Modulation of human neuronal
1E-type calcium channel by
2
-subunit
Ning
Qin1,
Riccardo
Olcese1,
Enrico
Stefani1,2, and
Lutz
Birnbaumer1,3
Departments of
1 Anesthesiology,
2 Physiology, and
3 Biological Chemistry and
Molecular Biology Institute, School of Medicine, University of
California, Los Angeles, California 90095
 |
ABSTRACT |
Calcium channels are composed of a pore-forming subunit,
1, and at least two auxiliary
subunits,
- and
2
-subunits. It is well known
that
-subunits regulate most of the properties of the channel. The
function of
2
-subunit is
less understood. In this study, the effects of the calcium channel
2
-subunit on the neuronal
1E voltage-gated calcium
channel expressed in Xenopus oocytes
was investigated without and with simultaneous coexpression of either
the
1b- or the
2a-subunit. Most aspects of
1E function were affected by
2
. Thus
2
caused a shift in the
current-voltage and conductance-voltage curves toward more positive
potentials and accelerated activation, deactivation, and the
installation of the inactivation process. In addition, the efficiency
with which charge movement is coupled to pore opening assessed by
determining ratios of limiting conductance to limiting charge movement
was decreased by
2
by
factors that ranged from 1.6 (P < 0.01) for
1E-channels to 3.0 (P < 0.005) for
1E
1b-channels. These results indicate that
2
facilitates the expression
and the maturation of
1E-channels and converts these
channels into molecules responding more rapidly to voltage.
calcium channel
2
-subunit; coupling
efficiency; facilitation of expression
 |
INTRODUCTION |
CALCIUM CHANNELS ARE a molecularly diverse group of
multisubunit proteins that are composed of a pore-forming and
voltage-sensing
1-subunit and
at least two auxiliary or regulatory subunits:
and
2
(4). The molecular
diversity of these channels is due to the existence of six distinct
genes encoding
1-subunits, one
gene encoding
2
, and four
-genes. Most of these subunits are differentially expressed in
neurons, skeletal muscle, smooth muscle, and a variety of secretory
cells. A further cause of molecular diversity in voltage-gated
Ca2+ channels is that most of
these subunits display variations in amino acid sequence because of
alternative splicing (2).
The regulatory roles of
-subunits have been extensively studied, and
it is now accepted that
-subunits facilitate channel assembly and
recruitment to the cell surface (5, 27, 33), increase the coupling of
voltage sensing to pore opening (19), and thereby facilitate activation
by voltage (17, 20). For neuronal type
1-subunits, e.g.,
1E,
-subunits also modulate voltage-induced inactivation in a subunit- and splice variant-specific manner. For example,
1b
promotes inactivation by accelerating its establishment and by shifting
the voltage-inactivation curve to a more negative potential, whereas
the
2a-subunit has the opposite
effect (22).
Studies on the actions of
2
have been less extensive, and its roles are less clear.
2
is encoded in a single
gene (8, 10) and is posttranslationally cleaved into
2 plus
, which however
remain linked by disulfide bonds (8, 13).
2
is widely distributed,
being present in all tissues expressing
Ca2+ channels studied thus far,
including skeletal, smooth, and cardiac muscles and neurons (6, 7, 10,
39, 41). Molecular cloning has identified splice variants in skeletal
muscle (10), brain (14, 39), and aorta (3) that are 95% identical and of as yet unknown functional significance. Recently, experiments by
Gurnett et al. (11) and Wiser et al. (40) showed that
2
, which has the potential
of traversing the membrane three times (10, 14), is a single
transmembrane protein anchored in the membrane by the COOH-terminally
placed
. As a consequence, all of
2 and 50% of
are
extracellular.
2
is highly
glycosylated (8, 13), a feature that appears to be necessary for its
regulatory function as seen in experiments in which removal of the
carbohydrate by digestion with protein
N-glycosidase F led to loss of channel activity (11). Coexpression of
2
has been shown to increase
1S and
1C dihydropyridine binding
sites in L and COS cells (31, 34, 36) and to a lesser extent of
1B
-conotoxin binding sites
in HEK 293 cells (38). In agreement with these findings,
2
increases ionic currents
of
1A and
1C in
Xenopus oocytes (12, 15, 16, 27) and
to a lesser extent also of
1B
(3, 38).
2
may thus
participate in the general process of multisubunit assembly and
transport to the plasma membrane (27, 36). However, exogenous
2
does not further increase
ionic current of
1E when expressed in Xenopus oocytes or COS-7
cells (30, 35).
At the functional level, Ca2+
channels without
2
seem to
differ from Ca2+ channels with
2
, pointing to a regulatory
function in addition to a structural role. Thus
2
modulates both
voltage-dependent activation and inactivation of
1A,
1C, and
1E, with the exact effect
varying somewhat depending on the heterologous expression systems (9,
28, 30, 35, 37).
In the present study, we further explored the effects of
2
on the functional
properties of neuronal
1E and
their interplay with those of
-subunits. In
Xenopus oocytes,
1E is largely independent of an
exogenous
or
2
for its
expression but is functionally regulated by exogenously supplied
-
and
2
-subunits (22, 24, 26,
33). Although there is constitutive expression of endogenous
-subunits (33) and probably of an
2
(29) in
Xenopus oocytes, they do not appear to
significantly affect the kinetics of the expressed exogenous channel
(29, 33). We report below that exogenous
2
regulates the coupling of
voltage sensing (charge movement) to pore opening as well as the
kinetics of activation and inactivation of these channels. Similar to
exogenous
-subunits,
2
does not affect
1E expression,
i.e., the average levels of measurable macroscopic currents. Except for
rates of activation and deactivation in response to voltage changes,
the effects of
2
oppose
those of
-subunits.
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METHODS |
Subcloning of
2
.
Rabbit skeletal muscle
2
, a
generous gift from the late Professor S. Numa, was subcloned from the
mammalian expression vector pKNH into pAGA2 (25). pAGA2 is a
transcription competent vector that is based on pGEM3 (Promega) and has
an engineered 5'-untranslated leader sequence from an alfalfa
mosaic virus RNA, an idealized Kozak consensus translation initiation
sequence, and a 3'-poly(A) tail of 92 adenylyls intended to
facilitate the translation and to improve the stability of the
transcript in Xenopus oocytes (25).
First, a 3.6-kb cDNA fragment encoding the complete
2
-subunit was purified from
an agarose gel after digestion with
EcoR I and subcloned into the
EcoR I site of pBluescript
(Stratagene) (pBS) to give
pBS-
2
. Second, the
NH2-terminal 300 bp of
2
were amplified with
Pfu thermostable DNA polymerase (Stratagene) using the
PCR, a sense primer starting with an
Nco I site (TAA TCC ATG GCT
GCG GGC CGC CCG), and an antisense primer (TGA AGG GTC GAC
CTC TCG) with a Sal I site. After
digestion with Nco I and
Sal I, the PCR fragment was subcloned
into Nco
I/Sal I digested pAGA2 to create pAGA2-
2
(N). Finally, a
3.2-kb DNA fragment encoding the COOH-terminal part of
2
was excised from
pBS-
2
by digestion with
Bgl II and
Sal I and subcloned into the
Bgl
II/Sal I of
pAGA2-
2
(N) to give
pAGA2-
2
.
In vitro synthesis of cRNA.
The expression constructs of human
1E (26) and rabbit
2a (23) were linearized with
Hind III, that of rabbit
1b (12) with
Not I, and that of
2
with
Xho I. The cRNAs were synthesized in
vitro with T7 RNA polymerase using reagents and protocols of the
mMESSAGE mMACHINE transcription kit (Ambion, Austin, TX), with the
exception that the LiCl precipitation step was repeated twice. The
cRNAs were suspended in diethylpyrocarbonate-treated H2O at a final concentration of
1-2 mg/ml.
Oocyte preparation.
Frogs were anesthetized by immersion in water containing
0.15-0.17% tricaine methanesulfonate for ~20 min or until full
immobility, and the ovaries were removed under sterile conditions by
surgical abdominal incision. The animals were then euthanized by
decapitation. The animal protocols were performed with the approval of
the Institutional Animal Care Committee of the University of
California, Los Angeles. Before injection, oocytes were defolliculated
by collagenase treatment (type I, 2 mg/ml for 40 min at room
temperature; Sigma, St. Louis, MO). Oocytes were maintained at
19.5°C in Barth solution (5 mM HEPES, pH 7.0, 100 mM NaCl, 2 mM
KCl, 1.8 mM CaCl2, and 1 mM
MgCl2). Recordings were done
4-12 days after the RNA injection.
Recording techniques.
The cut-open oocyte voltage-clamp technique (32) was used to record
ionic and gating currents from oocytes expressing
1C and
1E
Ca2+ channels, alone or in
combination with the regulatory
2a. The external solution
(recording chamber and guard compartments) had the following
composition: 10 mM Ba2+, 96 mM
Na+, and 10 mM HEPES, titrated to
pH 7.0 with MES. The lower chamber in contact with the fraction of the
oocyte permeabilized with 0.1% saponin contained 110 mM potassium
glutamate and 10 mM HEPES titrated to pH 7.0 with NaOH. Because oocytes
expressing Ca2+ channels showed
large outward Cl
current
even with Ba2+ as the charge
carrier, all the oocytes before recording were injected with
100-150 nl of 50 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-Na4 titrated to pH 7.0 with
MES, to prevent activation of Ca2+- and
Ba2+-activated
Cl
channels (18). To remove
contaminating nonlinear charge movement related to the oocytes
endogenous
Na+-K+-ATPase,
0.1 mM ouabain was added to all external solutions. Leakage and linear
capacity currents were compensated and subtracted on-line using
p/
4 protocol from
90 mV holding potential (SHP). Charge movement was detected for depolarizations more positive than
70 mV and that did not change with SHPs of
120 or
90 mV.
These results indicate that negative subtracting pulses from
90
mV SHP are adequate to subtract linear components.
Signals were filtered with an eight-pole Bessel filter to one-fifth of
the sampling frequency. All the experiments were performed at room
temperature (22-23°C).
 |
RESULTS AND DISCUSSION |
Neither exogenous
nor
2
enhances
appearance of macroscopic
1E
Ca2+ channel
currents.
Previous studies had shown that ionic currents produced in
Xenopus oocytes injected with
1C or
1A cRNA alone are very small, that coexpression with either
or
2
significantly increased the inward currents of both types of channels, and that currents are
further enhanced by combining all three subunits (9, 27). Unlike these
1-subunits, expression of the
human
1E alone in Xenopus oocytes produces relatively
large inward ionic currents (26). This was confirmed in the present
studies in which we observed peak inward currents that averaged 1.5 ± 1 (SE) µA (n = 15 oocytes from
7 batches; test potential, 10 mV from
90 mV holding potential)
in oocytes injected with
1E-cRNA alone. This was not
significantly affected by coexpression of exogenous
1b (2.0 ± 1.0 µA,
n = 12),
2
(1.7 ± 0.7 µA,
n = 7), or both
1b and
2
(2.4 ± 0.8 µA,
n = 16). In contrast, both
and
2
affected the kinetics of
1E activation, deactivation,
and inactivation and, as a result, the current-voltage
(I-V),
the conductance-voltage (G-V),
and the steady-state voltage-inactivation relationships. Figure
1 illustrates the effect of subunit
coexpression on the I-V
relationships of
1E;
1b shifted the
I-V
curve to more negative potentials, and
2
shifted the
I-V
curve to more positive potentials, indicating that
2
regulates the response to
voltage of this neuronal Ca2+
channel.

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Fig. 1.
Current-voltage relationships of
1 alone ( ),
1/ 2
( ),
1/ 1b
( ), and
1/ 1b/ 2
( ) channels. cRNAs were injected at molar ratio of
1E- 1b- 2 = 1:5:2. Each data point is mean peak normalized current from 5 or 6 oocytes. Currents were measured with 10 mM
Ba2+.
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2
modulates
1E
Ca2+ channel
activation by voltage.
Figure 2,
A-F,
shows that the rates of
1E
activation were markedly increased by
2
, both in the absence and
in the presence of coexpressed
-subunits. To compare the rates of
channel activation, we fitted the activation phase of time courses with
a first-order exponential function and calculated the time constants
for the different
1E-complexes
(Fig. 2G). As obtained with test
potentials to 0 mV,
2
in all
subunit combinations decreased the activation time constant
(
act). The
act of
1E decreased from 3.5 ± 0.1 ms (n = 6) to 1.7 ± 0.3 ms
(n = 7). Similarly, in the presence of
1b and
2a, two
-subunits with
opposing effects on inactivation (22), the coexpression of
2
decreased the
act from 4.5 ± 0.9 ms (n = 7)
(
1b) to 2.3 ± 0.2 ms
(n = 7)
(
1b +
2
) and from 3.9 ± 0.15 ms (n = 5)
(
2a) to 2.4 ± 0.6 ms
(
2a +
2d)
(n = 9). The rate of a channel
activation is the sum of the rates at which a channel changes from its
closed to its open states (
) and from open to closed states (
).
In addition to measuring the effects of
2
- and
-subunits on the
rates of channel activation at positive test potentials, we also
measured their effects on the rates of channel closure upon membrane
repolarization to
50 mV, since at this potential rates of
deactivation are dictated primarily by
(open to closed rate). A
composite plot of the calculated transition rates (Fig.
2G) showed that
2
increases not only the
rate at which the
1E-channel
activates but also the rate at which it deactivates and that the effect
on the open to closed rate is larger than that on the closed to open
rate. This last finding accounts for the shifts of the
G-V
and
I-V
curves to more positive potentials in the face of faster activation
kinetics (Fig. 3).

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Fig. 2.
Modulation of 1E channel
activation by 2 .
A-F:
Ba2+ currents elicited in oocytes
expressing 1E in different
combinations with and 2
by 50-ms depolarization pulses to 30, 0, and +30 mV from a
holding potential of 90 mV. Current signals were digitized at 10 kHz and filtered at 2 kHz. G: rates of
activation and deactivation as a function of test potential. Rates of
activation (1/ = 1/ + ) were determined by fitting activation
phase of current traces with a single exponential function
[I = Imax + A · exp( t/ )]
and rate of deactivation at 50 mV (1/ ~1/ ) was
determined by fitting deactivation phase of tail current obtained by
25-ms step from 16 to +36 mV with 4-mV increment, followed by
repolarization to 50 mV. Values obtained from individual fits
were averaged. Data shown on figure represent means ± SE
(n = 5 for
1E,
n = 7 for
1E/ 2 ,
n = 6 for
1E/ 1b,
n = 6 for
1E/ 1b/ 2 ,
n = 5 for
1E/ 2a,
and n = 7 for
1E/ 2a/ 2 ).
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Fig. 3.
Modulation of 1E channel
voltage-conductance relationship by
2 in absence of (A) and presence of either
1b
(B) or
2a
(C). Currents were evoked by 25-ms
depolarizing steps from 88 to +132 mV in 4-mV increments,
followed by repolarization to 50 mV. Relative fraction of
channels activated during 25-ms activating pulse was measured as peak
tail currents during repolarization to 50 mV. Data from
individual oocytes were fitted by
Im/Imax = I1/Imax/{1 + exp[z1(V1/2-1 Vm)F/RT]} + I2/Imax/{1 + exp[z2(V1/2-2 Vm)F/RT]},
where Im is
instantaneous tail current developed after activation at test pulse
potential (Vm),
which equals sum of
I1 and
I2;
V1/2 is midpoint;
z is effective valence;
Imax is current
at saturating voltage obtained from fit. Data points correspond to
means and error bars are ±SE of averaged normalized currents.
Continuous lines are sum of 2 Boltzmann distributions using averaged
I1,
I2,
V1/2-1,
V1/2-2,
z1, and
z2 parameters.
Dotted lines are first Boltzmann distribution (first component) using
averaged I1,
V1/2-1, and
z1 parameters.
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Previously, we reported that the
G-V
curves of
1E-channels obtained
in Xenopus oocytes injected only with
the
1E-cRNA could not be fitted
by a single Boltzmann distribution but that acceptable fits could be
obtained using the sum of two Boltzmann distributions with about equal
relative amplitudes having midpoints of activation (V1/2) at ~0
and 50 mV. In those studies, the primary effect of coexpressing an
exogenous
-subunit was to increase the relative amplitude of the
component with
V1/2 around 0 mV
from ~50 to 75-80%, without significantly changing the voltage
dependence and position along the voltage axis of the two individual
components (22). The mechanism by which exogenous
-subunits change
the ratio of the two components of the
G-V
curve is not clear so far. To account for the two components in the
G-V
curve, one could assume that there are two populations of
Ca2+ channels in oocytes injected
with
1E alone
(
1E alone and
1E with endogenous
-subunit); however, our previous results suggested that this is
unlikely (33). In the present studies, we reproduced the effects of the
-subunits on the
G-V
curves. Coexpression of
2
with
1E in the presence and
absence of exogenous
-subunits resulted in
G-V
curves that were shifted to more positive potentials along the voltage
axis. These shifts can be accounted for by a decrease in the proportion
of the more negative component of the G-V
curve and by a shift to more positive potentials of both midpoints of
the two Boltzmann distributions. These effects are consistent with the
results depicted in Fig. 1, showing that
2
shifts the peak of the
I-V
curve to more positive potentials by ~10 mV. As was the case for the
activation kinetics, the effect of
2
on the
G-V
relationship was also seen when a
-subunit was coexpressed. The
effects of either
1b (Fig.
3B) or
2a (Fig.
3C) were partially opposed by
2
so that the first
component of the
G-V
curve was decreased from 72 to 62% (in the presence of
1b) and 74 to 62% (in the
presence of
2a), respectively
(Table 1). Changes in the midactivation
potentials occurred as well.
2
markedly accelerates the installation of inactivation of
1E but affects steady-state
inactivation only slightly.
The second distinct regulatory effect of coinjection of
2
on
1E noticed by us was that
2
greatly accelerates the
rate of
1E inactivation. Figure
4 shows the inactivation time courses at a
test potential of 10 mV. These time courses could not be fitted with a
single exponential decay function. We thus analyzed the data
empirically by comparing times required to decrease channel activities
to 50% of peak and found the exogenous
2
to accelerate inactivation
regardless of coexpression of an exogenous
-subunit (Fig. 4). The
fastest rates of inactivation were obtained upon coexpression of
1E and
2
with exogenous
1b: half time at 10 mV = 43 ± 15 ms (n = 7). When steady-state
inactivation was determined as a function of inactivating test
potentials, we found no significant effect of
2
(Fig.
5 and Table 1). This suggests that
2
not only increases the
rate of inactivation but also increases the rate of recovery from
inactivation.

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Fig. 4.
2 -Subunit accelerates
inactivation of 1E.
Inactivation time courses of 1E
channels in combination with different - and
2 -subunits were obtained by
a 800-ms pulse to 10 mV from holding potential of 90 mV. Times
to decrease 50% of initial peak current during pulse
(t1/2) are
listed in parentheses as follows:
1 alone (150 ± 52 ms;
A),
1/ 2
(116 ± 11 ms; A),
1/ 1b
(107 ± 17 ms; B),
1/ 1b/ 2
(43 ± 15 ms; B),
1/ 2a
(401 ± 157 ms; C), and
1/ 2a/ 2
(282 ± 72 ms; C).
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Fig. 5.
2 -Subunit does not
significantly modulate steady-state inactivation of
1E-channel.
1E-Channels were inactivated by
10-s conditioning pulses ranging from 120 to +27 mV with 7-mV
increments, then deactivated for 4 ms at 90 mV and finally
activated by a 400-ms test pulse to +20 mV. Time between each episode
was 30 s. Sampling frequency was 25 Hz during conditioning pulse and
500 Hz during test pulse, and current signals were filtered at 200 Hz.
Relative fraction of channels available for activation was measured as
peak current during test pulses. Data from individual oocytes were
fitted by Im = Imin + Imax/{1 · exp[z(V1/2 Em)F/RT]}.
Data points correspond to means and error bars to ±SE of averaged
normalized currents. Continuous lines show Boltzmann distributions that
fitted averaged data of 1E.
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2
increases
1E-gating currents
without concomitant increases in ionic currents.
The coupling between the movement of the voltage sensor (i.e., charge
movement) and pore opening of a channel is the efficiency with which
voltage sensing is transduced into an increase in channel open
probability and is a parameter determined by both the identity of
1 and the effects that
regulatory subunits and other modifiers may have on the
1-subunit. We reported
previously that the
1E-channel shows a tight coupling between voltage sensing and pore opening when
compared with that of
1C (20,
21). We also reported that
-subunits significantly improve this
coupling efficiency, possibly by reducing the number of nulls (20). We
now tested the effect of
2
on the efficiency with which voltage sensing is coupled to pore
opening.
To study the effect of
2
on
coupling efficiency in
1E, we
determined both the limiting conductance
(Gmax) and
limiting charge movement
(Qmax) as a
function of voltage by using the same approach described previously
(21), and then related both values as seen for
1E-channels in
Xenopus oocytes injected with
1E alone or with combinations
of
- and
2
-subunits.
Previously, we reported that the charge moved reaches a maximum around
+40 mV for channels formed in oocytes injected with
1E alone or a mixture of
1E and
2a. To confirm that all of the
1E charge was moved at +40 mV
when coexpressed with
2
, we
determined charge movement as a function of increasing test potentials
(Q-V
curves) for channels formed by injection of
1E/
1b/
2
.
The results showed that the
Q-V
curves of
2
-containing
channels (Fig.
6A) are
identical to those of
1E- and
1E/
2a-channels
reported previously (21). Therefore, the
Qmax values were
computed by integrating the maximal gating current developing at the
reversal potential (approximately +60 mV, experimentally determined for
each oocyte). The
Gmax was then
obtained by dividing the peak tail current that developed at
50
mV after a 25-ms conditioning pulse to +132 mV by the driving force
E, given by the following equation:
E = Em
EBa =
50
mV
(+60 mV) =
110 mV, where
Em is membrane
potential and EBa
is the apparent reversal potential at 10 mM external
Ba2+. The
Gmax/Qmax
was then used as the measure of coupling efficiency, interpreting a
higher
Gmax/Qmax
as an indication of a higher coupling efficiency between charge
movement and pore opening. Figure 6 shows the
Gmax/Qmax
for
1E-channel by injection of
1E alone and in combination
with regulatory subunits. In contrast to
-subunits, which increased
the
Gmax/Qmax
of
1E by more than twofold, the
2
-subunit decreased the
1E
Gmax/Qmax
to less than one-half, from 0.42 ± 0.03 S/µC
(n = 5) to 0.25 ± 0.02 S/µC
(n = 7). Likewise,
2
decreased the
Gmax/Qmax
of
1E
2a
from 0.91 ± 0.10 S/µC (n = 5) to
0.46 ± 0.03 S/µC (n = 8) and
that of
1E
1b
from 1.07 ± 0.15 S/µC (n = 7) to
0.35 ± 0.03 S/µC (n = 7). We
found that the reduced coupling efficiency correlated with an increase
in the charge moved
(Qmax). For
example, the Qmax
of
1E/
1b
and
1E/
1b/
2
are 104 ± 30 pC (n = 6) and 438 ± 53 pC (n = 7), respectively. These data are consistent with published reports showing that
2
increases charge movement
of
1C or the amount of
1C-subunit in the plasma
membrane in either Xenopus oocytes or
HEK 293 cells (1, 27). Interestingly, unlike what happens with
1C channels, the increase in
1E charge movement is not
accompanied by a significant increase of ionic current. It will be
interesting to determine whether
2
affects charge movement in
other
1-subunits.

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Fig. 6.
2 reduces coupling
efficiency between charge movement and pore opening.
A: representative charge movement
(Q)-voltage
(V) relationship of
1C/ 1b/ 2 -channel.
Gating currents from oocytes coinjected with
1E,
1b, and
2 were recorded in an
extracellular solution containing 2 mM
Co2+ and 200 mM
La2+. Values of
Q were obtained by integrating gating
currents during pulse after baseline correction. Data represent means ± SE from 3 oocytes. B:
representative records of gating currents obtained at reversal
potential of approximately +60 mV. Records are part of data sets for
G-V
relationships shown in Fig. 3.
Qmax values are
time integrals of gating currents between 0 and 25 ms.
C: limiting conductance
(Gmax)/limiting
charge movement
(Qmax) of
1E-channel in combination with
different - and
2 -subunits.
Gmax values were
obtained by dividing peak tail currents in 10 mM
Ba2+ at 50 mV after a
voltage step to +132 mV, by Ba2+
driving force (Em EBa) = 110 mV, where
Em ( 50 mV)
is membrane potential and
EBa (60 mV) is
apparent (experimental) reversal potential for
Ba2+. Data points correspond to
means and error bars to ±SE of number of tests shown.
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In summary, our observations indicate that
2
modulates most aspects of
voltage-dependent responses of
1E: its activation and
deactivation kinetics and its inactivation. Thus, as seen in time
course studies,
2
increases
both the rate at which the channel opens in response to depolarization
and that at which it closes in response to repolarization. Indeed,
2
converted
1E into a channel that opens
more rapidly to changes in membrane potential at all potentials tested,
and this was independent of coexpression of an exogenous
-subunit.
As deduced from steady-state analysis, the
1E-channel under control of
2
requires higher potentials
for 50% activation (Fig. 3). It should be noted that the rate of
deactivation measured at
50 mV was increased more than the rate
of activation at +50 mV (Fig. 2G).
This accounts for the finding that
G-V
curves were shifted along the voltage axis to more negative potentials
in the face of facilitated activation (Fig. 3). We saw no evidence for
a strong interaction between the effects of
-subunits on activation
and those of the
2
, i.e.,
channel kinetics were regulated by
2
regardless of the presence
of a
-subunit and
-subunits displayed their characteristic effects on both activation and inactivation in the absence and in the
presence of
2
, with the
exception that even though
-regulated channels responded to
2
with faster inactivation
kinetics, at steady state the effects of
2
were almost undetectable
in
1E
1b-channels.
The regulatory effect of
2
is dependent on the type of
1-subunit. For some of the
1-subunits, it increases the
ionic current size (
1S,
1C,
1D,
1A, and
1B) (3, 12, 15, 16, 27, 38)
or drug binding sites (
1S,
1C,
1A, and
1B) (31, 34, 36), and for
other
1-subunits, it changes
the rate of activation and inactivation in the presence of
(
1A and
1C) or absence of
-subunit
(
1E and
1C) (28, 37). Based on these
results, we conclude that the function of
2
-subunit is not limited to playing a role in determining the subcellular distribution of Ca2+ channels or to facilitating
assembly and targeting of Ca2+
channels to the cell surface (27) but that it also functions as a
regulatory subunit modulating voltage-dependent activation and
inactivation and the coupling efficiency between charge movement and
pore opening. The most notable general effect of
2
on
1E is to increase the speed at
which the channel opens and closes in response to voltage. We conclude
that, like
-subunits,
2
contributes to the fine-tuning of
Ca2+ channel activity.
 |
ACKNOWLEDGEMENTS |
This study was supported in part by a National American Heart
Association Scientist Development Grant (to N. Qin), by a Greater Los
Angeles American Heart Association grant-in-aid (to R. Olcese), and by
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Grants AR-38970 (to E. Stefani) and AR-43411 (to L. Birnbaumer).
 |
FOOTNOTES |
Address for reprint requests: L. Birnbaumer, Dept. of Anesthesiology,
UCLA School of Medicine, BH-612, CHS, Box 951778, Los Angeles, CA
90095-1778.
Received 20 October 1997; accepted in final form 22 January 1998.
 |
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