From the Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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ABSTRACT |
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Voltage-gated calcium channels are composed of a main pore-forming 1 moiety, and one or more
auxiliary subunits (
,
2
) that modulate channel properties. Because modulatory properties may vary greatly with
different channels, expression systems, and protocols, it is advantageous to study subunit regulation with a uniform experimental strategy. Here, in HEK 293 cells, we examine the expression and activation gating of
1E calcium channels in combination with a
(
1-
4) and/or the
2
subunit, exploiting both ionic- and gating-current
measurements. Furthermore, to explore whether more than one auxiliary subunit can concomitantly specify gating properties, we investigate the effects of cotransfecting
2
with
subunits, of transfecting two different
subunits simultaneously, and of COOH-terminal truncation of
1E to remove a second
binding site. The main results are as follows. (a) The
2
and
subunits modulate
1E in fundamentally different ways. The sole effect of
2
is to increase current density by elevating channel density. By contrast, though
subunits also increase functional channel number, they also enhance maximum open probability (Gmax/Qmax) and hyperpolarize the voltage
dependence of ionic-current activation and gating-charge movement, all without discernible effect on activation
kinetics. Different
isoforms produce nearly indistinguishable effects on activation. However,
subunits produced clear, isoform-specific effects on inactivation properties. (b) All the
subunit effects can be explained by a
gating model in which subunits act only on weakly voltage-dependent steps near the open state. (c) We find no
clear evidence for simultaneous modulation by two different
subunits. (d) The modulatory features found here
for
1E do not generalize uniformly to other
1 channel types, as
1C activation gating shows marked
isoform dependence that is absent for
1E. Together, these results help to establish a more comprehensive picture of auxiliary-subunit regulation of
1E calcium channels.
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INTRODUCTION |
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Voltage-gated calcium channels are molecular transducers that trigger cellular processes ranging from
muscle contraction to neurotransmission. Modulation
of these channels thereby constitutes a key potential
mechanism for functional adaptation and plasticity. At
least three different subunits are believed to comprise
native calcium channels: a main, pore-forming 1 subunit, a cytoplasmic
subunit, and a disulfide-linked
2
subunit (for review, see Perez-Reyes and Schneider,
1994
; De Waard et al., 1996
). So far, seven different
genes encoding
1A,B,C,D,E,S,G subunits, and four different genes encoding
1,2,3,4 subunits have been identified, along with multiple splice variants. Given this heteroligomeric structure, regulation of channel properties
by variations in subunit composition have been widely
studied as a potential mechanism for tuning channel
gating properties to support a given physiologic role.
Despite the potential importance of modulation by
subunit combination, fundamental uncertainties remain about the effects of auxiliary subunits (for review
see Perez-Reyes and Schneider, 1994; De Waard et al.,
1996
; Walker and De Waard, 1998
). While coexpression studies have demonstrated that the addition of
auxiliary subunits (
,
2
) can have striking effects on
channel gating and/or channel expression, the specific
effects observed vary across studies, even using the
same
1 subunit. At least some of the differences in subunit effects may reflect isoform-specific variations in
the effects of distinct
subunit isoforms on
1 gating.
Further variability may arise from the use of diverse expression systems, electrophysiological methods, and experimental solutions. These points underscore the
need to explore subunit modulation of each
1 isoform
individually, and to undertake comprehensive studies
with uniform experimental conditions.
Although most previous work has focused on 1C, neuronal
1E channels (Soong et al., 1993
) have recently
emerged as important channels with which to attempt
such comprehensive investigation for several reasons.
First, subunit modulation of
1E has potential physiological relevance, as
1E (presumed "R-type") channels have been implicated in neuronal functions including neurotransmitter release (Wu et al., 1998
). Second,
1E demonstrates an exceptional capacity for high-level recombinant expression, which permits well-resolved measurements of both ionic and gating currents, even when the
1E subunit is expressed alone (which generally lowers
overall expression of current). This capability enables examination of changes in both peak open probability and
channel density (Olcese et al., 1996
), two critical measures for resolving how auxiliary subunits affect the overall level of calcium current. Third,
subunits may affect
1E expression in a uniquely different manner than observed with other pore-forming
1 subunits, providing a
potentially useful clue as to the underlying mechanism of
subunit modulation. Olcese et al. (1994
, 1996
) provide
the most biophysically detailed results in this regard, using
Xenopus oocytes. In contrast to other
1 subunits,
subunits caused little change or even a decrease in overall
1E
current density. This outcome resulted from decreased
channel density, as assessed by maximal gating charge,
countered by increased channel opening. By contrast, in mammalian expression systems,
subunits increased
overall
1E current density (Williams et al., 1994
; Stephens
et al., 1997
). Here, however, no
1E gating-current measurements have been made to permit assessment of underlying changes in channel density and open probability.
Finally,
1E is one of the channels in which a second
binding site has been explicitly identified (Tarelius et al.,
1997; Walker et al., 1998
). Characterization of mutant
1E
constructs lacking this site would allow determination of the functional importance of the secondary site.
Here, we therefore examine subunit modulation of
1E channels coexpressed with various combinations of
auxiliary subunits (
1-
4,
2
) in mammalian HEK 293 cells. The same recombinant expression system, along
with a consistent set of experimental solutions and protocols, is used throughout to facilitate direct comparison of channels with differing molecular composition.
Measurements of both ionic and gating currents permits in-depth analysis of subunit modulatory effects.
We focus on three key questions. (a) To what degree
does modulation of
1E current density reflect modulation of channel gating and/or number of functional
channels? (b) How do different auxiliary subunits compare with regard to modulation of activation gating? (c)
What is the functional impact of the secondary
binding site in
1E? Through addressing these questions,
this study helps to establish a more refined picture of
auxiliary-subunit modulation of
1E calcium channels.
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MATERIALS AND METHODS |
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Expression of N-Type Channels
HEK 293 cells, obtained from Dr. Jeremy Nathans (Johns Hopkins University; Gorman et al., 1990), were grown at 37°C in Dulbecco's modified Eagles medium (GIBCO BRL, Grand Island,
NY), 10% fetal calf serum (GIBCO BRL), 1% L-glutamine
(Sigma Chemical Co., St. Louis, MO), 1% penicillin-streptomycin (P0906; Sigma Chemical Co.), in 5% CO2. Low-passage number cells were used (<P20). cDNAs encoding channel subunits
1E (Soong et al., 1993
),
1C (Wei et al., 1991
),
1b (Pragnell et
al., 1991
),
2a (Perez-Reyes et al., 1992
),
3 (Castellano et al.,
1993b
),
4 (Castellano et al., 1993a
), and
2
(Tomlinson et al.,
1993
) were subcloned into mammalian expression plasmids
(pMT2; Genetics Institute, Cambridge, MA, for
4, pZEM229R;
ZymoGenetics, Inc., Seattle, WA, for
2
, pGW1; British Biotechnologies, Cowley, Oxford, UK for all others).
1E
was constructed by replacing the Bst 1107I (
1E: nucleotide 4299, given
start codon at nucleotide 1) and SalI (3' polylinker) region of
1E
in pGW1 with a shorter polymerase chain reaction fragment, including a premature stop codon after the codon for amino acid 1871. The portion of the channel derived from PCR was verified in its entirety with the use of the fluorescent dideoxy terminator method of thermocycle sequencing on an automated DNA sequencer (Applied Biosystems Division 373a; Perkin-Elmer Cetus
Instruments, Emeryville, CA). HEK 293 cells were transiently
transfected using a standard, calcium-phosphate precipitation
procedure (Brody et al., 1997
) with a total of 30 µg of DNA per
10-cm plate. 10 µg of a plasmid containing a pore forming subunit was included (
1E or
1C) and mixed with 10 µg of each desired auxiliary subunit (none, a
subunit, and/or the
2
subunit). If the amount of DNA totaled <30 µg, pBluescript was
added to make up the difference. For certain experiments, both
2a and
3 were simultaneously transfected either in a 1:1 ratio
(10 µg of each plasmid) or a 5:1 ratio (15 µg of
3, 3 µg of
2a).
More than 20% of cells transfected with a pore forming subunit
exhibited detectable high-threshold calcium currents.
"Mock-transfected" cells were transfected with 10 µg of 1b, 10 µg of
2
, and 10 µg of pBluescript. In our usual ionic current recording conditions (detailed below), we observed no high
threshold, voltage-gated, calcium-channel currents in such cells
(n = 32 cells, over two independent rounds of transfection), or
in cells transfected with the
2a subunit alone (n > 40 cells; Patil
et al., 1998
). In mock-transfected cells, we occasionally (~10% of cells) observed endogenous, low threshold calcium channel currents of small amplitude (peak ionic current ~20 pA in 10 mM
Ba2+), as reported previously by Sun et al. (1994)
. Although endogenous currents of such small amplitude would contribute
negligibly to our results, cells with low threshold activity were nevertheless rejected. At the biochemical level, Western blots performed on total membrane protein (30 µg/lane) from untransfected cells revealed no known high threshold
1 (A, B, C, D, E)
or
(1b, 2e, 3a, 4) subunits, and only low levels of
2
(personal
communication, Mark Williams, SIBIA Neurosciences Inc., La
Jolla, CA). Blots were probed individually with appropriate antibodies, and the lack of subunit proteins was gauged from the absence of bands that were clearly present using cells transfected
with corresponding recombinant subunits. The result that coexpression of
2
with
1E potentiated current by approximately
threefold suggests that trace expression of endogenous
2
did
not significantly influence our results.
Electrophysiology
Whole-cell recordings were obtained at room temperature 48-72 h
after transfection using an Axopatch 200A (Axon Instruments, Foster City, CA) and standard patch-clamp techniques. Cell capacitance ranged from 10-40 pF. Series resistance was typically
<5 M, and compensated 70-85%, resulting in a typical settling
time of ~80 µs. Voltage pulses were delivered every 15-20 s from
a holding potential of
110 mV, except for prepulse inactivation
protocols, where voltage pulses were given every minute from a
holding potential of
120 mV to allow recovery from inactivation. Data were typically acquired at 50 kHz and filtered at 10 kHz (
3 dB, four-pole Bessel). Displayed traces have generally
been additionally processed with a gaussian digital filter at 2 kHz.
Leak and capacity currents were subtracted by a P/8 protocol
(ionic currents) or P/
8 protocol (gating currents) from the
110-mV holding potential, unless otherwise noted (Armstrong
and Bezanilla, 1974
). To allow better resolution of small currents, we often subtracted a smooth curve fitted to the leak currents. In some cases, the first 200 µs after a voltage step contains
a large leak subtraction artifact, which was zeroed when present
before digital filtering.
The base external solution contained (mM) 155 N-methyl-D-glucamine (NMG) aspartate, 10 HEPES, 10 4-aminopyridine, 0.1 EGTA, pH 7.4 with NMG, 280-300 mOsm with no added charge
carriers. The internal solution contained (mM) 150 NMG-methanesulfonate (MeSO3), 1 MgCl2, 4 MgATP, 10 HEPES, 10 EGTA,
pH 7.3, with NMG, typically 280-290 mOsm. The h()-V relations shown in Fig. 11 for
1C were obtained with an internal solution in which the 150 mM NMG-MeSO3 was replaced by 150 mM
Cesium-MeSO3. For measurement of ionic currents, either 2 or
10 mM BaCl2 was added to the external solution. For typical gating current measurements, 0.2 mM LaCl2/2 mM MgCl2 was
added. External solution flowed continuously at a rate of 1-2 ml/
min during recording. The bath solution was grounded by a
0.5 M KCl agar bridge attached to a Ag-AgCl wire. Measurements were started after >5 min of dialysis with the internal solution. In
all cases, the junction potential between external and internal solutions was ~5 mV (Neher, 1992
). To determine the true applied potential, this value should be added to the voltages in the figures and text.
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For measurement of 1E activation curves, 2 mM BaCl2 was the
charge carrier throughout. Test depolarizations were 30 ms long and ranged from
70 to +70 mV (see Fig. 2 A, top) with repolarization to
50 mV to allow good resolution of tail currents. For
each cell, plots of peak tail current at
50 mV (Itail) vs. test pulse voltage (Vtest) were normalized by an estimate of maximal peak tail current (Itail,max). Itail,max was taken as the saturating value of
Boltzmann fits to the Itail-Vtest data. The resulting normalized relations are equivalent to normalized Po-V relations, and are referred to as G-V curves. G-V curves were then averaged across
cells. Such G-V curves were indistinguishable from G-V relations
obtained using 15-ms test depolarizations (data not shown). We
did not correct tail currents for the contribution of the "OFF"
gating current. To assay the magnitude of the error that such
OFF gating currents might produce, we corrected G-V relations
for six cells transfected with
1E
2a by subtracting the OFF gating
currents measured during repolarization to
50 mV. We found
that the average single-Boltzmann fit parameters for the corrected and uncorrected G-V curves were statistically indistinguishable (P < 0.05, Student's t test, uncorrected: z = 3.51 ± 0.4, V1/2 =
20.1 ± 3.2 mV; corrected: z = 3.57 ± 0.5, V1/2 = 19.7 ± 3.4 mV), although Itail,max was reduced by ~5% (
2,638 ± 568 pA
[uncorrected] vs. 2,516 ± 542 pA [corrected]). Gmax was calculated according to Gmax = Itail,max/(V
Vrev), where Vrev was +40
mV in 2 mM BaCl2. Therefore, the small error in Itail,max will lead
to a slight overestimate of the Gmax/Qmax ratio, which may vary
slightly for the different subunit combinations.
For gating currents, ionic currents were blocked by the external solution containing 0.2 mM LaCl3 (Bean and Rios, 1989).
The effective free La3+ concentration was 0.1 mM due to the
presence of 0.1 mM EGTA in all external solutions. The voltage
protocol was the same as for ionic currents, except that the test
pulse duration was decreased to 15 ms, and repolarization to
110 mV (see Fig. 5 A, top). Total charge moved during test depolarization (Qon) was obtained by integrating over the entire depolarizing epoch, taking as the zero baseline the average current
over the last 3 ms of the test pulse. Total charge moved during repolarization (Qoff) was calculated similarly. For each cell, Qon-V
and Qoff-V curves were normalized by an estimate of maximal mobile charge (Qmax), taken as the saturating value of the Boltzmann fit (detailed below) to the Qon-V or Qoff-V curves, as indicated in the text. Such normalized Qon-V and Qoff-V curves were
averaged across cells.
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To ensure that La3+ does not alter activation gating, we obtained Q-V relations both in the presence and absence of La3+
blockade. Fig. 1 A shows the results of the analysis, in which we
compared Qoff-V curves acquired in 2 mM MgCl2 () and 2 mM MgCl2/0.2 mM LaCl3 (
). The identity of the two curves, absent the expected surface-potential shift, provides additional strong support that La3+ does not perturb activation gating.
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To determine explicitly the surface-charge shift between solutions used for ionic and gating currents, we exploited the property that isolated gating currents can actually be measured in the solution for ionic current, so long as the voltage range is negative to the threshold (~65 mV, Fig. 1 B, inset) for ionic-current activation. We could then calculate the surface-charge shift by direct
comparison of the rising "foot" of Q-V curves obtained in ionic
and gating current solutions. Fig. 1 B shows the results of this approach. Before averaging across cells, Q-V data for a single cell
was normalized by the value of Qon at
65 mV in 2 mM Ba2+. In
the ionic-current solution containing 2 mM Ba2+, the Qon-V (Fig.
1 B, inset,
) and Qoff-V (inset,
) curves matched at potentials
negative to
65 mV, indicating that gating currents were isolated
below this potential. The main graph in Fig. 1 B demonstrates
that, over this range of voltages, Qon-V relations obtained in 2 mM Ba2+ (
) and 2 mM MgCl2/0.2 LaCl3 (
) are essentially indistinguishable, indicating that there is little if any surface-charge
shift between solutions. To quantitate the value of the shift, for
each cell the voltage shift required to fit the same dual-Boltzmann to both sets of Qon-V data was taken to be the surface potential difference. Averaging this value across cells gave a value of
3 ± 1 mV (n = 9). These results excluded the need for surface-charge correction between ionic and gating current measurements.
Steady state inactivation curves were approximated using a
protocol in which a 20-s prepulse was followed by a step to peak of the current-voltage (I-V)1 curve (typically 5 mV) to measure
the fraction of inactivated current. In some cases, a 10-ms normalizing prepulse at the test pulse potential was included before the
20-s prepulse to assay for the presence of cumulative inactivation or rundown. Steady state inactivation (h(
)-V) curves were
derived by normalizing test pulse currents by either the current
during the normalizing test pulse, or by the value of the test pulse
with no prepulse. All steady state inactivation curves were measured with 10 mM Ba2+ as charge carrier. Voltage commands
were given every minute from a holding potential of
120 mV.
Typically, prepulse voltages ranged from
120 to
20 mV in 10-mV
increments. Normalized h(
)-V relations were averaged across
cells. For cells transfected with two
subunits, the h(
)-V relation was fit with a dual-Boltzmann function to obtain parameters
for the low and high threshold components, in addition to the
relative contribution of each component.
Boltzmann fits to either G-V or Q-V relations were performed
with functions of the form B(V) = Bmax/{1 + exp[zF(V
V1/2)/ RT]}, where Bmax is the saturating value, z is the effective charge, and V1/2 is the midpoint of activation. Qon-V data above +40 mV were sometimes unreliable and were therefore excluded.
For dual-Boltzmann fits to h()-V relations, we used a function of
the form B(V) = flow{1 + exp[z low F(V
V1/2,low)/RT]}
1 + fhigh{1 + exp[z high F(V
V1/2,high)/RT]}
1, where V1/2,low and V1/2,high are midpoints of activation, zlow and zhigh are the effective valences, and
flow and fhigh are amplitudes of low and high threshold components. Fits were obtained using nonlinear, least-squares minimization. All reported values are mean ± SEM.
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RESULTS |
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Enhancement of Expressed Current Density by Auxiliary Subunits
Transfection of HEK 293 cells with the 1E subunit
alone, or in combination with various auxiliary subunits, led to the expression of well-resolved inward barium currents carried by recombinant calcium channels
(Fig. 2 A). The relative magnitudes of the various sets
of traces illustrate that addition of auxiliary subunits caused striking increases in the level of expressed current. To quantify the relative increase in current density, we calculated the maximum tail current upon repolarization to 50 mV [Gmax = nPo,max g(
50 mV)h],
where g is the unitary conductance, h is the fraction of
noninactivated channels at the end of the test pulse, n
is the number of channels, and Po,max is the maximum open probability. Fig. 2 B compares the average values
of Gmax for all different subunit combinations examined. The largest effect was the ~12-fold enhancement
of expressed current with the coexpression of
subunits. All
subunits were approximately equipotent in this regard, although the average
3 effect was slightly
smaller (approximately sevenfold). Addition of
2
to
1E produced a weaker increase in current (about
threefold), and the combination of
2
and
subunits
yielded no appreciable current enhancement over the
coexpression of
subunits alone. Since modulation of Gmax values may reflect not only changes in nPo,max, but
also differences in the number of noninactivated channels (h) with different subunits, we examined another
measure of current density (Fig. 2 C), Ipeak = nPo[Vpeak]
i[Vpeak], where Po[Vpeak] and i[Vpeak] are the open probability and unitary current at the voltage (Vpeak) yielding the maximum test-pulse current. This measure (Ipeak), which is less sensitive to test pulse inactivation, gave
similar results. Therefore, we are confident that Gmax
can henceforth be used as a quantitative indicator of
relative changes in current (nPo,max).
Isolation of Gating Currents from Channels Containing the
1E Subunit
To determine the origin of the increased current density (nPo,max), we wished to measure the maximum
amount of mobile gating charge (Qmax = nq, q is the
charge per channel), which provides a convenient assay
for the relative number of functional channels (n).
Measuring Qmax involves good resolution of the currents arising from gating charge movement (gating
currents; Armstrong and Bezanilla 1977; Sigworth,
1994
), which in turn requires a blocker that eliminates ionic currents without significantly perturbing channel
gating behavior. Previous work indicates that the highly
potent block by La3+ can be used to isolate gating currents of calcium channels containing the
1B subunit
(Jones et al., 1997a
), but not the
1C subunit (Kamp et
al., 1996
). To determine the feasibility of La3+ blockade
of calcium channels containing
1E, we examined gating currents with either 2 mM Ba2+ or 2 mM Mg2+/0.2
mM La3+ added to the bath solution (Fig. 3 A). Although 2 mM Mg2+/0.2 mM La3+ (solid traces) completely blocked ionic currents, the early outward transients that are dominated by gating current were unchanged, arguing strongly that La3+ does not alter the
voltage sensor movement that underlies activation gating. Furthermore, the block of ionic current was completely reversible (Fig. 3 B,
), and did not alter Qrev
during repetitive stimulation in the presence of La3+
(
, obtained by integrating outward transients at the
reversal potential). The lack of change of Qrev argues
that La3+ does not promote channel inactivation, which
would be apparent as a reduction in Qrev (Jones et al.,
1997b
) known as gating-charge immobilization (Armstrong and Bezanilla, 1977
; Bezanilla et al., 1991
). Similar results to those for
1E +
2a (Fig. 3, A and B) were
obtained with the other subunit combinations (data
not shown). Further experiments (see MATERIALS AND
METHODS) demonstrated that La3+ did not affect the
voltage dependence of charge movement, and that the
surface charge shift between solutions used for ionic and
gating current measurements was ~3 mV.
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With assurance that La3+ does not detectably alter
gating or surface-charge properties, we turned to analysis of extensive sets of currents recorded during La3+
block for 1E
2a (Fig. 4 A). These traces represent genuine calcium-channel gating currents for several reasons. First, no such currents are observed in mock-transfected cells (Fig. 4 B). Second, no nonlinear charge movement is present in the range of our leak
pulses (Fig. 4 C). Third, the measured charge movement is not affected by the choice of the leak subtraction protocols (data not shown). Finally, these "nonlinear displacement" currents have the standard properties typically associated with gating currents (Fig. 4 D):
time integrals of outward (Qon) and inward (Qoff) displacement currents saturated with increasing test depolarization; Qoff ~ Qon in the absence of inactivation
(Fig. 4 D); charge movement (Qon-V or Qoff-V curves) occurs before, and then parallels, ionic-current activation (G-V curve); and finally, the maximal amount of
gating-charge (Qmax) is linearly correlated with maximal current density (Gmax) (Fig. 4 E).
Mechanism of Current Potentiation by Auxiliary Subunits
With the ability to isolate gating currents, we could now
compare auxiliary subunits with regard to their mechanism for current potentiation. Fig. 5 A displays representative gating-current records for most of the different subunit combinations. These traces illustrate that
all auxiliary subunits boost the maximum amount of
gating charge (Qmax), which is taken as the saturating value of the Boltzmann fit to the Qon-V relation. Fig. 5 B
compares the average values of Qmax for all different
subunit combinations examined. Qmax for channels expressed from 1E alone was characteristically small, with
a mean of 0.8 ± 0.1 fC/pF (n = 9).
Subunits induced the strongest enhancement of Qmax, ranging from fourfold for
3 to sevenfold for
4. Coexpression of
2
also
produced clear augmentation of Qmax, though the effect was less potent than for
subunits. Table I summarizes the complete details of the analysis. Given that gating-charge per channel (q) does not appear to be affected by auxiliary subunits (Noceti et al., 1996
), the
rise in Qmax likely reflects an increase in the number of
functional channels (n). Hence, our results indicate
that the enhancement of current density by auxiliary
subunits arises, at least in part, from an increase in the
number of functional channels. Such an increase in the
number of functional channels may reflect either improved processing and trafficking of
1E channels (increasing total amount of
1E protein), or an increase in
the fraction of functional
1E protein in the membrane
(with no increase in total amount of
1E protein) by the
2
and
subunits.
To determine whether an increase in the maximal
open probability (Po,max) also contributes to higher
ionic-current densities, we calculated the ratio Gmax/
Qmax, which is directly proportional to Po,max, so long as
auxiliary subunits do not alter permeation properties
of the channel (as in Fig. 6 D and Noceti et al., 1996). Fig. 5 C shows that all
subunits approximately doubled Gmax/Qmax, but
2
left the ratio unchanged. Table I reports further details of the calculations. The
data in Fig. 5 suggest that
subunits enhance
1E current density by jointly increasing the number of functional channels (as reported by Qmax) and the maximal
open probability (as reflected by Gmax/Qmax). The enhancement of current by the
2
subunit appears to be
fundamentally different: there may be a pure increase
in the number of functional channels, without change
in Po,max.
Subunit Modulation of Activation Gating
A second goal of this study was to compare auxiliary
subunit effects on the kinetics and voltage dependence
of channel activation. To qualitatively compare activation kinetics for channels with different subunits, we
normalized the rising phases of exemplar ionic-current
records (Fig. 2 A) evoked by voltage steps to
30, 10, and +10 mV (Fig. 6 A). The identical trajectories of
traces from all four
subunits suggest that
subunits
produce channels with similar activation kinetics. Fig. 6
B shows the identical analysis for channels expressed
from
1E alone (solid trace) or from
1E +
2
(dashed
trace). The records for
1E +
1b (gray traces) are reproduced for comparison. Here again, the close correspondence between traces suggests that auxiliary subunits do not significantly modulate activation kinetics.
To examine whether auxiliary subunits affect the
steady state voltage dependence of activation (G-V), we
tested for subunit-dependent changes in G-V curves derived from peak tail currents (see MATERIALS AND
METHODS) (Fig. 6 C). Coexpression of the 2
subunit
had little effect on the G-V (Fig. 6 C) or I-V (Fig. 6 D)
relations. The lack of effect of
2
on the kinetics and
voltage dependence of activation, as well as on Gmax/
Qmax (Fig. 5 C), suggests that this subunit is functionally
uncoupled from any aspect of activation in
1E. In contrast, single-Boltzmann function analysis (Fig. 6 C, solid
curves, and Table II) clearly demonstrates that coexpression of
subunits produces an ~7-mV hyperpolarizing shift and a modest increase in the steepness of
G-V relations (e.g., the Boltzmann valence (z) increases
from 2.4 for
1E to 3.6 for
1E
2a). As expected from the
shift in the G-V relation, coexpression of
subunits
shifted the peak of the I-V relation leftward (Fig. 6 D)
without altering the reversal potential.
The results in Fig. 6, C and D, are compatible with
earlier work on subunit effects in Xenopus oocytes
(Olcese et al., 1994
), in which G-V relations were fitted
with dual-Boltzmann functions. In agreement with the
earlier report, application of dual-Boltzmann analysis
to our G-V data (Fig. 6 C, dashed curves) suggests that the apparent hyperpolarization and steepening of activation by
subunits could arise from an increase in the
proportion of the low threshold Boltzmann component from ~30 to ~70%, without change in valence or
midpoint parameters of individual Boltzmann functions. More in-depth interpretation of the data, like
that introduced by dual-Boltzmann analysis, is deferred
to the DISCUSSION, where explicit fits of a multistate kinetic model will be employed. For simplicity, in the remainder of the RESULTS, we retain single-Boltzmann analysis for first-order characterization of experimentally
resolvable changes in activation. Regardless of the particular analytical functions used to describe the data, the
results thus far (Figs. 5 and 6, A-D) clearly indicate that
subunits increase ionic current by simultaneously
modulating the G-V relation and doubling the Gmax/
Qmax ratio, in agreement with the findings of Olcese et
al. (1994
, 1996
).
To explore the mechanistic basis of the subunit effects on activation, we investigated how auxiliary subunits influenced Q-V curves derived from gating currents (Fig. 6 E). The rising phase of Q-V curves is very
sensitive to modulation of the early events in the activation pathway, and the interrelation of Q-V and G-V
curves lends insight into steps that couple voltage sensor movement to channel openings (Jones et al.,
1997a
). Fig. 6 E illustrates that all
subunits produced
essentially identical effects on the Q-V relation: a small
hyperpolarizing shift in the midpoint (~5 mV) with little change in the steepness (Boltzmann valence [z]
ranges from 2.5 to 2.8, Table II). The effects of
subunits on Q-V curves are smaller than on G-V curves,
thereby narrowing the gap between Q-V and G-V relations along the voltage axis. As expected from previous
null results,
2
had no effect on the Q-V relation. All the
subunit effects on gating (Figs. 5 C and 6, C-E), particularly the contraction between Q-V and G-V curves, fit
nicely with the idea that all
subunits act primarily to modulate a single locus of weakly voltage-dependent
steps late in the activation pathway (see DISCUSSION).
Functional Stoichiometry of Subunit Interaction
Previous reports in Xenopus oocytes indicate that 1E
channels containing different
subunits have strikingly different inactivation characteristics, despite very
similar activation gating (Olcese et al., 1994
). Here, we
sought to confirm this effect in HEK 293 cells so that
we could exploit this property to test whether multiple
subunits can simultaneously define the functional behavior of a calcium channel. To assay inactivation properties, we used a 20-s prepulse followed by a test pulse
to the peak of I-V relations (Fig. 7 A). Typical currents
for
1E
2a and
1E
3 channels illustrate the extremes of
inactivation behavior observed with the different subunit combinations.
2a dramatically slowed inactivation,
while
3 accelerated inactivation.
1b and
4 subunits
also accelerated inactivation during the test pulse (not
shown), though not as strongly as
3. To provide a robust indication of the differences in inactivation properties, we used such records to calculate steady state inactivation curves (h(
)V curves; Fig. 7 B). While addition of
2
did not affect the h(
)V relation, coexpression of
subunits induced striking modulation of steady state inactivation:
1b,
3, and
4 all left-shifted h(
)V curves
by ~10, 15, and 10 mV, respectively;
2a imparted a
right shift of ~15 mV. The profound distinction between the effects of
2a and the other
subunits has
been reported in previous studies of
1E (Olcese et al.,
1994
), and of other neuronal calcium channels, including
1A (Stea et al., 1994
) and
1B (Patil et al., 1998
).
Table III summarizes the Boltzmann analysis of h(
)V
data.
To investigate whether multiple subunits can concomitantly specify the functional properties of a single
calcium channel, we took advantage of the vast difference between the h(
)V relations for
1E
3 and
1E
2a
channels. If there are multiple
subunit sites on
1E
that specify inactivation properties, then cotransfection of both
2a and
3 subunits should result in mixed-composition channels (e.g.,
1E
2a
3) whose inactivation
behavior should be distinct from that of pure
1E
2a- or
1E
3-like channels. However, if there is only one functionally active
subunit site per channel, the aggregate
h(
)V relation should possess only two components. Fig. 8, A and B, shows the results for one such experiment in which
3 and
2a were cotransfected in a 1:1
weight ratio. This example demonstrates that inactivation is clearly biphasic, with a low threshold, readily inactivating component, as well as a high threshold, inactivation-resistant component. Only two Boltzmanns are
required to produce an excellent fit of the data since
the average residual for the dual-Boltzmann fit is close
to zero (Fig. 8 C). Furthermore, the average fit parameters to the h(
)V data correspond closely to the steady
state inactivation properties of pure
1E
2a and
1E
3
channels (Fig. 7 B and Tables III and IV). Cotransfection of
3 and
2a in a 5:1 weight ratio merely decreased
the relative amplitude of the low threshold component (Fig. 8 D), while preserving the intrinsic properties of
the two components (Table IV). The only apparent deviation from parameters obtained with pure-composition channels is a small 7-9-mV increase in the V1/2 for
the high threshold component (compare Tables III and IV). Although this increase could reflect a minor
contribution of mixed-composition channels, the overall results are consistent with the functional dominance
of pure
1E
2a and
1E
3 channels.
|
|
As a further test for the possible functional role of a
second subunit site (Tareilus et al., 1997
), we examined how auxiliary subunits modulated the properties
of a COOH-terminal truncation of the
1E construct
(
1E
, amino acids 1-1871 of
1E [1-2251]) that lacks
the secondary binding site. Fig. 9 A displays ionic currents for channels composed of
1E
+
2
or
1E +
2a +
2
subunits. Coexpression of
2a with
1E
increased
Gmax from
306 ± 115 pS/pF (n = 11) to
1,708 ± 251 pS/pF (n = 4), a 5.6-fold increase similar to the
4.1-fold increase in Gmax seen for wild-type
1E (Fig. 2 B).
Similarly, modulation of activation by
2a is unchanged
by the COOH-terminal deletion, as demonstrated in
Fig. 9 B by the identical subunit modulation of G-V relations for
1E
(circles) and wild-type
1E (squares). Finally, we compared
subunit modulation of the steady
state inactivation properties of
1E
(Fig. 9, C, traces,
and D, symbols; Table III) with data obtained with wild-type
1E (Fig. 9 D, lines). The
1E data have been shifted
uniformly by
7 mV in the Fig. 9 D overlay to account
for a difference in inactivation that is present even without
subunit coexpression (e.g.,
1E
+
2
in Fig. 9
D); this small shift likely reflects a difference in the intrinsic inactivation behavior of the
1 backbone (Soldatov et al., 1997
), rather than a change in the modulatory action of
subunits. The close correspondence between h(
)-V relations for
1E (lines) and
1E
(symbols)
in the Fig. 9 D overlay illustrates that
subunit modulation of inactivation is similar for the two constructs. Although the small difference between modulation of
1E
and
1E inactivation (most apparent for
1E
3
2
, Fig. 9 D,
) could reflect a minor contribution of a second
subunit binding site, all the results in Figs. 8 and
9 support the view that a single
subunit binding site
predominates in specifying inactivation properties. If
present, the potential contribution of a second site appears to be small by comparison.
|
|
![]() |
DISCUSSION |
---|
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---|
Although auxiliary subunits clearly have a role in defining channel properties, specific modulatory effects vary
widely across studies, underscoring the need to examine comprehensively the modulation of each 1 subunit
under the same experimental conditions. Here, we
have performed a systematic evaluation of auxiliary
subunit regulation of expression and gating of
1E calcium channels in HEK 293 cells. The experiments lead
to three main conclusions. (a) The
2
and
auxiliary
subunits differ fundamentally in the manner by which
they induce an overall increase in current density. Coexpression of
2
with the pore-forming
1E moiety produced a clear-cut enhancement of current, arising purely
from an increase in the number of functional channels
(n), without significantly affecting channel gating behavior. By contrast, coexpression of
subunits induced
stronger potentiation of current by joint elevation of
channel number (n) and maximal open probability
(Po,max), suggesting effects on both channel assembly
and gating. (b) While
2
had no appreciable effect on
activation gating,
subunits produced significant hyperpolarizing shifts in the voltage dependence of ionic-current activation and gating-charge movement, all
without discernible change in activation kinetics. Importantly, different
isoforms produced nearly indistinguishable effects in regard to both current potentiation and activation gating. (c) Little functional evidence for a secondary
subunit binding site was found, fitting with earlier biochemical evidence for a
1:1 stoichiometry of
1 and
subunits for skeletal (De
Waard and Campbell, 1995
) and neuronal N-type
(Witcher et al., 1993
) channels. Together, these findings represent an important contribution to clarifying both the mechanism and structural determinants of
auxiliary-subunit modulation of calcium channels.
In the sections to follow, we will first relate each of
the conclusions to previous studies of 1E and, where
relevant, other calcium channels. For clarity, we will
discuss
2
and
subunit effects sequentially, as independent parts. A kinetic mechanism is then developed
to explain how
subunits can produce all the observed
changes in gating, simply by alteration of the equilibrium between a single, weakly voltage-dependent transition near the open state. Finally, we consider the generality of our conclusions to other
1 isoforms.
Modulation of 1E by
2
The 2
subunit produced an approximately threefold
increase in
1E current, which arose almost exclusively
from elevated channel expression (Qmax). The
2
subunit had no other clear modulatory effects, except to
slightly antagonize the effect of
3 on inactivation (Fig.
7 B). All measures of activation gating, including the
maximal open probability (Gmax/Qmax), the voltage dependence of charge movement (Q-V), and the voltage
dependence of ionic activation (G-V) were similar to
1E alone. Similar effects on inactivation and expression were reported for doe1 (marine ray analog of
1E)
expressed in Xenopus oocytes (Ellinor et al., 1993
).
However, in contrast to our results, in studies of rat
1E
in COS-7 cells (Stephens et al., 1997
) and human
1E in
Xenopus oocytes (Wakamori et al., 1994
), coexpression
of
2
was found to produce a depolarizing shift in the
G-V without modifying expressed current levels; however,
these studies agree with our findings concerning the
slight antagonism of
3 effects on inactivation (Fig. 7 B).
The applicability of our results to other channel types
is unclear. However, it is interesting to note that the reported effects of the 2
subunit on other
1 subunits
also varies widely, sometimes agreeing with our findings, other times not. For example, with regard to modulation of
1C channel density, the
2
subunit was found to increase ligand binding sites (Welling et al.,
1993
), protein levels (Shistik et al., 1995
), and gating
currents (Bangalore et al., 1996
). By contrast, in other
studies (Wei et al., 1995
; Gurnett et al., 1997
) of
1C,
maximal dihydropyridine binding is not increased by
2
coexpression. Similarly, activation kinetics of
1C
accelerate in some studies (Singer et al., 1991
; Bangalore et al., 1996
), but not others (Mikami et al., 1989
;
Welling et al., 1993
). The sources of this variability have
yet to be determined.
Subunits Act Differently than the
2
Subunit
By contrast to the 2
subunit, coexpression of
subunits (
1-
4) enhanced current density (Gmax) by increasing not only the number of functional channels
(Qmax) but also the maximum open probability (Gmax/
Qmax). Similar effects on current density have been reported in COS 7 (Stephens et al., 1997
) and HEK 293 cells (Williams et al., 1994
), but not in Xenopus oocytes
(Soong et al., 1993
; Olcese et al., 1994
, 1996
). Furthermore, in a study of
1E gating currents in Xenopus oocytes (Olcese et al., 1996
), coexpression of
2a with
1E
actually decreased the number of functional channels
(Qmax), although surprisingly they found a twofold increase of Gmax/Qmax that is qualitatively similar to our
result. Additional support for the role of the
subunit
in modulating Po,max comes from a separate study that
used fluctuation analysis to determine the effects of
2a
and
1a on
1E open probability (Noceti et al., 1996
).
Fitting with the doubling of the Gmax/Qmax ratio, the
subunit also induced hyperpolarizing shifts of both
G-V and the Q-V relations and slightly reduced the gap
between the two, all while producing little effect on activation kinetics. Here, the action of
subunits is also
somewhat controversial. Although most studies of
1E
report effects on G-V relations and activation kinetics that are similar to ours (Witcher et al., 1993
; Olcese et
al., 1994
; Stephens et al., 1997
), in one case (Wakamori
et al., 1994
),
1b coexpression with the human
1E in Xenopus oocytes was found to slow activation kinetics substantially. With respect to gating currents, the only
other study of
1E charge movement (Olcese et al.,
1996
) also found that the
subunit reduced the gap
between the G-V and Q-V. However, in contrast to the
small but statistically significant (P < 0.01, Student's t
test, comparing cells with and without a
) shift in the
Q-V reported here, Olcese et al. (1996)
found that the
subunit produced no significant change in the Q-V.
Again, these discrepancies may reflect differences between clones (human versus rat
1E) or expression systems (Xenopus oocytes versus HEK 293 cells). Despite
these minor differences, all the results indicate a role
for
subunits in modulating activation gating.
In other calcium channels, the reported effects of the
subunits vary even more widely than for the
2
subunit. However, at least in some respects,
subunit
modulation of other
1 subunits appears similar to
what we find for
1E. For example, there are reported
shifts in the voltage dependence of ionic activation for
1A (Stea et al., 1994
; De Waard and Campbell, 1995
)
and
1C (Wei et al., 1991
; Neely et al., 1993
). Increased
current density has also been observed for many of the
1
subunits including
1A (Mori et al., 1991
),
1B (Williams
et al., 1992a
),
1C (Perez-Reyes et al., 1992
),
1D (Williams et al., 1992b
), and
1S (Ren and Hall, 1997
). Furthermore, for
1C, studies of gating currents in both
HEK 293 cells (Kamp et al., 1996
; Josephson and
Varadi, 1996
) and Xenopus oocytes (Neely et al., 1993
)
find that
subunit modulation of ionic current activation is not associated with much shift in the Q-V, similar
to what we find for
1E. On the other hand, even these
few gating current studies disagree in other regards.
While coexpression of
1a (Kamp et al., 1996
) or
3 (Josephson and Varadi, 1996
) with
1C increased both current density and Qmax in HEK 293 cells similar to our
results for
1E,
2a increased current without changing
Qmax in Xenopus oocytes (Neely et al., 1993
). Therefore,
as with
1E, the specific effects observed with
coexpression appear to depend on as yet unknown distinctions between expression systems, perhaps the endogenous expression of
XO subunits in Xenopus oocytes (Tareilus et al., 1997
).
Different Isoforms Have Similar Effects on Activation and
Expression, but Not on Inactivation
There was little isoform dependence to the modulation
of all the above measures of activation gating, suggesting that different subunits act by a similar mechanism
to modulate activation and expression of
1E, despite
very different effects on inactivation. While no other
study has compared gating currents of
1E channels containing different
subunits, measurements of
1E
ionic-current G-V curves in Xenopus oocytes support this
finding (Olcese et al., 1994
). However, for other
1 subunits, modulation of expression and activation may differ across
subunits. For example, there clearly is isoform specificity in the
subunit modulation of current
potentiation in
1A (Stea et al., 1994
; De Waard et al., 1994
) and
1S (Ren and Hall, 1997
). This fits with the
binding affinity differences in vitro of various
subunits to
1A (De Waard et al., 1995
). However, binding
of various
subunits to the I-II linker of
1B occurs with
the same affinity (Scott et al., 1996
). Whether differences in in vitro binding affinities translates into discernible gradations of functional effects remains to be
established.
One Subunit May Predominate in Directing Baseline
Channel Properties
Most previous studies have implicitly assumed that only
one subunit is involved in modulating channel properties, consistent with biochemical evidence for a 1:1
stoichiometry of
1 and
subunits for skeletal (De
Waard et al., 1996
) and N-type (Witcher et al., 1993
)
channels. However, a recent report by Tareilus et al.
(1997)
identified a second
subunit binding site on
the COOH terminus of
1E, raising the possibility that
two or more
subunits might collectively determine
channel gating properties. Here, we found little evidence that two
subunits modulate the properties of
the
1E channel, either in regard to expression or gating.
Mechanism of Subunit Modulation of
1E Gating
To account for subunit effects on G-V and Q-V
curves, previous studies have proposed that the
subunit acts mainly on the weakly voltage-dependent steps
that "couple" channel opening to voltage sensor movement (Neely et al., 1993
; Olcese et al., 1996
). Here, we
demonstrate that this mechanism may explain not only the modulation of G-V and Q-V curves, but also the
doubling of maximum open probability. Fig. 10 depicts
a channel gating model that closely resembles those
previously used in the study of potassium channel gating (Zagotta and Aldrich, 1990
; Schoppa et al., 1992
). There are three independent, voltage-dependent transitions between the closed states (C0, C1, C2, and C3),
each associated with an appropriately scaled equilibrium constant K0. These transitions are followed by a
weakly voltage-dependent transition (C2-C3) with equilibrium constant K1 and a final voltage-independent
step with equilibrium constant K2. K0 and K1 are voltage
dependent according to a Boltzmann distribution, Ki = exp{[ziF(V
Vi)]/(RT)}. To obtain baseline model parameters (z0, z1, V0, V1, K2), we fit
1E alone Q-V and G-V
data (Fig. 10 B). Then, to simulate the observed twofold
change in the maximum open probability, we modified
only K2, the equilibrium constant for the last voltage-
independent transition leading to channel opening.
This simple change reproduced well both the shift in
the G-V relationship and the shift in the Q-V relationship (Fig. 10 C). In fact, such simulations indicate that
modifying the coupling of charge movement to channel opening (K2) usually also perturbs the Q-V relation
and, therefore, charge movement. Yet in several studies
(Kamp et al., 1996
; Josephson and Varadi, 1996
; Neely et
al., 1993
; Olcese et al., 1996
), shifts in ionic activation
have been seen with little or no modification of charge
movement. In these studies, it may be that the shift in
charge movement is too small to be well resolved.
|
To determine the effect of modifying K2 on the time
course of activation, we modeled the kinetics of activation. The choice of rate constants is constrained by the
equilibrium constants, according to Ki(V) = i(V)/
i(V), where
i(V) is the forward rate constant and
i(V) is the backward rate constant (seconds
1). We
chose
i(V) = fi exp{[di ziF(V
Vi)]/(RT)}, and
i(V) =
i(V)/Ki(V), giving us free parameters f0, d0, f1, d1, and
f2, which we could vary to fit the activation kinetics. Fig.
10 D shows a model fit (thin solid line) consistent with
1E alone steady state model parameters (z0, z1, V0, V1,
K2; Fig. 10 B). Representative ionic current data (Fig.
10 B, thick gray line) are derived by subtracting the gating currents (Fig. 5 A) from the
1E
1b whole cell
records in Fig. 6 A. Because of the subunit invariance of activation kinetics, these ionic currents also represent
the expected time course of
1E alone. Changing K2 to
accord with the twofold increase in maximal open
probability produced by
subunits can modify the activation kinetics (Fig. 10 E, dashed line), but this may be
compensated for by altering only f1 and f2. This amounts to subtle changes in the absolute rate constants of the
last two transitions, but only an alteration of the equilibrium constant of the last transition. Although no
change in parameters corresponding to the more voltage-dependent steps (z0 and f0) is necessary, we found
that we could not well reproduce the invariance of activation kinetics by just modifying f2, corresponding to
the last voltage-independent step. Therefore, this simulation argues that all the effects of
subunit modulation
of
1E (increased open probability, hyperpolarization of G-V and Q-V curves, and invariant activation kinetics) can be attributed to actions on one or a few weakly
voltage-dependent steps before opening.
Generalizability of Results to Other 1 Isoforms?
From the previous discussion, it is clear that generalization across 1 subtypes is a difficult proposition. Nevertheless, we wondered whether some of the most robust
properties of
1E modulation by subunits would translate to a different
1 subunit. In particular, there was
striking adherence to two "rules" for
1E modulation by
subunits: (a) all
subunits produce no change in the
kinetics of activation, but induce identical but relatively small hyperpolarizing shifts of the G-V curve, and (b)
distinct
subunits impart vastly different steady state
inactivation curves. Do these features of subunit modulation hold true as general tenets for other
1 subunits?
Fig. 11 tests this proposition for the 1C calcium
channel. The results indicate a complete reversal of the
behavior found with
1E. Now the kinetics of activation
are clearly different for
3 and
2a subunits. In addition, the different
isoforms led to large differences in
G-V curves. On the other hand, steady state inactivation
curves show only small isoform-dependent distinctions.
The diametrically opposite behaviors exhibited by
1E and
1C subunits have interesting implications for
the structure-function relations underlying
1-
modulation. The leading candidates for structural interaction between these two subunits are a small motif on
the I-II linker of
1 subunits known as the "alpha interaction domain" or AID (Pragnell et al., 1994
), and another small motif in the middle of
subunits known as
the "
interaction domain" or BID (De Waard et al.,
1995
). AID and BID peptides bind with high affinity (tens
of nanomolar), and the BID region has documented importance for modulation of
1 subunits (De Waard et al.,
1995
). The key points with regard to our findings are that
the BID is highly (~70%) homologous across
subunits,
and the AID is also highly conserved across different
1
subunits (Pragnell et al., 1994
; De Waard et al., 1995
).
The profound differences in
isoform selectivity for entirely different gating properties, depending on the
1
subtype, suggest that either the AID-BID interaction is
exquisitely sensitive to small sequence variations in the
AID (Scott et al., 1996
), or there are other features that
contribute to
subunit modulation of
1 (Chien et al.,
1996
). Distinguishing between these two possibilities and
identifying any secondary interaction sites will be important challenges for the future.
![]() |
FOOTNOTES |
---|
Address correspondence to David T. Yue, Program in Molecular and Cellular Systems Physiology, Departments of Biomedical Engineering and Neuroscience, Johns Hopkins University School of Medicine, Ross Building, Room 713, 720 Rutland Avenue, Baltimore, MD 21205. Fax: 410-955-0549; E-mail: dyue{at}bme.jhu.edu
Original version received 12 March 1998 and accepted version received 15 June 1998.
We thank K.P. Campbell for the 1b clone, T.P. Snutch for the
1E and
2
clones, E. Perez-Reyes for the
1C,
2a,
3, and
4
clones, M. deLeon for construction of
1E
, J.G. Mulle for technical assistance, and David Brody and Carla DeMaria for discussion and comments.
This work was supported by the National Institutes of Health (NIH) to D.T. Yue, the National Science Foundation Presidential Faculty Fellowship (D.T. Yue), a Maryland American Heart Association Postdoctoral Fellowship (S.K. Wei), and an NIH Medical Scientist Training Program Award (L.P. Jones).
![]() |
Abbreviation used in this paper |
---|
I-V, current-voltage.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. |
Armstrong, C.M., and
F. Bezanilla.
1974.
Charge movement associated with the opening and closing of the activation gates of the
Na channels.
J. Gen. Physiol.
63:
533-552
|
2. | Armstrong, C.M., and F. Bezanilla. 1977. Inactivation of the sodium channel. II. Gating current experiments. J. Gen. Physiol. 70: 567-590 [Abstract]. |
3. |
Bangalore, R.,
G. Mehrke,
K. Gingrich,
F. Hofmann, and
R.S. Kass.
1996.
Influence of L-type Ca channel alpha 2/delta-subunit on
ionic and gating current in transiently transfected HEK 293 cells.
Am. J. Physiol.
270:
H1521-H1528
|
4. | Bean, B.P., and E. Rios. 1989. Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating. J. Gen. Physiol. 94: 65-93 [Abstract]. |
5. | Bezanilla, F., E. Perozo, D.M. Papazian, and E. Stefani. 1991. Molecular basis of gating charge immobilization in Shaker potassium channels. Science. 254: 679-683 [Medline]. |
6. | Brody, D.L., P.G. Patil, J.G. Mulle, T.P. Snutch, and D.T. Yue. 1997. Bursts of action potential waveforms relieve G-protein inhibition of recombinant P/Q-type Ca2+ channels in HEK 293 cells. J. Physiol.(Camb.). 499: 637-644 [Abstract]. |
7. |
Castellano, A.,
X. Wei,
L. Birnbaumer, and
E. Perez-Reyes.
1993a.
Cloning and expression of a neuronal calcium channel beta subunit.
J. Biol. Chem.
268:
12359-12366
|
8. |
Castellano, A.,
X. Wei,
L. Birnbaumer, and
E. Perez-Reyes.
1993b.
Cloning and expression of a third calcium channel beta subunit.
J. Biol. Chem.
268:
3450-3455
|
9. |
Chien, A.J.,
K.M. Carr,
R.E. Shirokov,
E. Rios, and
M.M. Hosey.
1996.
Identification of palmitoylation sites within the L-type calcium channel beta2a subunit and effects on channel function.
J.
Biol. Chem.
271:
26465-26468
|
10. | De Waard, M., and K.P. Campbell. 1995. Subunit regulation of the neuronal alpha 1A Ca2+ channel expressed in Xenopus oocytes. J. Physiol. (Camb.). 485: 619-634 [Abstract]. |
11. | De Waard, M., C.A. Gurnett, and K.P. Campbell. 1996. Structural and functional diversity of voltage-activated calcium channels. In Ion Channels. T. Narahashi, editior. Plenum Publishing Corp., New York. 41-87. |
12. | De Waard, M., M. Pragnell, and K.P. Campbell. 1994. Ca2+ channel regulation by a conserved beta subunit domain. Neuron. 13: 495-503 [Medline]. |
13. |
De Waard, M.,
D.R. Witcher,
M. Pragnell,
H. Liu, and
K.P. Campbell.
1995.
Properties of the alpha 1-beta anchoring site in voltage-dependent Ca2+ channels.
J. Biol. Chem.
270:
12056-12064
|
14. | Ellinor, P.T., J.F. Zhang, A.D. Randall, M. Zhou, T.L. Schwarz, R.W. Tsien, and W.A. Horne. 1993. Functional expression of a rapidly inactivating neuronal calcium channel. Nature. 363: 455-458 [Medline]. |
15. | Gorman, C.M., D.R. Gies, and G. McCray. 1990. Transient production of proteins using an adenovirus transformed cell line. DNA and Protein Engineering Techniques 2: 3-10 . |
16. |
Gurnett, C.A.,
R. Felix, and
K.P. Campbell.
1997.
Extracellular interaction of the voltage-dependent calcium channel alpha-2
delta and alpha1 subunits.
J. Biol. Chem.
272:
18508-18512
|
17. | Jones, L.P., P.G. Patil, T.P. Snutch, and D.T. Yue. 1997a. G-protein modulation of N-type calcium channel gating current in human embryonic kidney cells (HEK 293). J. Physiol. (Camb.). 498: 601-610 [Abstract]. |
18. | Jones, L.P., P.G. Patil, J.G. Mulle, M.B. Sachs, and D.T. Yue. 1997b. Inactivation of recombinant N-type calcium channels probed by gating current analysis. Soc. Neurosci. Abstr. 23: 475.4 . |
19. | Josephson, I.R., and G. Varadi. 1996. The beta subunit increases Ca2+ currents and gating charge movements of human cardiac L-type Ca2+ channels. Biophys. J. 70: 1285-1293 [Abstract]. |
20. | Kamp, T.J., M.T. Perez-Garcia, and E. Marban. 1996. Enhancement of ionic current and charge movement by coexpression of calcium channel beta 1A subunit with alpha 1C subunit in a human embryonic kidney cell line. J. Physiol. (Camb.). 492: 89-96 [Abstract]. |
21. | Mikami, A., K. Imoto, T. Tanabe, T. Niidome, Y. Mori, H. Takeshima, S. Narumiya, and S. Numa. 1989. Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. Nature. 340: 230-233 [Medline]. |
22. | Mori, Y., T. Friedrich, M.S. Kim, A. Mikami, J. Nakai, P. Ruth, E. Bosse, F. Hofmann, V. Flockerzi, T. Furuichi, et al . 1991. Primary structure and functional expression from complementary DNA of a brain calcium channel. Nature. 350: 398-402 [Medline]. |
23. | Neely, A., X. Wei, R. Olcese, L. Birnbaumer, and E. Stefani. 1993. Potentiation by the beta subunit of the ratio of the ionic current to the charge movement in the cardiac calcium channel. Science. 262: 575-578 [Medline]. |
24. | Neher, E.. 1992. Correction for liquid junction potentials in patch clamp experiments. Methods Enzymol. 207: 123-130 [Medline]. |
25. | Noceti, F., P. Baldelli, X. Wei, N. Qin, L. Toro, L. Birnbaumer, and E. Stefani. 1996. Effective gating charges per channel in voltage-dependent K+ and Ca2+ channels. J. Gen. Physiol. 108: 143-155 [Abstract]. |
26. | Olcese, R., A. Neely, N. Qin, X.Y. Wei, L. Birnbaumer, and E. Stefani. 1996. Coupling between charge movement and pore opening in vertebrate neuronal alpha(1E) calcium channels. J. Physiol. (Camb.). 497: 675-686 [Abstract]. |
27. | Olcese, R., N. Qin, T. Schneider, A. Neely, X. Wei, E. Stefani, and L. Birnbaumer. 1994. The amino terminus of a calcium channel beta subunit sets rates of channel inactivation independently of the subunit's effect on activation. Neuron. 13: 1433-1438 [Medline]. |
28. | Patil, P.G., D.L. Brody, and D.T. Yue. 1998. Preferential closed-state inactivation of neuronal calcium channels. Neuron. 20: 1027-1038 [Medline]. |
29. |
Perez-Reyes, E.,
A. Castellano,
H.S. Kim,
P. Bertrand,
E. Baggstrom,
A.E. Lacerda,
X.Y. Wei, and
L. Birnbaumer.
1992.
Cloning and
expression of a cardiac/brain beta subunit of the L-type calcium
channel.
J. Biol. Chem.
267:
1792-1797
|
30. | Perez-Reyes, E., and T. Schneider. 1994. Research overview: calcium channels: structure, function and classification. Drug Dev. Res. 33: 295-318 . |
31. | Pragnell, M., M. De Waard, Y. Mori, T. Tanabe, T.P. Snutch, and K.P. Campbell. 1994. Calcium channel beta-subunit binds to a conserved motif in the I-II cytoplasmic linker of the alpha 1-subunit. Nature. 368: 67-70 [Medline]. |
32. | Pragnell, M., J. Sakamoto, S.D. Jay, and K.P. Campbell. 1991. Cloning and tissue-specific expression of the brain calcium channel beta-subunit. FEBS Lett. 291: 253-258 [Medline]. |
33. |
Ren, D.J., and
L.M. Hall.
1997.
Functional expression and characterization of skeletal muscle dihypropyridine receptors in Xenopus oocytes.
J. Biol. Chem.
272:
22393-22396
|
34. | Schoppa, N.E., K. McCormack, M.A. Tanouye, and F.J. Sigworth. 1992. The size of gating charge in wild-type and mutant Shaker potassium channels. Science. 255: 1712-1715 [Medline]. |
35. |
Scott, V.E.,
M. De Waard,
H. Liu,
C.A. Gurnett,
D.P. Venzke,
V.A. Lennon, and
K.P. Campbell.
1996.
Beta subunit heterogeneity in
N-type Ca2+ channels.
J. Biol. Chem.
271:
3207-3212
|
36. | Shistik, E., T. Ivanina, T. Puri, M. Hosey, and N. Dascal. 1995. Ca2+ current enhancement by alpha 2/delta and beta subunits in Xenopus oocytes: contribution of changes in channel gating and alpha 1 protein level. J. Physiol. (Camb.). 489: 55-62 [Abstract]. |
37. | Sigworth, F.J.. 1994. Voltage gating of ion channels. Q. Rev. Biophys. 27: 1-40 [Medline]. |
38. | Singer, D., M. Biel, I. Lotan, V. Flockerzi, F. Hofmann, and N. Dascal. 1991. The roles of the subunits in the function of the calcium channel. Science. 253: 1553-1557 [Medline]. |
39. |
Soldatov, N.M.,
R.D. Zuhlke,
A. Bouron, and
H. Reuter.
1997.
Molecular structures involved in L-type calcium channel inactivation. Role of the carboxyl-terminal region encoded by exons
40-42 in alpha1S subunit in the kinetics and Ca2+ dependence of
inactivation.
J. Biol. Chem.
272:
3560-3566
|
40. | Soong, T.W., A. Stea, C.D. Hodson, S.J. Dubel, S.R. Vincent, and T.P. Snutch. 1993. Structure and functional expression of a member of the low voltage-activated calcium channel family. Science. 260: 1133-1136 [Medline]. |
41. |
Stea, A.,
W.J. Tomlinson,
T.W. Soong,
E. Bourinet,
S.J. Dubel,
S.R. Vincent, and
T.P. Snutch.
1994.
Localization and functional
properties of a rat brain alpha 1A calcium channel reflect similarities to neuronal Q- and P-type channels.
Proc. Natl. Acad. Sci.
USA.
91:
10576-10580
|
42. | Stephens, G.J., K.M. Page, J.R. Burley, N.S. Berrow, and A.C. Dolphin. 1997. Functional expression of rat brain cloned alpha-1e calcium channels in COS7 cells. Pflügers Arch. Eur. J. Physiol. 433: 523-532 [Medline]. |
43. | Sun, D., F. Chang, A. Chien, X.-L. Xhao, R. Shirokov, E. Rios, and M. Hosey. 1994. Expression of functional cardiac L-type Ca2+ channels in transiently transfected HEK (293) cells. Biophys. J. 66: A320 . (Abstr.) . |
44. |
Tareilus, E.,
M. Roux,
N. Qin,
R. Olcese,
J.M. Zhou,
E. Stefani, and
L. Birnbaumer.
1997.
A Xenopus oocyte beta subunit![]() |
45. | Tomlinson, W.J., A. Stea, E. Bourinet, P. Charnet, J. Nargeot, and T.P. Snutch. 1993. Functional properties of a neuronal class C L-type calcium channel. Neuropharmacology. 32: 1117-1126 [Medline]. |
46. | Wakamori, M., T. Niidome, D. Furutama, T. Furuichi, K. Mikoshiba, Y. Fujita, I. Tanaka, K. Katayama, A. Yatani, A. Schwartz, et al . 1994. Distinctive functional properties of the neuronal BII (class E) calcium channel. Receptors Channels 2: 303-314 [Medline]. |
47. |
Walker, D.,
D. Bichet,
K.P. Campbell, and
M. De Waard.
1998.
A ![]() ![]() |
48. | Walker, D., and M. De Waard. 1998. Subunit interaction in voltage-dependent Ca2+ channels: role in channel function. Trends Neurosci. 21: 148-154 [Medline]. |
49. |
Wei, X.,
S. Pan,
W. Lang,
H. Kim,
T. Schneider,
E. Perez-Reyes, and
L. Birnbaumer.
1995.
Molecular determinants of cardiac Ca2+
channel pharmacology. Subunit requirement for the high affinity and allosteric regulation of dihydropyridine binding.
J. Biol.
Chem.
270:
27106-27111
|
50. |
Wei, X.Y.,
E. Perez-Reyes,
A.E. Lacerda,
G. Schuster,
A.M. Brown, and
L. Birnbaumer.
1991.
Heterologous regulation of the cardiac Ca2+ channel alpha 1 subunit by skeletal muscle beta and
gamma subunits. Implications for the structure of cardiac L-type
Ca2+ channels.
J. Biol. Chem.
266:
21943-21947
|
51. | Welling, A., E. Bosse, A. Cavalie, R. Bottlender, A. Ludwig, W. Nastainczyk, V. Flockerzi, and F. Hofmann. 1993. Stable co-expression of calcium channel alpha 1, beta and alpha 2/delta subunits in a somatic cell line. J. Physiol. (Camb.). 471: 749-765 [Abstract]. |
52. | Williams, M.E., P.F. Brust, D.H. Feldman, S. Patthi, S. Simerson, A. Maroufi, A.F. McCue, G. Velicelebi, S.B. Ellis, and M.M. Harpold. 1992a. Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. Science. 257: 389-395 [Medline]. |
53. | Williams, M.E., D.H. Feldman, A.F. McCue, R. Brenner, G. Velicelebi, S.B. Ellis, and M.M. Harpold. 1992b. Structure and functional expression of alpha 1, alpha 2, and beta subunits of a novel human neuronal calcium channel subtype. Neuron. 8: 71-84 [Medline]. |
54. |
Williams, M.E.,
L.M. Marubio,
C.R. Deal,
M. Hans,
P.F. Brust,
L.H. Philipson,
R.J. Miller,
E.C. Johnson,
M.M. Harpold, and
S.B. Ellis.
1994.
Structure and functional characterization of neuronal
alpha 1E calcium channel subtypes.
J. Biol. Chem.
269:
22347-22357
|
55. | Witcher, D.R., M. De Waard, J. Sakamoto, C. Franzini-Armstrong, M. Pragnell, S.D. Kahl, and K.P. Campbell. 1993. Subunit identification and reconstitution of the N-type Ca2+ channel complex purified from brain. Science. 261: 486-489 [Medline]. |
56. |
Wu, L.G.,
J.G. Borst, and
B. Sakmann.
1998.
R-type Ca2+ currents
evoke transmitter release at a rat central synapse.
Proc. Natl. Acad.
Sci. USA.
95:
4720-4725
|
57. | Zagotta, W.N., and R.W. Aldrich. 1990. Voltage-dependent gating of Shaker A-type potassium channels in Drosophila muscle. J. Gen. Physiol. 95: 29-60 [Abstract]. |