Eli Lilly and Company Limited, Erl Wood Manor, Windlesham, Surrey GU20 6PH, United Kingdom
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ABSTRACT |
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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
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
1E together with
2b and
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
1E,/
2b
/
1b mediated
currents (IC50 = 4 and 24.6 µM, respectively).
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
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,
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
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.
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INTRODUCTION |
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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
1E gene coded for the
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
(
1G,
1H, and
1I). On the other hand, several recent studies
have suggested that
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
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
1E/
2b
/
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
1E gene together with
2b
and
1b by using the whole cell configuration of the
patch-clamp technique. We evaluated the biophysical properties of the
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
1E/
2b
/
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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METHODS |
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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 1
69-3/
2b
/
1b.
The cell line was generated in collaboration with the Salk Institute
Biotechnology Industrial Associates (SIBIA).
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 m when filled with the
internal solution. The typical series resistance
(Rs) values were 5.9 ± 0.4 (SE) m
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 kg1). 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.
PROTOCOLS.
Activation, inactivation, and deactivation curves were acquired using
the protocols described in the relevant figure legends. In experiments
addressing 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
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 ![]() |
RESULTS |
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General properties
ACTIVATION OF 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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Steady-state inactivation
The steady-state inactivation of 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 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
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
1E channels.
RECOVERY FROM INACTIVATION.
To further characterize the inactivation properties of the
1E/
2b
/
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
1E/
2b
/
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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PHARMACOLOGY.
The 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
-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).
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Ca2+ and/or voltage dependence of inactivation of
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
(
f) and a slow (
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 (
f and
s) did not differ significantly whatever
charge carrier was used (33.72 ± 2.35 ms and 34.02 ± 2.97 ms for
f in Ca2+ and
Ba2+, respectively, and 140.35 ± 15.96 ms
and 147.15 ± 25.11 ms for
s, in
Ca2+ and Ba2+,
respectively).
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The kinetics of inactivation, , 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
s and
f,
respectively, with 10 mM Ca2+ (n = 11) and 106.02 ± 14.6 ms and 24.6 ± 4.38 ms for
s and
f, respectively, with 10 mM Ba2+ (n = 8).
MANIPULATION OF CA2+ ENTRY.
The above data suggest a similar inactivation of
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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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 (f and
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,
s were 147.15 ± 25.15 ms
(n = 18) and 126.30 ± 9.36 ms (n = 10), respectively, and
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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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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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
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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Mock action potentials
To evaluate the consequences of the
1E/
2b
/
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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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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DISCUSSION |
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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
1E/
2b
/
1b-encoded
channels can be categorized as HVA Ca2+ channels.
Indeed,
1E/
2b
/
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
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
1E/
2b
/
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
1E/
2b
/
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
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
1E, whereas Williams et al.
(1994)
have shown a greater permeability of
Ba2+ than Ca2+ through
human
1E.
Finally, the human
1E/
2b
/
1b
currents were not blocked significantly by
-conotoxin GVIA,
-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
1E/
2b
/
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
1E currents
(Soong et al. 1993
; Williams et al.
1994
).
Our results reveal properties consistently found by several authors
studying 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
subunits (
1B vs.
2A,
1A, or no
), 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
1E/
2b
/
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 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
1E-containing channels produces fast
inactivating Ba2+ currents whether
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
).
1E activation and inactivation
kinetics are known to be modulated by both
and
2
subunits. Parent et al.
(1997)
have demonstrated that the rate of inactivation
generally increases after coexpression with the different
subunits:
3 >
1 >
4 (from fastest to slowest). Interestingly,
the
2a subunit actually slowed down
1E inactivation kinetics. We found a fast
kinetic of inactivation of
1E-mediated currents in the presence of
1B subunit, which
is in line with the fact that coexpression with
1 subunits has been shown previously to cause
faster inactivation kinetics of both the rat and human
1E-containing calcium channels (Olcese
et al. 1994
; Soong et al. 1993
). However, in the
case of rabbit
1E-channels, the
1 subunit
led to slower kinetics (Wakamori et al. 1994
).
Interestingly,
1A-containing channels could
also be modulated by
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
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
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
1E/
2b
/
2a
and
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
1E/
2b
/
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
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
1E/
2b
/
1b.
However, our data have also shown that
1E/
2b
/
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
1E/
2b
/
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
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
1E/
2b
/
1b, however with
a much lower affinity for Ca2+ than in other HVA calcium
channel, like the
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 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,
1E/
2b
/
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 1E/
2b
/
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 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.
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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.
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FOOTNOTES |
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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.
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REFERENCES |
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