Inactivation Properties of Human Recombinant Class E Calcium Channels

Anne Jouvenceau, Federica Giovannini, Cath P. Bath, Emily Trotman, and Emanuele Sher

Eli Lilly and Company Limited, Erl Wood Manor, Windlesham, Surrey GU20 6PH, United Kingdom


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Jouvenceau, Anne, Federica Giovannini, Cath P. Bath, Emily Trotman, and Emanuele Sher. Inactivation Properties of Human Recombinant Class E Calcium Channels. J. Neurophysiol. 83: 671-684, 2000. The electrophysiological and pharmacological properties of alpha 1E-containing Ca2+ channels were investigated by using the patch-clamp technique in the whole cell configuration, in HEK 293 cells stably expressing the human alpha 1E together with alpha 2b and beta 1b accessory subunits. These channels had current-voltage (I-V) characteristics resembling those of high-voltage-activated (HVA) Ca2+ channels (threshold at -30 mV and peak amplitude at +10 mV in 5 mM Ca2+). The currents activated and deactivated with a fast rate, in a time- and voltage-dependent manner. No difference was found in their relative permeability to Ca2+ and Ba2+. Inorganic Ca2+ channel blockers (Cd2+, Ni2+) blocked completely and potently the alpha 1E,/alpha 2bdelta /beta 1b mediated currents (IC50 = 4 and 24.6 µM, respectively). alpha 1E-mediated currents inactivated rapidly and mainly in a non-Ca2+-dependent manner, as evidenced by the fact that 1) decreasing extracellular Ca2+ from 10 to 2 mM and 2) changing the intracellular concentration of the Ca2+ chelator 1.2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), did not affect the inactivation characteristics; 3) there was no clear-cut bell-shaped relationship between test potential and inactivation, as would be expected from a Ca2+-dependent event. Although Ba2+ substitution did not affect the inactivation of alpha 1E channels, Na+ substitution revealed a small but significant reduction in the extent and rate of inactivation, suggesting that besides the presence of dominant voltage-dependent inactivation, alpha 1E channels are also affected by a divalent cation-dependent inactivation process. We have analyzed the Ca2+ currents produced by a range of imposed action potential-like voltage protocols (APVPs). The amplitude and area of the current were dependent on the duration of the waveform employed and were relatively similar to those described for HVA calcium channels. However, the peak latency resembled that obtained for low-voltage-activated (LVA) calcium channels. Short bursts of APVPs applied at 100 Hz produced a depression of the Ca2+ current amplitude, suggesting an accumulation of inactivation likely to be calcium dependent. The human alpha 1E gene seems to participate to a Ca2+ channel type with biophysical and pharmacological properties partly resembling those of LVA and those of HVA channels, with inactivation characteristics more complex than previously believed.


    INTRODUCTION
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Several subtypes of voltage-dependent calcium channels (VDCCs) have been identified in excitable cells on the basis of their physiological and pharmacological properties (De Waard et al. 1996). They have been classified into two main categories: low-voltage activated (LVA), represented by T-type Ca2+ channels, and high-voltage activated (HVA), represented by L-, N-, P-, Q-, and R-type Ca2+ channels.

VDCCs vary in terms of voltage dependence of activation and inactivation, relative permeability to different divalent cations, rate, extent and mechanism of inactivation, as well as single-channel conductance and distribution of open and closed times. Numerous Ca2+ channel gene subtypes are now well characterized: class A genes code for channels of the P/Q-type, classes C and D for L-type, and class B for N-type channel families. However, the class E genes have not yet been unambiguously attributed to a category of VDCC. Soong et al. (1993) and Bourinet et al. (1996) suggested that the alpha 1E gene coded for the alpha 1-subunit of a member of the LVA Ca2+ channel family similar to that of the T-type Ca2+ channel (Piedras-Renteria et al. 1997). However, Perez-Reyes et al. (1998) have recently shown that there are at least three different genes encoding for the LVA T-type Ca2+ channel (alpha 1G, alpha 1H, and alpha 1I). On the other hand, several recent studies have suggested that alpha 1E currents are HVA-like and very similar to the R-type current found in cerebellar granule neurons (Piedras-Renteria and Tsien 1998; Rock et al. 1998; Tottene et al. 1996; Williams et al. 1994; Zhang et al. 1993). However, the question is still open as to how many different calcium channel subtypes are encoded by the alpha 1E gene, and its different isoforms in different cells.

Defining the biophysical and physiological properties of R-type and/or class E-mediated currents in different cell types is becoming increasingly important in light of the accumulating evidence that these channels are not only present on neuronal somata, where they can play important roles in the control of cell excitability, but are also present in nerve terminals where they participate to the control of neurotransmitter release (Lim and Lim-Shian 1997; Wu et al. 1998, 1999).

The aim of this study was to characterize the alpha 1E/alpha 2bdelta /beta 1b-mediated calcium currents, focusing on the inactivation properties of class E and their possible implication in neuronal physiology. To achieve this goal, we studied the cell line E52-3 stably expressing the alpha 1E gene together with alpha 2bdelta and beta 1b by using the whole cell configuration of the patch-clamp technique. We evaluated the biophysical properties of the alpha 1E-mediated currents (activation, deactivation, and inactivation) with emphasis on the Ca2+ and/or voltage dependency of the inactivation process. Finally, we studied the characteristics of the alpha 1E/alpha 2bdelta /beta 1b-mediated currents in response to action potential-like voltage-clamp protocols mimicking neuronal action potentials (McCobb and Beam 1991; Wheeler et al. 1996).


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Cell culture

CELL LINE. The HEK 293 cells used in this work (cell line E52-3) express Ca2+ channels generated by the stable transfection of plasmids bearing a drug-resistant gene and the human Ca2+ channel subunits alpha 1 69-3/alpha 2bdelta /beta 1b. The cell line was generated in collaboration with the Salk Institute Biotechnology Industrial Associates (SIBIA).

E52-3 cells were maintained at 37°C and 5% CO2 and grown in plastic Falcon dishes in DMEM (GIBCO, Paisley, UK) containing 5% defined bovine serum (HyClone, Logan, UT), 1% penicillin G/streptomycin sulfate/glutamine (GIBCO), and 1% geneticin (50 mg/ml). One day before recording, the cells were dissociated by gentle trituration with a fire-polished glass Pasteur pipette and replated onto poly-L-lysine-coated glass coverslips. The culture dishes were maintained at 37°C in a humidified atmosphere of 5% CO2 in air for the next 24 h.

Electrophysiology

The patch-clamp technique in the tight-seal whole-cell configuration was used to record Ca2+, Ba2+, and Na+ currents. Recordings were made at room temperature (21-24°C). Currents were recorded using Clampex 6 software and an Axopatch-1D amplifier (Axon Instruments, Foster City, CA), filtered at 10 kHz by the built-in filter of the amplifier and stored on a Compaq computer. The resistance of the patch electrodes was between 2 and 5 mOmega when filled with the internal solution. The typical series resistance (Rs) values were 5.9 ± 0.4 (SE) mOmega with a whole cell capacitance (Cm) of 10.8 ± 0.5 pF (n = 150). Leak currents were substracted by the P/4 procedure.

SOLUTIONS. Calcium currents were recorded in the following extracellular solution (in mM): 160 tetraethylammonium chloride (TEACl), 5 CaCl2 or BaCl2, 5 MgCl2, 10 4-(2-hydroxyethyl)-1-piperazine-ethanesulfonic acid (HEPES), and 10 glucose, with pH adjusted to 7.4 with TEA hydroxide (standard osmolarity: 315 mOsm kg-1). The intracellular solution consisted of 135 mM CsCl, 1 mM MgCl2, 10 mM HEPES, 14 mM ditrisphosphocreatine, 3.6 mM MgATP, 50 U/ml creatinine phosphokinase, and 150 µM or 15 mM 1.2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA), pH 7.1, osmolarity 285-290 mOsm.

Monovalent currents through the alpha 1E Ca2+ channel were recorded with the following solutions (in mM): 10 TEACl, 150 NaCl, 10 EGTA, 10 HEPES, 10 glucose, and 1 MgCl2. The intracellular solution consisted of 148.5 mM N-methyl-D-glucamine, 1 mM MgCl2, 10 mM HEPES, 14 mM ditrisphosphocreatine, 3.6 mM MgATP, and 50 U/ml creatine phosphokinase.

PROTOCOLS. Activation, inactivation, and deactivation curves were acquired using the protocols described in the relevant figure legends. In experiments addressing alpha 1E pharmacology, currents were elicited by test voltage steps to the peak current potential from a holding potential of -90 mV. The pharmacological inhibition of the peak current amplitude was measured after achieving control conditions in 5 mM Ca2+ buffer. The drugs were perfused by whole bath application. In experiments designed to analyze how Ca2+ channel alpha 1E might respond to physiological stimuli, voltage protocols reminiscent of neuronal action potentials were generated using a series of five voltage ramps. These consisted of the following: 1) a fast upstroke, mimicking Na+ channel activation (-70 to +50 mV in 0.2 ms), 2) a rapid phase of repolarization, mimicking Na+ channel inactivation (+50 to +20 mV in 0.4 ms), 3) a more slowly repolarizing "shoulder" potential of variable duration (+20 to -10 mV in 0.4-6 ms), 4) a further repolarization to an afterhyperpolarization level (-10 to -90 mV in 4 ms), and 5) recovery to the resting potential (-90 mV in 2 ms). Such waveforms were normally applied every 10 s.

DATA ANALYSIS. Data acquisition, analysis, fitting, averaging, and presentation were carried out using a combination of pClamp6 (Axon Instruments, Foster City, CA), Excel (Microsoft), and Statview (4.5 for the Macintosh). Unless otherwise stated, all data are presented as means ± SE. Statistical testing was carried out with two-tailed unpaired t-tests. To quantify the time course of inactivation, the decaying phase of Ca2+, Ba2+, or Na+ currents was empirically measured, i.e., we measured the duration of the Ca2+, Ba2+, or Na+ current at nine different levels of the current amplitude (from 90 to 10% of the total amplitude) for each potential used.

The voltage dependence of steady-state inactivation or peak conductance activation was described as a single Boltzman equation: I(V) = (Imax - Imin)/1 + exp[(- V1/2)/k] + Imin, where Imax is maximal activable current, Imin is minimal activable current, V1/2 is the voltage of half activation or half inactivation, and k (units of mV) represents the voltage dependence of the distribution.


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General properties

ACTIVATION OF alpha 1E-ENCODED CHANNELS. Figure 1 illustrates typical sets of Ca2+ (Fig. 1A) and Ba2+ (Fig. 1B) current traces elicited by four depolarizing pulses (-70, -20, 0, and +10 mV) from a holding potential of -90 mV. For each cell, the peak current generated at a test potential was measured and then normalized to the largest inward current observed in the range of potentials used. The normalized data were then pooled to produce the average current-voltage (I-V) curves shown in Fig. 1C. With 5 mM Ca2+ as a charge carrier (n = 8), inward currents were first observed at approximately -30 mV and maximum inward currents obtained at approximately +10 mV (Fig. 1C). When experiments were performed using 5 mM Ba2+ as a charge carrier (n = 8), the I-V curves were slightly shifted in the hyperpolarized direction compared with that in 5 mM Ca2+. Inward currents were first detectable on depolarization to -40 mV, clearly visible at -30 mV, and reached maximal amplitude around 0 mV (Fig. 1C).



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Fig. 1. Current-voltage relationship of Ca2+ and Ba2+ currents in HEK alpha 1E cells. Selected traces from current-voltage series recorded from single cells bathed in solutions containing 5 mM Ca2+ (A) and 5 mM Ba2+ (B). Currents were elicited by increasingly depolarizing pulses from a holding potential of -90 mV. Depolarizations were applied every 10 s. C: averaged peak current-voltage relationships for 5 mM Ca2+ (n = 8) and 5 mM Ba2+ (n = 8).

Estimates of the voltage dependence of activation of alpha 1E-mediated channels were obtained analyzing I-V curves after appropriate allowance for gradations in driving force. The normalized conductance was calculated from I-V data and plotted against the command potential as shown in Fig. 2A. These mean curves yielded estimates for the voltage of half-maximal activation (V1/2) of -12.6 mV for 5 mM Ca2+ (n = 8), and -20.5 mV for 5 mM Ba2+ (n = 8). Therefore both kinds of current show similar steep voltage dependency; however, their midpoint voltages differed by ~10 mV as expected from the I-V curves.



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Fig. 2. Comparison of 5 mM Ca2+ and 5 mM Ba2+ currents with respect to voltage- and time-dependent kinetics. A: voltage dependence of peak conductance of alpha 1E type channels recorded with solutions containing 5 mM Ca2+ (; n = 8) or Ba2+ (triangle ; n = 8). Conductance is defined as I peak/(- Erev), with Erev taken as +72 mV for 5 mM Ca2+ and +62 mV for 5 mM Ba2+. Curves were fitted with the Boltzman equation with k = 8.98 and 6.55 for Ca2+ and Ba2+, respectively. B: time dependence of activation, calculated from the tau of activation. Note that no difference was observed between 5 mM Ca2+ (n = 8) and 5 mM Ba2+(n = 8). C: graph of normalized conductance for 5 mM Ca2+ (n = 15) and 5 mM Ba2+ (n = 10), against membrane potentials. Curves were fitted with the Boltzman equation with k = 7.59 and 13.47 for Ca2+ and Ba2+, respectively. D: mean deactivation time constant (tau  of deactivation) plotted against a wide range of repolarization potentials (Vtail) for recordings obtained in 5 mM Ca2+ (n = 8) and 5 mM Ba2+ (n = 10).

The voltage dependency of the kinetics of activation of Ca2+- and Ba2+-mediated currents (n = 8 and 8, respectively) is compared in Fig. 2B. In both cases, increasing the strength of depolarization speeded the activation rate until the time constant approached a lower limit. At -30 mV, for example, the tau of activation in 5 mM Ca2+ was 7.09 ± 1.5 ms, as compared with 0.54 ± 0.11 ms at +50 mV, representing relatively fast activation kinetics. In addition, the activation kinetics of the alpha 1E currents obtained with 5 mM Ca2+ or 5 mM Ba2+ as charge carriers were found to be similar. For example, the taus of activation at +10 mV were 1.64 ± 0.38 ms and 1.02 ± 0.11 ms for Ca2+ and Ba2+ ions, respectively.

Steady-state inactivation

The steady-state inactivation of alpha 1E currents was investigated by using a standard protocol (Fig. 2C). Long prepulses (1 min) to different voltages were applied to allow inactivation to reach a steady state. This was followed by a 100-ms pulse to a potential near the peak of the I-V curve to assess the amount of available channels.

As shown in Fig. 2C, the point of half-maximal inactivation (V1/2) occurred at -49.7 mV in 5 mM Ca2+ (n = 15), and -69.1 mV in 5 mM Ba2+ (n = 10). This shift of 20 mV in midpoint potentials was in the same direction as the shift in V1/2 of activation (Fig. 2A). These data show that both Ca2+ and Ba2+ currents have a steep voltage dependence of inactivation.

DEACTIVATION PROPERTIES OF alpha 1E-MEDIATED CHANNELS. Ca2+ channels can also be characterized by the kinetics of channel closing or deactivation, which can be determined by the time course of tail current relaxation. We measured deactivation for a range of repolarizations between -110 and -70 mV in the presence of 5 mM Ca2+ (n = 8) and 5 mM Ba2+ (n = 10; Fig. 2D). In both cases the deactivation time constants were voltage dependent: the more negative the repolarization potential, the smaller the deactivation time constant. Moreover, the decay of these tail currents was rapid and well fitted with a single exponential with an asymptotic value approx 400 µs. The mean deactivation rates were slower, although not significantly, in Ba2+ than in Ca2+, most likely as a consequence of the general shift in the voltage dependency of activation. Similar results were obtained when deactivation time constants were measured from cells recorded consecutively in 5 mM Ca2+ and 5 mM Ba2+. At -90 mV for example, taus of deactivation were of 0.49 ± 0.08 and 0.56 ± 0.09 for Ca2+ and Ba2+ (n = 5), respectively.

CALCIUM/BARIUM SELECTIVITY. Voltage-activated Ca2+ channels can also be discriminated by their permeability properties. We found that E52-3 cells express, during depolarizing steps, peak inward currents of similar amplitude in Ba2+ or in Ca2+ (Fig. 1, A and B). The same results were obtained by measuring the tail current amplitude during repolarization. Indeed, the tail current amplitudes recorded in 5 mM Ba2+ were not significantly larger than those obtained in 5 mM Ca2+. For example, at -100 mV, the amplitudes of tail currents were -54.26 ± 7.76 (n = 6) and -66.90 ± 8.58 (n = 8) for Ca2+ and Ba2+ as charge carriers, respectively, confirming the similar permeability of Ca2+ and Ba2+ ions through alpha 1E channels.

RECOVERY FROM INACTIVATION. To further characterize the inactivation properties of the alpha 1E/alpha 2bdelta /beta 1b currents, we examined the rate of recovery of ICa2+ (n = 9) and IBa2+ (n = 7) from inactivation, because this is another process that can be influenced by Ca2+ (Brehm et al. 1980; Gutnick et al. 1989; Yatami et al. 1983). Figure 3 shows the ratio of amplitudes of Ca2+ and Ba2+ currents elicited by two consecutive test pulses with varying interpulse intervals. According to this figure, the recovery from inactivation of alpha 1E/alpha 2bdelta /beta 1b channels is time dependent. In fact, at -120 mV, the amplitude of the Ca2+ current recovers exponentially with a time constant of 500 ms. Moreover, using the same interpulse intervals (300 ms), but changing the holding potential of this interpulse (from -140 to 0 mV), we found that recovery from inactivation is also voltage dependent. Indeed, the recovery is more complete at -120 mV (96%) than at -60 mV (32%). Note that the recovery was relatively fast for both Ca2+ and Ba2+ ions, and there was no significant difference in the rate of recovery between the two cations.



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Fig. 3. Recovery from inactivation of ICa2+ and IBa2+ measured with a 250-ms prepulse at -10 mV followed by 250-ms test pulse, both applied from a holding potential of -120 mV. Prepulse and test pulses were separated by increasing intervals starting from 50 ms, up to 1,000 ms, with a 50-ms step. Lines indicate single exponential fits to the recovery time course. Inset: application of prepulse and test pulses separated by a constant interval of 300 ms, and applied at different holding potential (starting from -140 mV, up to 0 mV, with a 10-mV step).

PHARMACOLOGY. The alpha 1E-mediated currents were not significantly affected by any of the organic drugs that we tested (300 nM Agatoxin IVA, a P/Q-type Ca2+ channel antagonist; 300 nM omega -conotoxin GVIA, an N-type Ca2+ channel antagonist; and 10 µM nitrendipine, an antagonist of L type Ca2+ channels). In fact, only nitrendipine at this high dose gave a nonsignificant 12% inhibition of the currents (not shown).

However, the alpha 1E-mediated currents were highly sensitive to inorganic cations. Dose-response curves for Ni2+ (n = 13) and Cd2+ (n = 10) block are shown in Figs. 4, B1 and 2, respectively. Both ions inhibited the whole cell Ca2+ currents (5 mM Ca2+) in E52-3 cells in a concentration-dependent manner. Sigmoidal fitting to the mean toxin-blocking trajectory of the higher Ni2+ and Cd2+ applications yielded estimates for the dose of half-maximal block (IC50 values) for Cd2+ and Ni2+, of 4 and 24.6 µM, respectively.



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Fig. 4. A1 and A2: calcium currents (5 mM Ca2+) evoked by a 52-ms step depolarization at -10 mV from a holding potential of -80 mV before (control), during, and after (wash) application of 30 µM nickel (Ni2+) and 5 µM cadmium (Cd2+), respectively. B1 and B2: dose-response curves for nickel (n = 13) and cadmium (n = 10), respectively. Data were fitted with sigmoidal equations.

Ca2+ and/or voltage dependence of inactivation of alpha 1E currents

Normalized Ca2+and Ba2+currents (both at 10 mM) elicited by 340-ms pulses from -90 mV to the peak membrane potential of the particular cell under investigation are shown in Fig. 5A1. Ca2+ and Ba2+ currents show a rapid decay immediately after the peak inward current followed by a slower rate of decline during the depolarizing step. Indeed, currents elicited by a 340-ms pulse appeared to inactivate with a fast (tau f) and a slow (tau s) phase. The fast component of inactivation of Ca2+ or Ba2+ currents comprised ~60% of the total current recorded, whereas the slow component was ~40% (n = 18 and n = 10, for Ca2+ and Ba2+, respectively). Interestingly, the kinetics of inactivation of the Ca2+ currents did not appear to be altered by Ba2+ substitution; Fig. 5A2 shows that the time of decay (tau f and tau s) did not differ significantly whatever charge carrier was used (33.72 ± 2.35 ms and 34.02 ± 2.97 ms for tau f in Ca2+ and Ba2+, respectively, and 140.35 ± 15.96 ms and 147.15 ± 25.11 ms for tau s, in Ca2+ and Ba2+, respectively).



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Fig. 5. Effects of Ba2+ (10 mM) and Ca2+ (10 mM) as charge carriers on current inactivation. A1: example of peak-normalized sweeps of Ca2+ and Ba2+ currents elicited by a 340-ms depolarization at the membrane potential corresponding to the maximal response of the cell. Holding potential was -90 mV. External solutions were as follows (in mM): 155 TEACl, 10 CaCl2 or BaCl2, 5 MgCl2, 10 4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid (HEPES), and 10 glucose, with pH adjusted to 7.4 with TEA hydroxide (standard osmolarity: 315 mOsmkg-1). A2: slow and fast time constants for inactivation of Ca2+ and Ba2+ channels are shown for pulses corresponding to the maximal response of the cell. The values (mean ± SE) between Ca2+ and Ba2+ did not differ significantly for tau f as well as tau s (P > 0.05). B: voltage dependence of slow (B1) and fast (B2) time constants for inactivation of Ca2+ (n = 11) and Ba2+ currents (n = 8).

The kinetics of inactivation, tau , were voltage dependent in the range of command potentials between -30 and +60 mV. We observed that both the slow and fast time constants of inactivation of Ca2+ and Ba2+ currents decreased progressively with increasing test potential (Fig. 5, B1 and B2). The limiting decay time constants at strong depolarization were 128.37 ± 14.02 ms and 31.9 ± 4.72 ms for tau s and tau f, respectively, with 10 mM Ca2+ (n = 11) and 106.02 ± 14.6 ms and 24.6 ± 4.38 ms for tau s and tau f, respectively, with 10 mM Ba2+ (n = 8).

MANIPULATION OF CA2+ ENTRY. The above data suggest a similar inactivation of alpha 1E currents in Ca2+ and in Ba2+. To further evaluate a possible Ca2+-dependent inactivation mechanism, we also analyzed whether decreasing the amount of Ca2+ entry affected the inactivation properties. Changing the concentration of extracellular Ca2+ from 10 to 2 mM resulted in a significant decrease in the Ca2+ current amplitude as shown in the example of Fig. 6A1. This was confirmed by the comparison of the mean amplitudes (3,800 ± 110 pA and 1,650 ± 252 pA, n = 10) in these two situations (Fig. 6A2). Normalizing and superimposing the currents (Fig. 6B1) showed that the depression of the current did not lead to significant changes in the kinetics of inactivation. Similarly, averaged inactivation time constants (n = 10) did not differ significantly in the 2- and 10-mM Ca2+ conditions (Fig. 6B2).



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Fig. 6. Effects of extracellular Ca2+ concentration on current inactivation. A1: calcium currents were obtained at the membrane potential corresponding to the maximal response of the cell. Extracellular Ca2+ concentration was lowered from 10 to 2 mM Ca2+. External solutions were as follows (in mM): 163 TEACl, 2 CaCl2 or BaCl2, 5 MgCl2, 10 4-(2-hydroxyethyl)-1-piperazine-ethane-sulfonic acid (HEPES), and 10 glucose, with pH adjusted to 7.4 with TEA hydroxide (standard osmolarity: 315 mOsmkg-1). A2: histograms showing the reduction of the current amplitude (mean ± SE, n = 10; ***P < 0.001). B1: peak-normalized traces from the cell illustrated in A1. B2: averaged time constants for inactivation of Ca2+ channels recorded with solutions containing 2 or 10 mM Ca2+ at the voltages of maximal current. Note that both tau s and tau f were similar for both concentrations used. Error bars represent SE.

EFFECTS OF INTRACELLULAR BUFFERING ON CURRENT INACTIVATION. The lack of obvious Ca2+-dependent inactivation reported above could possibly be due to the high buffering capacity of intracellular solutions. To address this point, we lowered the concentration of BAPTA in the patch pipette from 15 mM to 150 µM. In the example illustrated in Fig. 7A, normalized traces showed clearly that the current obtained in the presence of 150 µM BAPTA inactivated similarly to that obtained in the presence of 15 mM BAPTA. The inactivation time constants (tau f and tau s), at the voltage corresponding to the peak current in either condition did not differ significantly (Fig. 7B). For high and low concentrations of BAPTA, tau s were 147.15 ± 25.15 ms (n = 18) and 126.30 ± 9.36 ms (n = 10), respectively, and tau f were 33.72 ± 2.35 ms (n = 18) and 43.510 ± 4.08 ms (n = 10), respectively. We also quantified the percentage of inactivation produced during 310-ms depolarizing steps to potentials between -40 and +60 mV and expressed the percentage as a function of the current amplitude at both high and low concentration of BAPTA (Fig. 7, C1 and C2). The results obtained showed that the degree of inactivation was not dependent on the current amplitude in either case (correlation coefficient, r < 0.5). This further indicates that, despite the greater level of Ca2+ entry, the extent of inactivation was similar.



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Fig. 7. Effects of intracellular buffering on current inactivation. A: examples of normalized peak sweeps of Ca2+ current (10 mM) elicited by a 340-ms depolarization at the membrane potential corresponding to the maximal response of the cell. Currents were obtained with an intracellular solution containing either 15 mM or 150 µM BAPTA. B: averaged time constants for inactivation recorded with high and low concentration of BAPTA (n = 18 and n = 10 cells, respectively). Note that both tau s and tau f were not significantly different between these 2 conditions. C: illustration of 2 different scatter plots of Ca2+ current amplitude vs. percentage of inactivation obtained during a 310-ms depolarization. Percentage of inactivation is defined by the ratio between the minimum and the maximum amplitude. Data were measured at test potentials of -40 mV through +60 mV, using solutions containing 10 mM Ca2+ with pipette intracellular solution of 15 mM or 150 µM BAPTA (C1 and C2, respectively). Data points were fitted with a regression line. The correlation coefficients (r) of the fits are shown.

Altogether, these data suggest that Ca2+ ions do not play a major role in alpha 1E current inactivation.

DOUBLE PULSE CA2+ CURRENT INACTIVATION. Another method to examine the Ca2+ and voltage dependency of inactivation is to apply a double pulse protocol as shown in Fig. 8A (top panel). The duration of the prepulse is 100 ms. The test pulse is to a test potential corresponding to the maximum opening of Ca2+ channels and also lasted 100 ms. The interval between cycles is 10 s. Representative current traces with prepulse potentials between -90 and +60 mV, with high and low intracellular BAPTA concentrations, are shown in Fig. 8A (middle and bottom panel, respectively). In both high (n = 14) and low (n = 11) BAPTA conditions, the test pulse current exhibited a bell-shaped inverse relation to the prepulse potential (Fig. 8B). Also, the maximal current reduction of test pulse currents was similar in high and low BAPTA (68.26 and 60.89%, respectively), suggesting no difference in the inactivation between the two conditions. Although an interpulse interval of 12.5 ms to -90 mV was routinely used, the same results were obtained in a set of experiments where this interval was omitted. Moreover, similar results were observed with Ba2+ ions (10 mM), as charge carriers either with high or low BAPTA concentrations (data not shown, n = 5 and 7, respectively). These results, obtained in the double-pulse experiments in high or low BAPTA and in Ca2+ versus Ba2+, are in line with the apparent lack of Ca2+-dependent inactivation observed in the other experiments described above.



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Fig. 8. A: Calcium currents, in the presence of 15 mM or 150 µM of BAPTA, were elicited by a double pulse voltage protocol. One hundred-millisecond depolarizing prepulses were applied in 10-mV increments from -90 mV to +60 mV. After a 50-ms pulse interval at -90 mV, each prepulse was followed by a 100-ms test pulse at a test potential corresponding to the maximum opening of the Ca2+ channels (A1). Representative current traces recorded from HEK 293 cells transfected with alpha 1E are shown underneath the pulse protocol. Recordings were made in 10 mM Ca2+ with 15 mM or 150 µM BAPTA in the intracellular solution (A2 and A3, respectively). B: peak current amplitude at prepulse () and test pulses (triangle ) were normalized to the prepulse peak current of each cell and plotted against the prepulse potential.

NA+ CURRENTS. To further examine whether a divalent rather than a Ca2+-dependent mechanism could be responsible for our "bell-shaped" results, we used Na+ (150 mM) instead of Ca2+ or Ba2+, as the permeant ion. Na+ currents (n = 6) activated at potentials ~20 mV more negative than those recorded with 5 mM Ca2+. Inward currents were first observed at approximately -60 mV and were maximal at approximately -30 mV (Fig. 9B). Using the same double-pulse protocol employed above, the inactivation of this Na+ current (n = 6) was no longer bell shaped and displayed only a voltage-dependent relationship to the prepulse potential (Fig. 9, A and B). A scaled Ca2+ current (5 mM) trace plotted together with Na+ current (150 mM, Fig. 9C) illustrated that Na+ current decayed more slowly when Ca2+ was removed. This evidence was confirmed by the fact that the averaged mean fast and slow tau  recorded with solution containing 150 mM Na+ (n = 6) were significantly longer than with 10 mM Ca2+ (n = 8) as a charge carrier (P = 0.0086 and P = 0.018, respectively) for a test potential where the cells were near their maximal peak (Fig. 9C). This confirms that the inactivation process is slower in Na+ than in Ca2+. Similar results were obtained at membrane potentials between -60 and +50 mV (Fig. 9D). Interestingly, the inactivation time constant plotted as a function of command potential showed that the kinetics of inactivation were mostly voltage dependent (even if weakly) for both conditions, not showing typical properties of a current dependent process such as a bell-shaped curve for example. Note that with 150 mM Na+ as charge carrier, the voltage dependence of inactivation was shifted ~30 mV to more hyperpolarized voltages with respect to 10 mM Ca2+.



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Fig. 9. Activation and inactivation properties of Na+ currents. A: Na+ currents were elicited by the same double pulse protocol described previously (see Fig. 8). Representative current traces recorded from HEK 293 cells transfected with alpha 1E are shown underneath the pulse protocol. Recordings were made in 150 mM Na+. B: peak current amplitude at prepulse () and test pulses (triangle ) were normalized to the prepulse peak current of each cell and plotted against the prepulse potential (n = 6). C: voltage dependence of slow and fast time constants for inactivation of Ca2+ (n = 8) and Na+ currents (n = 6). D: currents obtained from separate cells recorded with solutions containing Na+ (150 mM) or Ca2+ (10 mM), at voltages of maximal currents. Time constants for inactivation of Na+ (n = 6) and Ca2+ (n = 8) currents. Note that the parameter values of the Na+ fit are significantly different from the average values for Ca2+ currents (*P < 0.05; **P < 0.01).

Mock action potentials

To evaluate the consequences of the alpha 1E/alpha 2bdelta /beta 1b calcium channel properties to a physiological event, we used a mock action potential protocol. As shown in Fig. 10A, the protocols consist of a series of voltage ramps, each representing a distinct phase of the theoretical action potential (see METHODS). The capacitative and leak currents remaining after block by Cd2+ were subtracted to yield to the traces shown in Fig. 10A. Interestingly, action potential-like voltage protocols (APVP)-induced currents were never detected during the upstroke phase of the action potential protocol but always during the declining phase (see dashed line Fig. 10A). Increasing the APVP shoulder duration augmented the peak current amplitude, which approached an asymptotic maximum level of 1.45-fold for Ca2+ (Fig. 10B, n = 7), not significantly different from the first Ca2+ current. Additionally, this increase in amplitude was accompanied by a change in the Ca2+ current waveform. Indeed, when the APVP shoulder duration was in the 0.4- to 2.0-ms range, all generated calcium fluxes were approximately U-shaped, whereas when the APVP shoulder duration was beyond 2.4 ms, waveforms were then made up of two components.



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Fig. 10. Changes in pattern of divalent cation entry caused by prolongation of the plateau phase of mock action potentials. A, top: waveforms of mock action potentials, induced in 10 mM Ca2+ and imposed by voltage clamp. A, bottom: Ca2+ currents evoked by the above voltage clamps and obtained by Cd2+ (200 µM) subtraction. B: graph plotting the mean current amplitude against action potential-like voltage protocol (APVP) shoulder duration for 7 experiments such as those shown in A. C: dependence of relative charge transfer on duration of action potential shoulder. Integrated charge for various shoulder durations (Q) were normalized by that obtained with a 0.4-ms shoulder (Q0.4). D: averaged results for time-to-peak Ca2+ entry, measured from the beginning of the mock action depolarization (n = 7). E: example of 3 traces of the 15 evoked by 15 APVPs delivered at 100 Hz. Currents recorded with 150 µM intracellular BAPTA. F: graphic plot of the mean normalized amplitude of each response obtained after a 15 APVPs train. Before averaging across cells, data were normalized to the current amplitude of the 1st response in the train.

The total charge transferred was also increased with the increase in mock action potential duration. Quantitatively, the charge transferred increased threefold as the APVP shoulder was increased from 0.4 to 6 ms (P = 0.0203; Fig. 10C, n = 7). This increase was significantly different from the first current for APVP shoulder durations above 3.6 ms.

An increase in time-to-peak of Ca2+ current paralleled the increase in phase 3 of the APVP (Fig. 10D, n = 7), suggesting a relationship between the length of the shoulder and the time-to-peak. Nevertheless, it is worth noting that the peak always occurred during the strong hyperpolarization phase (phase 4) of the protocol and never during the shoulder phase.

When we carried out 15 successive APVPs applied at 100 Hz with a shoulder duration of 2.0 ms (Fig. 10E), an exponential decline in the current amplitude occurred as the number of APVPs applied increased (Fig. 10F, n = 8). This suggested the existence of an accumulation of the inactivation or a "wind down" of activation. For example, after the 15th pulse at 100 Hz, the amplitude of the peak was decreased to 30% of the control value.


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We have examined the electrophysiological and pharmacological properties of class E-encoded Ca2+ channels, stably expressed in HEK 293 cells (E52-3 cells).

General properties

We have found that on the basis of their activation and deactivation properties, human alpha 1E/alpha 2bdelta /beta 1b-encoded channels can be categorized as HVA Ca2+ channels. Indeed, alpha 1E/alpha 2bdelta /beta 1b-encoded channels have a voltage threshold for activation at -30 mV in 5 mM Ca2+, half-activation at -12.6 mV and activate rapidly (tau of activation 1.64 ± 0.38 ms at +10 mV), similarly to the R-type calcium channel (Carbone et al. 1997; Randall and Tsien 1997). Second, the deactivation rate of alpha 1E channels was faster in our hands than that seen in T-type Ca2+ channels (Huguenard 1996) and closer to that observed in HVA Ca2+ channels, in general, and in R-type Ca2+ channels, in particular (Randall and Tsien 1997). The recovery from inactivation of our alpha 1E/alpha 2bdelta /beta 1b-mediated currents is time and voltage dependent as reported by Nakashima et al. (1998), resembling that observed for T-type calcium channels (Carbone and Lux 1987; Huguenard 1996). However, according to the literature, some HVA calcium channels also show a voltage-dependent recovery from inactivation (Hirano et al. 1989; Lacinova and Hofmann 1998; Morril et al. 1998; Nakayama and Brading 1993).

Because the permeability to Ca2+ and Ba2+ ions through alpha 1E/alpha 2bdelta /beta 1b channels was found to be the same, it seems that the channels expressed by our cells behave, in terms of permeability, more like T-type VDCCs (Huguenard 1996). This result is relevant because the permeability properties of the alpha 1E channel and its isoforms are still under discussion. Indeed, Bourinet et al. (1996) have demonstrated, using single-channel recordings, an equal permeability of Ca2+ and Ba2+ through rat alpha 1E, whereas Williams et al. (1994) have shown a greater permeability of Ba2+ than Ca2+ through human alpha 1E.

Finally, the human alpha 1E/alpha 2bdelta /beta 1b currents were not blocked significantly by omega -conotoxin GVIA, omega -agatoxin IVA, or nitrendipine. Our results are in agreement with earlier studies (Bourinet et al. 1996; Wakamori et al. 1994; Williams et al. 1994) and suggest that the pharmacological profile of this alpha 1E/alpha 2bdelta /beta 1b channel is distinct from that of L-, N-, and P/Q-type HVA channels (Soong et al. 1993), and similar to both T- and R-type VDCCs. Inorganic ions are known to block both HVA and LVA channels with different potencies. Both Cd2+ and Ni2+ inhibited whole cell E52-3 Ca2+ currents in a concentration-dependent manner, with IC50 values close to those previously reported for other alpha 1E currents (Soong et al. 1993; Williams et al. 1994).

Our results reveal properties consistently found by several authors studying alpha 1E channels (Bourinet et al. 1996; Williams et al. 1994), accounting for differences in the expression systems and concentrations of ions used. Indeed, recordings have been made from a number of different cell types (HEK 293 vs. oocytes or COS-7), often in the presence of different beta  subunits (beta 1B vs. beta 2A, beta 1A, or no beta ), and by using solutions of varying ionic concentration (Bourinet et al. 1996; Meir and Dolphin 1998; Parent et al. 1997; Soong et al. 1993; Williams et al. 1994).

Taken altogether, these observations show that the fundamental properties of alpha 1E/alpha 2bdelta /beta 1b-encoded channels correspond to an intermediate phenotype between "classical" LVA and HVA Ca2+ channels and possibly, to a particular R-type still to be described in native preparations.

Ca2+ dependence and/or voltage dependence of inactivation

The inactivation of alpha 1E-mediated currents is fast when compared with that of other HVA calcium channels, in particular when studied in Ba2+. This is in agreement with previous studies showing that functional expression of alpha 1E-containing channels produces fast inactivating Ba2+ currents whether alpha 1E is expressed in Xenopus oocytes (Ellinor et al. 1993; Schneider et al. 1994; Soong et al. 1993; Wakamori et al. 1994) or in mammalian cells (Williams et al. 1994). alpha 1E activation and inactivation kinetics are known to be modulated by both beta  and alpha 2delta subunits. Parent et al. (1997) have demonstrated that the rate of inactivation generally increases after coexpression with the different beta  subunits: beta 3 > beta 1 > beta 4 (from fastest to slowest). Interestingly, the beta 2a subunit actually slowed down alpha 1E inactivation kinetics. We found a fast kinetic of inactivation of alpha 1E-mediated currents in the presence of beta 1B subunit, which is in line with the fact that coexpression with beta 1 subunits has been shown previously to cause faster inactivation kinetics of both the rat and human alpha 1E-containing calcium channels (Olcese et al. 1994; Soong et al. 1993). However, in the case of rabbit alpha 1E-channels, the beta 1 subunit led to slower kinetics (Wakamori et al. 1994). Interestingly, alpha 1A-containing channels could also be modulated by beta  subunit coexpression with the same rank of potency (De Waard and Campbell 1995; Stea et al. 1994). In contrast, Lambert et al. (1997) have shown that beta  subunit depletion does not affect the decay of inactivation of the native T-type calcium channel. Consequently, class E channels have inactivation properties that are closer to that of T-type calcium channels in terms of extent and kinetics, but closer to that of HVA calcium channel, if we consider their modulation by the coexpression of beta  subunits.

The kinetics of inactivation of the Ca2+ or Ba2+ currents appeared to be made up of two components (fast and slow), as reported earlier (Parent et al. 1997). We observed a strong and similar inactivation of both Ca2+ and Ba2+ currents, which was also described in alpha 1E/alpha 2bdelta /beta 2a and alpha 1E alone encoded channels (Parent et al. 1997). These observations could be interpreted as a lack of Ca2+-dependent inactivation and the presence of a pure voltage-dependent inactivation for this channel type (De Leon et al. 1995). However, these results could also suggest that these channels have an inactivation mechanism that is activated by both Ca2+ and Ba2+, because recent studies have demonstrated significant and often quite rapid Ba2+-dependent inactivation of Ca2+ channels (Branchaw et al. 1997; Ferreira et al. 1997). Because of this, caution should be paid in relying only on Ba2+ substitutions to discriminate Ca2+- from voltage-dependent inactivation of calcium channels.

We provide four lines of evidence that indicate that alpha 1E/alpha 2bdelta /beta 1b channels do inactivate mainly in a voltage-dependent manner. 1) Lowering the concentration of Ca2+ from 10 to 2 mM did not affect the kinetics of inactivation, whereas the current amplitude is significantly reduced. 2) Reducing intracellular BAPTA concentration from 15 mM to 150 µM had no effect on the kinetics or extent of inactivation. The fast and slow time constants of inactivation were identical, and the percentage of inactivation did not vary as a function of the Ca2+ current amplitude whatever the BAPTA concentrations. 3) The recovery from inactivation of Ca2+ and Ba2+ currents was similar. Because it has been demonstrated that Ca2+ may have an inhibitory effect on the rate of recovery from inactivation (Brehm et al. 1980; Gutnick et al. 1989; Yatani et al. 1983), this result also argues against Ca2+-dependent inactivation of alpha 1E-containing channels. 4) The kinetics (fast and slow time constants) of inactivation of Ca2+, Ba2+, and Na+ currents did not have a bell-shaped dependency on the test potential, which further strongly supports the lack of Ca2+-dependent inactivation in alpha 1E/alpha 2bdelta /beta 1b.

However, our data have also shown that alpha 1E/alpha 2bdelta /beta 1b inactivation is not purely voltage dependent. Indeed, in double-pulse experiments with either low or high BAPTA intracellular concentrations and with either Ca2+ or Ba2+ as the charge carrier, the relationship between conditioning pulse potential and current inactivation had, surprisingly a U-shape, with maximum inactivation occurring at the peak point of inward current. As originally demonstrated by Brehm and Eckert (1978), such a U-shape is consistent with a current-dependent inactivation mechanism. However, it has been demonstrated that such a U-shaped inactivation curve can also be explained by a strictly voltage-dependent model of inactivation in which the rate constant for inactivation decreases, rather than increasing, with more positive depolarizations (Jones and Marks 1989). These results, therefore, cannot provide conclusive evidence for current dependency. However, our experiments using Na+ as the permeant ion have shown that Na+ currents through alpha 1E/alpha 2bdelta /beta 1b calcium channels did not exhibit the same kinetics of inactivation of Ca2+ or Ba2+ currents, decaying significantly more slowly, in particular the slow component of the inactivation, whatever the membrane potential applied. These results are in line with previous evidence that also Li+, when used as the charge carrier, gives rise to slower inactivation of alpha 1E subunits expressed alone (Parent et al. 1997). Moreover, the inactivation of Na+ current observed with a double-pulse protocol is more consistent with a pure voltage-dependent mechanism and significantly different from the Ca2+ current inactivation obtained with the same protocol. Taken together, this result suggests that the calcium-binding site, responsible for calcium-dependent inactivation may also exist on alpha 1E/alpha 2bdelta /beta 1b, however with a much lower affinity for Ca2+ than in other HVA calcium channel, like the alpha 1C calcium channel (De Leon et al. 1995). On the other hand, a different site, responsible for the "divalent cation-dependent" inactivation we described here, might also exist.

In general, the inactivation of alpha 1E current in HEK cells seems rather complex. Most of our results on class E inactivation are best explained by a voltage-dependent process, but some of them clearly point to a current-dependent and, more precisely, to a "divalent current-dependent" inactivation mechanism. These results revise the general notion of a pure voltage-dependent inactivation for class E calcium channel, which was principally based on the lack of inactivation change following Ba2+ substitution and decrease in intracellular Ca2+ buffering (De Leon et al. 1995; McNaughton et al. 1997).

Mock action potentials

Increasing APVP shoulder duration did not cause a much larger increase in the amplitude of currents carried by these channels. In this way, alpha 1E/alpha 2bdelta /beta 1b calcium channel behaves more similarly to the HVA calcium channel than to the LVA calcium channel (McCobb and Beam 1991; Randall and Tsien 1997). This result can be partially explained by the deactivation properties of our calcium channels. According to McCobb and Beam (1991) and Wheeler et al. (1996), the kinetics of deactivation have important consequences for transduction. For short shoulder duration, the total phase of hyperpolarization (+20, -90 mV; phases 3 and 4) remains fast, leading to a rapid and large entry of Ca2+, from which we obtained a U-shaped curve for the calcium current. Even though long shoulders at potentials close to the peak would lead to an increase in Ca2+ entry, these long-lasting hyperpolarizing voltages would slow down the Ca2+ driving force. We have shown that overall the amount of Ca2+ entry was lower than that expected from a long period of activation of the channels.

Even if the deactivation kinetics of our channels are faster than that of the T-type and more similar to that of the R-type, we found a slow time-to-peak for the APVP-induced currents through alpha 1E/alpha 2bdelta /beta 1b-mediated channels, very close to that of the T-type described by Randall and Tsien (1997).

We finally showed that when APVPs are evoked repetitively, the amplitude of the Ca2+ current declines. Similar results were already observed in alpha 1B cells by McNaughton et al. (1998). Continued repetitive activation of APVP at 100-Hz frequency showed that the decline in Ca2+ current amplitude with high-frequency was the dominant feature and suggests that accumulation of current inactivation may have physiological consequences.

As mentioned above, class E and/or R-type channels have been now found in several neurons and in both pre- and postsynaptic locations. They can contribute, with their peculiar biophysical properties, to the generation and shaping of action potentials (Forsythe et al. 1998). At nerve terminals, they contribute to neurotransmitter release (Lim and Lim-Shian 1997; Westenbroek et al. 1998), although with lower efficacy with respect to other channels, probably because of their specific localization far from the release sites (Wu et al. 1998). Although their contribution to neurotransmitter release could be small, their contribution to other Ca2+-dependent processes occurring at the nerve terminals and relevant to synaptic plasticity, could be much greater. The fact that these presynaptic R-type channels are also modulated by the activation of metabotropic glutamate and GABAB receptors (Wu et al. 1998) also suggests a relevant role in synaptic plasticity. In this context, a deeper characterization of the specific properties (including their inactivation) of the different calcium channels often coexpressed in the same somata on nerve terminals could help in achieving a better understanding of calcium channel heterogeneity and its functional consequences.


    ACKNOWLEDGMENTS

We thank Drs. H. Bester and D. Kullmann for helpful comments on the manuscript.

A. Jouvenceau was supported by a research grant from the Cino et Simone Del Duca association.


    FOOTNOTES

Present address and address for reprint requests: A. Jouvenceau, Dept. of Clinical Neurology, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK.

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 19 April 1999; accepted in final form 28 September 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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