 7 days in 35-mm dishes, they were transfected with expression plasmids encoding the alpha 1E-subunit and GFP, with or without the beta 2b- and alpha 2/delta -subunits. Effectene (Qiagen, Valencia, CA), which we found most effective for transient transfection in primary cultured cardiac myocytes, was used as a carrier. The plasmid DNAs were mixed with 25 µl of Effectene reagent and added into the 35-mm dishes containing 1.6 ml of culture medium to perform transfection according to the manufacturer's instructions. The plasmid DNA mass ratio was adjusted to 1:1:1:0.1 for alpha 1E:beta 2b:alpha 2/delta :GFP. Three or four days after transfection, electrophysiological experiments were performed, usually by selecting GFP-luminescent cells.

Electrophysiology. Macroscopic currents were measured by using the whole cell variant of the patch-clamp technique (10). Patch pipettes have resistances of 1-3 MOmega when filled with pipette solution containing (in mM) 140 CsCl, 10 EGTA, 3 MgATP, and 5 HEPES, pH 7.3 with CsOH. Ca2+ current (ICa) amplitude was measured at room temperature (22-25°C) while cells were bathed in the external solution consisting of (in mM) 135 tetraethylammonium chloride, 5.4 KCl, 10 CaCl2, 1 MgCl2, and 5 HEPES, pH 7.4 with Tris. Peak ICa amplitudes were estimated as the maximal inward deflection from holding current. Current amplitudes were normalized to the cell capacitance, estimated by analyzing the charging transients elicited by a 10-mV pulse, and were represented as current densities. All recordings were made with an EPC-7 patch-clamp amplifier (List-Electronik, Darmstadt, Germany); pCLAMP software (version 6; Axon Instruments, Foster City, CA) was used for both command-pulse delivery and data analysis.

Data analysis. The peak conductance of Ca2+ channels (G) was calculated as G = ICa/(ECa - Em), where ECa is the apparent reversal potential and Em is the test potential. G, normalized to maximal G (Gmax), was plotted against Em and then fitted by the Boltzmann equation of the form G = Gmax/[1 + exp(Em - V1/2,act)/k], where V1/2,act is the voltage evoking 50% activation and k is the slope factor. In addition, steady-state inactivation was determined from the channel availability after 1-s prepulses. The current amplitudes (I) elicited by depolarizing pulses to 30 mV from various prepulse potentials (V) were normalized to the maximum test current amplitude (Imax). Individual experimental points in each group were fitted to the Boltzmann equation I/Imax = 1/[1 + exp(V - V1/2,inact)/k], where V1/2,inact is the midpoint of inactivation.

Kinetics of Ca2+ current inactivation (tau inact) were estimated by fitting the trace elicited by 1-s depolarizing pulses to a single exponential function, except for the L-type current trace, which was fitted to a double exponential function. On the other hand, the activation kinetics (tau act) were estimated as the time from 10% to 90% of peak (tau 10-90).

Data are expressed as means ± SE. Statistical analysis was carried out with Bonferroni's multiple t-test, and differences at P < 0.05 were deemed significant.


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Expression of GFP in ventricular myocytes. Culture of adult rabbit ventricular myocytes results in an ~70% decrease of the inward rectifier K+ current (data not shown) but little change in the density of ICa,L, as reported previously (23). Exogenous cDNAs were expressed in cultured cardiac myocytes with an efficiency of 1-10%, as assessed from the number of GFP-luminescent cells. An example of a GFP-expressing luminescent myocyte is shown in Fig. 1. Significant changes in cell capacitance were not observed with the expression of exogenous cDNAs; the mean value of the cell capacitance is ~45 pF in each group. Figure 2A, left, depicts a typical ICa,L elicited in the presence of 10 mM Ca2+. PN-200-110 (PN; 10 µM), a DHP-type L-type Ca2+ channel blocker, almost completely suppressed the ICa,L (Fig. 2A, left, closed circle). ICa,L was first detected at depolarization to -20 mV from the holding potential and reached a peak at 30 mV (Fig. 2B, open circles). A typical ICa,L trace obtained from GFP-luminescent cells is represented in Fig. 2A, right. Average peak ICa,L in GFP-transfected cells (n = 11) was ~60% of that in the control (n = 13; Fig. 2B). Voltage-dependent activation, steady-state inactivation, and time courses of ICa,L in GFP-transfected cells are all similar to those in the control (Fig. 2C and Table 1). The pharmacological properties of ICa,L were also not affected by GFP expression. PN decreased the magnitude of ICa,L to <5% in GFP-transfected myocytes (Fig. 2A, right, closed square; Table 1). From these results, transfection and expression of GFP decreased ICa,L amplitudes (P < 0.05) without affecting L-type channel function and without yielding a PN-resistant component of the Ca2+ current.


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Fig. 1.   Cardiac myocyte transfected with the expression plasmid encoding green fluorescence protein (GFP). A: image of cardiac myocyte obtained by bright-field microscopy. B: fluorescent image of the same cell.



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Fig. 2.   Effects of GFP expression on L-type Ca2+ current (ICa,L). Expression of GFP decreased the density of ICa,L but did not shift the current-voltage (I-V) curve. A: typical traces of Ca2+ current (ICa) recorded from control (left) and GFP-expressing cells (right) in the absence (open symbols) and presence (filled symbols) of 10 µM PN-200-110 (PN). B: I-V relationship for whole cell ICa,L in control (circles; n = 13) and GFP-expressing cells (squares; n = 11) in the absence (open symbols) and presence (filled symbols) of 10 µM PN. Expression of GFP significantly decreased peak ICa,L. C: voltage dependence of ICa,L in control () and GFP-expressing cells (open circle ). Activation curve (I/Imax) of ICa,L, obtained from I-V relationship, was not affected by GFP expression. Steady-state inactivation (G/Gmax) was determined as channel availability after 1-s prepulses. Channel availability in cells expressing GFP was not different from that of control (cf. Table 1). Each symbol represents the mean ± SE.


                              
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Table 1.   Properties of Ca2+ channel activity

Expression of alpha 1E gene in ventricular myocytes. Expression of alpha 1E-, beta 2b-, and alpha 2/delta -subunits increased the total ICa compared with that in the control and in cells transfected with GFP alone (P < 0.05; Table 1). This increase was caused by the appearance of a large PN-resistant Ca2+ current (Fig. 3A, left, closed circle). Further addition of 100 µM Ni2+ markedly inhibited this current (Fig. 3A, left, closed square). Changing the charge carrier from Ca2+ to Ba2+ resulted in a current-voltage (I-V) relationship shift toward negative potentials, no increase of the current amplitude, and no slowing of the current decay (Fig. 3B). These results indicated that the channel consisted of the alpha 1E-subunit (ICa,E; Refs. 37 and 39) is responsible for the PN-resistant inward current. For comparison, T-type current (ICa,T) was recorded from freshly dissociated guinea pig ventricular myocytes. Figure 3A, right, represents the typical ICa,T in the presence of 10 mM Ca2+, which rapidly activates and inactivates. ICa,T was clearly observed at a test potential of -40 mV and reached a peak at approximately -20 mV (Fig. 4A, bottom right). ICa,T was not inhibited by PN (Fig. 3A, left, closed circle), where the peak amplitude of ICa,T was 2.2 ± 0.2 and 2.0 ± 0.4 pA/pF (n = 12) in the absence and presence of 10 µM PN, respectively. ICa,T was partially or almost completely inhibited by 0.1 or 1 mM Ni2+, respectively (Fig. 3A, right, closed or open squares, respectively).


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Fig. 3.   Properties of alpha 1E current (ICa,E) expressed in rabbit ventricular myocytes and native T-type current in guinea pig ventricular myocytes. A: effect of PN and Ni2+ on total ICa. Left: Ca2+ current obtained by 50-ms depolarization to +20 mV from a holding potential of -80 mV in alpha 1Ebeta 2balpha 2/delta -transfected cells. Right: T-type Ca2+ current (ICa,T) obtained by a depolarization to -20 mV for 50 ms from a holding potential of -80 mV in freshly isolated guinea pig ventricular myocytes. Bath solution was tetraethylammonium solution containing 10 mM Ca2+. Currents were recorded in the absence (open circle ) or presence () of PN-200-110 (10 µM) and in the presence of PN plus 100 µM Ni2+ () or PN plus 1 mM Ni2+ (). B: effect of exchange of Ca2+ to Ba2+ on PN-insensitive currents. Left: Ca2+ and Ba2+ current traces in the presence of PN (10 µM) were superimposed. tau inact = 22.1 ± 4.7 and 19.0 ± 2.7 ms for Ca2+ and Ba2+, respectively, at 20-mV test potential. Right: I-V relationship of ICa,E with Ca2+ (open circle ) or Ba2+ () as a charge carrier in alpha 1Ebeta 2balpha 2/delta -transfected cells obtained in the presence of 10 µM PN (n = 6). Each symbol represents the mean ± SE.



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Fig. 4.   Properties of ICa,E expressed in rabbit ventricular myocytes and ICa,T in guinea pig ventricular myocytes. A: I-V relationships obtained from alpha 1Ebeta 2balpha 2/delta - (n = 12), alpha 1Ebeta 2b- (n = 6), and alpha 1E-transfected myocytes (n = 11) and from guinea pig ventricular myocytes (T-type; n = 12) before (open circle ) or after () application of PN (10 µM). B: voltage dependence of activation and steady-state inactivation of ICa,E and ICa,T. Left: steady-state activation curves obtained by plotting the peak Ca2+ channel conductance (G) normalized to the maximal conductance (Gmax) at each test-potential: alpha 1E (, n = 10), alpha 1Ebeta 2b (, n = 8); alpha 1Ebeta 2balpha 2/delta (, n = 11), and T-type channel (open circle , n = 12). In each case, the data were well fit by a Boltzmann equation (see values in Table 1). Each symbol represents the mean ± SE. Right: steady-state inactivation (I/Imax) was measured with same protocol described in text: alpha 1E (, n = 4), alpha 1Ebeta 2b (, n = 4), alpha 1Ebeta 2balpha 2/delta (, n = 8), and T-type channel (open circle , n = 8). Data were well fit by a Boltzmann equation (see values in Table 1). Each symbol represents the mean ± SE.

ICa,E was detected in 78% (15/19) of the alpha 1Ebeta 2balpha 2/delta -transfected and GFP-luminescent cells. ICa,E was also observed in alpha 1E- or alpha 1Ebeta 2-transfected and GFP-luminescent cells with an efficiency of 45% (9/20) or 60% (9/15), respectively. No PN-insensitive currents were recorded from the alpha 1Ebeta 2b-transfected but GFP-negative cells (n = 10), which largely supports the statement that the GFP is a good marker to detect cells with successful expression of the alpha 1E channel. ICa,E in alpha 1E- and alpha 1Ebeta 2b-transfected cells were qualitatively similar to those recorded from alpha 1Ebeta 2balpha 2/delta -transfected cells, although they were substantially smaller in amplitude (Fig. 4A and Table 1).

The parameters of voltage-dependent activation and inactivation of alpha 1E channel were quantified by I-V relationship and steady-state inactivation curve, respectively (Fig. 4B). On the basis of the voltage dependence of activation, ICa,E expressed in cardiac myocytes was classified as an HVA current (Fig. 4B, left, open circles; Table 1). The voltage for half-maximal inactivation (V1/2,inact) of alpha 1E channel was not much different from that expressed in Xenopus oocytes (Fig. 4B, right; Ref. 37). However, ICa,E decayed more rapidly than previously reported for heterogeneously expressed rabbit alpha 1E channel (Fig. 3A, left; Refs. 22 and 37). At 20 mV, tau inact of ICa,E was ~20-30 ms, which was substantially shorter than that of alpha 1E channel expressed in Xenopus oocytes (37) or HEK-293 (22) cells, although it was widely variable among cells transfected with alpha 1E alone (Table 1).

Modulation of Ca2+ channel by isoproterenol. The beta -adrenergic agonist isoproterenol (Iso; 1 µM) potentiated the L-type Ca2+ channel (Fig. 5). Iso increased ICa,L amplitude 2.5-fold, shifted the I-V curve toward negative potentials, and slowed the decay of ICa,L (Fig. 5, A, top, and B). These changes induced by Iso were also observed in GFP-expressing cells; Iso increased ICa,L amplitude 2.2-fold (n = 4; Fig. 5B). However, Iso basically had no effect on ICa,E (Fig. 5A, bottom, and B). The maximal amplitude of ICa,E was 11.9 ± 3.2 and 11.8 ± 2.5 pA/pF (n = 4) in the absence and presence of Iso, respectively.


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Fig. 5.   Effects of isoproterenol (Iso) on ICa,E and ICa,L. A: ICa traces obtained by depolarization to -10, 0, 10, and 20 mV for 50 ms from a holding potential of -80 mV in time-matched culture cells before (top) or after (bottom) application of Iso (1 µM). B: I-V curves of control, GFP-expressing, and alpha 1Ebeta 2balpha 2/delta -expressing cells observed before (open circle ) or after () application of Iso. Ordinate represents current density, and abscissa represents test potential. Each symbol represents the mean ± SE. All data for alpha 1Ebeta 2balpha 2/delta -expressing cells were obtained in the presence of 10 µM PN.

Prolongation of ICa,L inactivation by alpha 1E expression. ICa,L, designated as a PN-sensitive current, was decreased by the expression of alpha 1E-subunit. Moreover, inactivation of ICa,L was remarkably slowed in cells transfected with alpha 1E-subunit (Fig. 6A). We have quantified the degree of inactivation slowing by comparing r50 (ratio of current remaining after 50 ms to that at the peak × 100%). At 20 mV, r50 was 23.5 ± 4.0% (n = 12) in the control but was 53.2 ± 5.7% (n = 9) in cells expressing alpha 1E-subunit. Furthermore, the degree of inactivation slowing of ICa,L was found to depend on the expression level of alpha 1E-subunit, where r50 was 70.0 ± 4.1% (n = 4) in cells with ICa,L/total ICa <40%, while r50 was 40 ± 3% (n = 5) in ICa,L/total ICa >40% (Fig. 6B, open circles). Thus, the less ICa,L shares in total ICa, the slower the ICa,L decays. This inactivation slowing of ICa,L was attenuated substantially by the coexpression of beta 2b-subunit (Fig. 6A), where r50 was 36 ± 8% (n = 4) in cells with ICa,L/total ICa <40% (Fig. 6B, closed circles). Therefore, the inactivation rate of ICa,L is regulated by beta 2b, which might be occupied by the coexpressed alpha 1E-subunit.


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Fig. 6.   Effect of alpha 1E-subunit expression on inactivation rate of ICa,L. A: representative Ca2+ current traces obtained by 50-ms depolarizations ranging between -10 and +20 mV from a holding potential of -80 mV in cells expressing alpha 1E (left) and alpha 1Ebeta 2b (right). Top: total ICa. Middle: ICa,E (ICa in presence of 10 µM PN). Bottom: ICa,L (total ICa - PN-resistant ICa). Decay of ICa,L was much slower in alpha 1E-transfected cells than in cells transfected with alpha 1E and beta 2b. B: inactivation kinetics of ICa,L in cells transfected with alpha 1E-subunit depend on the expression level of alpha 1E. r50 (ratio of current remaining after 50 ms to that at the peak × 100%) at +20 mV was plotted against the percentage of ICa,L in the total ICa: open circle , alpha 1E-expressed cells; , alpha 1E- and beta 2b-expressed cells. Expression of alpha 1E-subunit caused reduction of ICa,L inactivation, which was attenuated by the coexpression of beta 2b.


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GFP construct, which contains membrane-sorting signal, was used as a marker for detecting myocytes expressing the exogenous cDNAs. To avoid the possible unknown effect of GFP tagging to channel subunits (38), we expressed marker GFP together with channel subunits (alpha 1E, beta 2b, and alpha 2/delta ). The molar ratio of GFP was reduced to 1/10 compared with channel subunits to increase the percentage of channel availability in GFP-positive cells. The validity of the use of GFP was verified by the fact that no ICa,E was detected in 10 GFP-negative cells transfected with alpha 1Ebeta 2b. The expression of GFP significantly decreased ICa,L amplitude with little effect on channel biophysical properties. (Fig. 2 and Table 1). This finding indicates that GFP competes with endogenous protein, including alpha 1-, beta -, and alpha 2/delta -subunits, during the processes of transcription, translation, and membrane insertion, which may lead to reduction of ICa,L. HVA alpha 1-subunits could generate T-like current in some conditions (21) and could exhibit different character, depending on the expression environment (2, 34, 40, 41). This might be caused by cell-specific Ca2+-associated proteins such as ryanodine receptor or calmodulin, which alter the availability of channels (24), or by Ca2+-dependent inactivation and facilitation of Ca2+ channel (17, 43). We thus examined whether alpha 1E channel expressed in cardiac myocytes generates T-like current. Expression of alpha 1E-subunit produced the PN-resistant Ca2+ current that was abolished by 100 µM Ni2+ (Fig. 3A, left). Earlier studies demonstrated that alpha 1E channel is insensitive to DHPs but sensitive to lower concentrations of Ni2+ (IC50 = 30 µM; Refs. 37 and 39). Changing the charge carrier from Ca2+ to Ba2+ caused no increase of current amplitude, a finding that is also agreeable with the previous report for the characteristic of alpha 1E channel (3, 37). In addition, Iso potentiated the ICa,L in cells expressing GFP, but the PN-resistant current was insensitive to beta -adrenergic modulation. These pharmacological properties are similar to those of T-type current of rabbit sinoatrial cell (9). However, the biophysical properties are quite different from those of T-type current. V1/2,act of the alpha 1E channel (5 mV) was far more positive than that for T-type channel (-33 mV) in 10 mM Ca2+. The voltage range that the alpha 1E channel activated was similar to the range of alpha 1E channel expressed in Xenopus oocytes and HEK-293 cells (25, 37) but different from that of T-type channel (Fig. 4B). A subtype of alpha 2/delta -subunit (alpha 2/delta -3) is known to shift the voltage dependence of alpha 1E channel activation toward negative potentials; however, this subtype is not expressed in heart (15). Therefore, it can be concluded that the alpha 1E channel does not represent T-like current, suggesting that the alpha 1E gene is not responsible for the cardiac T-type channel.

alpha 1E channel expressed in cardiac myocytes was almost completely inhibited by 100 µM Ni2+, which is different from that expressed in oocytes (37) and GH3 cells (IC50 = 140 µM; Ref. 39). Moreover, ICa,E in cardiac myocytes decayed more rapidly than in oocytes, GH3, or HEK-293 cells, where the expressed alpha 1E current remained >50% at 160 ms after depolarization (22). On the other hand, the decay was rather similar to that observed in neuronal R-type currents (tau inact = 20 ms; Ref. 31). These differences among ICa,E could be caused by the differences of beta -subunit coexpressed. For example, coexpression of beta 2b accelerated the decay of ICa,E and shifted the steady-state inactivation curve toward negative potentials (Fig. 4B, right; Table 1), while beta 2a, a splicing variant of beta 2b, prolonged the decay and shifted the curve toward positive potentials (28).

Expression of alpha 1E-subunit decreased ICa,L and altered inactivation properties of ICa,L. The current decrease is explicable by the aforementioned competition between endogenous channel subunits and exogenous GFP and alpha 1E-subunit during the channel expression. Furthermore, it may be caused by the reduced availability of auxiliary subunits to alpha 1C, especially the beta -subunit, which is necessary for membrane targeting of the alpha 1C-subunit (5, 7) and to alter the L-type channel activation and inactivation (19). The reduced availability of beta -subunit for alpha 1C was supported by the fact that properties of ICa,E were not much different among cells expressing alpha 1E, alpha 1Ebeta 2b, and alpha 1Ebeta 2balpha 2/delta . These results suggest that exogenously expressed alpha 1E-subunit interacts with and scrambles for intrinsic beta - and alpha 2/delta -subunits. Furthermore, ICa,L inactivation was markedly slower in cells with a robust expression of alpha 1E-subunit, and the inactivation became faster with the coexpression of beta 2b-subunit. Thus beta -subunit regulates the inactivation of L-type channel also in ventricular myocytes (8, 35, 38). Inactivation of L-type channel in cardiac myocytes was accelerated by beta 2b, while it was slowed by overexpression of beta 2a (38). This may suggest that inactivation of L-type channel may be largely regulated by beta 2b-subunit in heart. In addition, deficiency of beta -subunit for alpha 1C could affect the L-type channel function and cardiac contractility (12). On the other hand, coexpression of beta 2b-subunit increased ICa,L little, though it did increase ICa,E. This is probably because of the competition between exogenous cDNA and endogenous genes during the process of transcription and translation. It is interesting to speculate that the slowing of inactivation of L-type current observed in this study may represent the state in the hypertrophied heart (20, 27). This idea is reinforced by the previous observation that the IGF-1-induced Ca2+ current is inhibited by the alpha 1E-specific antisense oligonucleotide (30). If this is the case, alpha 1E channel may be a key element to consider for the treatment of cardiac hypertrophy. Further rigorous study will be necessary to prove this hypothesis in atrial myocytes.


    ACKNOWLEDGEMENTS

We thank Y. Mori for pSPCBII-2, F. Hofmann and V. Flockerzi for pBH17, C. Akazawa for pCAGS65A, and M. T. Akuzawa for technical assistance.


    FOOTNOTES

This research was supported by grants from the Ministry of Education, Science, Sports and Culture (to R. Ochi and T. Tanabe), Japanese Vehicle Corporate (to R. Ochi), and CREST, Japan Science Technology Cooperation (to T. Tanabe).

Address for reprint requests and other correspondence: T. Tanabe, Dept. of Pharmacology and Neurobiology, Graduate School of Medicine, Tokyo Medical and Dental Univ., Yusima 1-5-45, Bunkyo-ku, Tokyo 113-8519, Japan (E-mail: t-tanabe.mphm{at}med.tmd.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 24 May 2000; accepted in final form 22 August 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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