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The Journal of Neuroscience, May 15, 1998, 18(10):3689-3698
Structural Features Determining Differential Receptor Regulation
of Neuronal Ca Channels
Arthur A.
Simen and
Richard J.
Miller
Department of Pharmacological and Physiological Sciences, Committee
on Neurobiology, University of Chicago, Chicago, Illinois 60637
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ABSTRACT |
Dihydropyridine-insensitive Ca channels are subject to direct
receptor G-protein-mediated inhibition to differing extents. 
1B channels are much more strongly modulated than
1E channels. To understand the structural basis for this
difference, we have constructed and expressed various 1B
and 1E chimeric Ca channels and examined their
regulation by 
-opioid receptors. Replacement of the first
membrane-spanning domain of 1E with the corresponding region of 1B resulted in a chimeric Ca channel that was
modulated by -opioid receptors to a significantly greater extent
than 1E. Transfer of the N terminus and I/II loop from
1B in addition to domain I resulted in a chimeric
channel that was modulated to the same extent as 1B.
Other regions of the molecule do not appear to contribute significantly
to the degree of inhibition obtained, although the C terminus may
contribute to facilitation.
Key words:
calcium channels; G-proteins; opioid receptors; modulation; structure-function; inverse agonism
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INTRODUCTION |
Voltage-sensitive Ca channels are
one of the most important classes of molecules used by excitable cells
for the transduction of cellular excitability into the intracellular Ca
signals that regulate events such as muscle contraction and the release
of neurotransmitters. Inhibition of Ca influx through these channels by
G-protein-linked receptors is an important means by which
neurotransmitters and drugs can regulate the strength of synaptic
transmission (Miller, 1990
; Rhim et al., 1994). Elucidation of the
primary structure of Ca channels has demonstrated that they consist of
a family of related proteins that has two branches. Each branch
consists of three different proteins that differ in their pharmacology and, in particular, in their sensitivity to dihydropyridine (DHP) drugs. It is thought that the subfamily of DHP-insensitive Ca channels
can be regulated by receptors through the direct interaction of
G-protein 
subunits with the major pore-forming
(1) subunit of the channel (Herlitze et al.,
1996; Ikeda, 1996; De Waard, 1997; Zamponi et al., 1997). Expression
studies have indicated that all three types of DHP-insensitive Ca
channels (1A, 1B, and
1E) are regulated by G-proteins, although there
are large differences in their susceptibility to modulation. Thus,
1B is the most sensitive to G-protein modulation, and
1E is the least sensitive (Toth et al., 1996; Yassin et
al., 1996).
The structural basis for the G-protein regulation of Ca channels has
received considerable attention, and recent studies have started to
reveal the sites of interaction between G-protein subunits and
Ca channels. Initially, it was shown that G-protein subunits
could interact with two distinct sites in the intracellular loop
connecting the first two domains of the 1 subunit (I/II loop) (De Waard et al., 1997; Qin et al., 1997; Zamponi et al., 1997).
One of these sites contains the QXXER motif, which has been shown to be
important in the binding of subunits to other effectors
(Pragnell et al., 1994; Chen et al., 1995). However, although there is
little doubt that subunits can interact with this region of the
channel, controversy exists as to the significance of this interaction.
Experiments designed to demonstrate the importance of this interaction
for G-protein regulation of Ca channels have yielded mixed results.
Further studies have indicated that subunits can also bind to a
region in the C-terminal tail of sensitive Ca channels (Qin et al.,
1997), and there are data suggesting that this interaction may be
important in G-protein modulation (Qin et al., 1997; Zhang et al.,
1996). In either case, because binding to these sites is a common
element to all three DHP-insensitive Ca channels, further structural
features are likely to be required to determine differential
sensitivity to G-proteins. To identify structural elements involved in
determining the differential sensitivity of 1B compared
with 1E channels, we have created a variety of chimeric
Ca channels in which different parts of 1B-1 were
transferred into an 1E-3 background. Exchange of domain
I alone yields a chimeric channel with considerably increased
sensitivity to modulation, but the further exchange of the I/II linker
and the N terminus is required to obtain a chimeric channel with
sensitivity to modulation equivalent to native 1B-1.
Transfer of the C terminus of 1B-1 appears to enhance
facilitation but not inhibition.
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MATERIALS AND METHODS |
Cell culture. tsA-201 cells (a gift from Dr. W. A. Horne, Stanford University) were cultured in MEM (Life Technologies,
Gaithersburg, MD), 10% fetal bovine serum (Life Technologies), and 1%
penicillin-streptomycin (Life Technologies) at 37°C in 5%
CO2. When cells reached 40-70% confluency they were
replated in 35 mm dishes and were transfected 4-10 hr later with a
total of 5-7 µg of DNA (2-5 µg of 1, 1.7 µg of 2B, 0.8 µg of 1B,
1.5 µg of 1R or 
R, and 1 µg of CD8-) by
polyethyleneimine-mediated transfection (Boussif et al., 1996). Cells
were washed with culture medium 1-2 hr after transfection, and fresh
10% serum-containing medium was added. Cells were detached from dishes
with HBSS (Ca- and Mg-free; Life Technologies) 30-40 hr later and were
replated on poly-L-lysine-coated coverslips. Cells were
recorded from 36 to 80 hr after transfection. cDNA for CD8 was a
gift from Dr. J. Bluestone (University of Chicago). The
1R and R constructs were gifts from Dr. G. I. Bell (Howard Hughes Medical Institute, University of Chicago) and have
been described previously (Yasuda et al., 1993). Expression of
2B and 1B were verified by reverse
transcription-PCR and Northern blotting (data not shown).
Electrophysiology. Currents were recorded in a solution
containing 15 mM BaCl2 (5 mM
BaCl2 in the case of the current-voltage data), 150 mM TEA-Cl, and 10 mM HEPES, pH 7.4, with TEA-OH
or N-methyl-D-glucamine. Pipette solutions were
made according to the method of Bean (1992). U69593 (Sigma, St. Louis,
MO; or Research Biochemicals, Natick, MA) and norbinaltrophimine
(norBNI; Research Biochemicals) were made as concentrated stock
solutions in 100% ethanol. ICI-174,864 was made as a 10 mM
stock solution in DMSO. [D-Pen2,5]-enkephalin (DPDPE) was made
as a 10 mM stock solution in sterile water. U69593, DPDPE,
and ICI-174864 were added to the bath solution at concentrations of 1 µM, and norBNI was added at 100 nM. Data were
recorded under the control of pClamp6 software (Axon Instruments, Foster City, CA) with an Axon 200A amplifier at an acquisition rate of
10 kHz and were filtered with a four-pole Bessel filter at 2 kHz.
Pipettes were typically 1-3 m
in resistance, and series resistance
was compensated >80% in all cases. To assay G-protein modulation,
currents were elicited by a dual-pulse protocol consisting of two 50 msec depolarizations to 0 mV from a holding potential of 
90 mV,
separated by 800 msec at the holding potential, with a 30 msec, 90 mV
depolarization (prepulse) ending 5 msec before the second pulse. Cells
expressing CD8 were identified by incubation with anti-CD8-coated
beads (Dynal, Great Neck, NY), and bead-decorated cells were chosen
exclusively for recordings.
Data analysis. Data were analyzed off-line using
Clampfit (Axon Instruments), Systat (SPSS Inc., Chicago, IL),
MATLAB (MathWorks Inc.), and custom-written software. Current
inhibition was estimated as follows. Peak current amplitudes were
measured and were adjusted to remove any slow linear "runup" or
"rundown" by subtraction of linear function fits to the baselines.
A series of adjacent current amplitudes was averaged just before drug
exposure, during U69593 exposure at the peak of the drug effect, and
during the peak of the norBNI response. Currents in the presence of
U69593 were taken as an estimate of current in the presence of
maximally activated receptor I(R*). When norBNI caused
amplitudes to increase beyond baseline values before drug exposure,
currents were taken as an estimate of current in the presence of
maximally inactive receptor I(R). Otherwise, baseline values
were used to estimate I(R). "Total modulation" was then
defined as [I(R) I(R*)]/I(R).
Relief of inhibition by the depolarizing prepulse was quantified by
calculating a "corrected prepulse ratio" to allow for comparisons
between constructs while correcting for differences in inactivation
caused by the prepulse. Current ratios in the presence of U69593,
I(+pp)/I(pp), were calculated and corrected for
differences in inactivation between constructs by multiplying by the
ratio I(pp)/I(+pp) in the presence of norBNI.
Facilitation was expressed as a simple current ratio,
I(+pp)/I(pp), for the purpose of describing the
intrinsic receptor activity of the 1R with respect to
1B-1 currents.
Unless otherwise noted, comparisons between the constructs in terms of
current inhibition and relief of inhibition were conducted by means of
a one-way ANOVA followed by post hoc analysis by the Tukey
multiple-comparison procedure (Neter et al., 1985). Averages were
expressed as mean ± SEM.
Construction of cDNAs.The chimeric contructs were created
using overlap extension PCR (Ho et al., 1989; Horton et al., 1989, 1990) and PCR-mediated site-directed mutagenesis. PCR products were
cloned into pGEM-T (Promega, Madison, WI), pGEM-T-EZ (Promega), or
pT7-Blue (Novagen, Madison, WI) and sequenced using semiautomated DNA
sequencing (ABI 377; Perkin-Elmer, Oak Brook, IL). All 1 subunit expression contructs were created by ligation of recombinant and native sequences into the pcDNA3.1 expression vector (Invitrogen, San Diego, CA). Final constructs were confirmed by a combination of
restriction analysis and DNA sequencing. The native 1B-1
construct bBbBbBbBb consisted of residues 1-2340 of GenBank accession
number 284339 (a gift from Dr. R. Harpold, SIBIA Neurosciences). The native 1E-3 construct eEeEeEeEe consisted of residues
1-2271 of GenBank accession number 1082919 (a gift from Dr. R. Harpold, SIBIA Neurosciences). The construct bBbBbEeEb consisted of
1B-1 1-1145, 1E-3 1149-1724, and
1B-1 1710-2340. The construct bBbBbEeEe consisted of
1B-1 1-1145 and 1E-3 1149-2271. The
construct bBbEeEeEe consisted of 1B-1 1-482 and
1E-3 477-2271. The construct eBbEeEeEe consisted of
1E-3 1-89, 1B-1 96-482, and
1E-3 477-2271. The construct eBeEeEeEe consisted of
1E-3 1-89, 1B-1 96-355, and 1E-3 351-2271. The construct eEbEeEeEe consisted of
1E-3 1-350, 1B-1 356-482, and
1E-3 477-2271. The construct eEbBbEeEe consisted of
1E-3 1-350, 1B-1 356-1145, and
1E-3 1149-2271. The construct eEeEbEeEe consisted of
1E-3 1-703, 1B-1 710-1145, and
1E-3 1149-2271. The construct eEbEbEeEe consisted of
1E-3 1-350, 1B-1 356-482,
1E-3 477-703, 1B-1 710-1145, and
1E-3 1149-2271. The construct e(E90-309
B315-355)bEeEeEe consisted of 1E-3 1-309,
1B-1 315-482, and 1E-3 477-2271. The
2B/ and 1B Ca channel subunit
cDNAs were gifts from Dr. R. Harpold (Sibia Neurosciences) and were
subcloned into the pCMV6c expression vector.
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RESULTS |
Intrinsic activity of the -opioid receptor and effects on
1B and 1E
After transfection of tsA201 cells (Margolskee et al., 1993) with
the Ca channel subunits 1-B1,
1B, and 2B, the
1-opioid receptor (1R), and CD8, we
observed that 1-B1 currents often showed a degree of
prepulse facilitation, suggestive of G-protein-mediated inhibition,
even without previous exposure to 1R agonists (Fig. 1A,B). Overall, 62% of
1B-1- and 1R-transfected cells responsive to 1R agonists showed some degree of facilitation before
drug exposure. These effects were not seen in cells incubated overnight with 100 ng/ml pertussis toxin (n = 5) or in cells that
were not transfected with the 1R receptor
(n = 6), suggesting a receptor-mediated, G-protein
(Gi and/or Go)-dependent mechanism (data
not shown). Similar G-protein receptor-dependent modulation of Ca
channels in the absence of agonist has been described by other groups
(Ikeda, 1992; Zhang et al., 1996). The effects seen in the present
study did not diminish when cells remained in a rapidly flowing bath for longer than 20 min, suggesting that agonist-independent modulation was not caused by the secretion of opioid agonists by the cells or the
presence of opioid agonists in the culture media (Doupnik et al.,
1994). U69593 is a 1R-selective agonist with an affinity for 1 receptors in the range of 1-2 nM
(Avidor-Reiss et al., 1995; Simonin et al., 1995) and an
EC50 for adenylyl cyclase inhibition of ~8 nM
(Avidor-Reiss et al., 1995). When U69593 was added to the bath solution
at a supramaximal concentration of 1 µM,
1-B1-expressing cells showed a marked reduction in
current (Fig. 1A) (47 ± 6.4%; n = 13), similar to the results of a number of other
groups (Kaneko et al., 1994; Tallent et al., 1994; Carpenter et al.,
1996; Toth et al., 1996). When the 1R-selective
"antagonist" norBNI (Portoghese et al., 1987) was added directly
after U69593 exposure at a concentration of 100 nM, the
inhibition produced by the agonist was always relieved within 3-4 min
(Fig. 1A,B), and currents often increased in
magnitude beyond those seen before U69593 exposure (Fig.
1A,B) (n = 13). When U69593 was added
to cells without subsequent addition of norBNI, current inhibition and
facilitation washed out in ~10 min, and no enhancement of current
amplitudes was observed (data not shown) (n = 5). When
norBNI was added to 1R expressing cells that showed
facilitation without previous agonist exposure, this facilitation was
always reversed, and currents were always observed to increase in
amplitude (Fig. 1C) (n = 5). However, when
the opioid receptor antagonist naloxone (20 µM) was added
to such cells, no current increase was observed (n = 3)
(data not shown), suggesting that naloxone, unlike norBNI, is a neutral
antagonist. norBNI is known to bind at 1 receptors with
affinities in the range of 0.5-1.2 nM (Prather et al.,
1995; Simonin et al., 1995) and completely blocks opioid agonist
stimulation of adenylyl cyclase at 100 nM (Avidor-Reiss et
al., 1995). Because norBNI was applied in the absence of agonist and
reduced prepulse ratios to that seen in untransfected cells, we believe
that maximal relief of agonist-independent modulation was obtained with
100 nM norBNI.
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Figure 1.
Opioid agonist and inverse agonist effects on Ba
currents for 1B-1- and 1R-expressing
tsA-201 cells. A, Peak current amplitudes and
representative currents before prepulse (green)
and after a 30 msec depolarizing prepulse (black),
illustrating the effects of the addition to the bath of 1 µM U69593 or 100 nM norBNI. Currents were
taken from the points indicated. Currents were evoked by a 50 msec
depolarization to 0 mV from a holding potential of 90 mV.
Calibration: 250 pA, 10 msec. B, Representative currents
illustrating relatively small current inhibition after U69593 exposure
and relatively large current enhancement after norBNI exposure in cells
with a large degree of modulation (as indicated by a relatively large
increase in current amplitudes after a prepulse) before agonist
exposure. C, Representative currents illustrating the
effects of 100 nM norBNI added before 1 µM
U69593. D, Currents evoked from a 1B-1-
and R-expressing cell in the presence of 100 nM norBNI,
1 µM ICI-174864, and 1 µM DPDPE.
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Both the U69593 (inhibitory) and norBNI (enhancing) effects in these
experiments could be completely blocked by pertussis toxin
(n = 5), and cells responsive to one drug were always
responsive to the other. When 50 µM GF109203, a specific
PKC inhibitor, was included in the intracellular solution
(n = 3), the response of 1B-1- and
1R-expressing cells to U69593 and norBNI was unaltered relative to cells without GF109203 in the intracellular solution, suggesting that PKC-mediated phosphorylation plays no role in the
observed effects on the currents (data not shown).
To further verify that norBNI does in fact act by relieving
agonist-independent G-protein modulation, the effects of receptor expression, U69593, and norBNI on current facilitation as determined by
current ratios were examined (Ikeda, 1991) and were defined as the
ratio of the current amplitude after a prepulse, I(+pp) to
that before a prepulse, I(pp). Baseline current ratios
(before drug exposure) for 1B-1- and
1R-expressing cells (ratio, 1.05 ± 0.08;
n = 13) were significantly greater than for cells not expressing 1R (data pooled from
1R-untransfected cells and U69593- and
norBNI-unresponsive cells; ratio, 0.89 ± 0.05; n = 13; p < 0.05, one-tailed t test). These
ratios were significantly correlated with the degree of current
enhancement obtained after the addition of norBNI (baseline relative to
peak of norBNI effect; r = 0.84; p < 0.001; n = 13) (Fig. 1, compare A,B),
suggesting that cells most responsive to norBNI expressed the highest
levels of agonist independent modulation. The effects of U69593 and
norBNI on current amplitudes were mirrored in terms of the current
ratios. At baseline, before drug exposure, a small degree of
facilitation was seen (ratio, 1.05 ± 0.08), facilitation
increased in the presence of U69593 (ratio, 1.70 ± 0.13), and
currents inactivated to a small extent after a prepulse in the presence
of norBNI (ratio, 0.78 ± 0.03; n = 13 in all
cases) (Fig. 1). This current inactivation after a prepulse, as a
consequence of a reduction in active G-protein, is similar to the
results of Ikeda (1992), who found that internal perfusion of rat
sympathetic neurons with GDPS caused inactivation after a prepulse
at most prepulse potentials. In summary, these results suggest that the
currents were inhibited to an intermediate degree before drug exposure,
that the agonist U69593 rapidly caused additional current inhibition,
and that norBNI is actually an inverse agonist, relieving both the
agonist-independent and agonist-dependent inhibition of the
current.
Because this agonist-independent receptor activity is likely to
complicate estimates of receptor effects on Ca channels, we used U69593
to observe currents in the presence of maximum receptor activity,
I(R*), and norBNI to observe currents in the presence of
inactive receptor, I(R). An index of total modulation was
estimated from the peak of the U69593 response [an estimate of
I(R*)] and the peak of the norBNI response (in the case of
cells showing agonist-independent modulation) or baseline currents (in
the case of cells without agonist-independent modulation) to
estimate I(R). Total modulation was then defined as
[I(R) I(R*)]/I(R). In the case of
1B-1- expressing cells, modulation was 68.9 ± 3.7% (n = 13) (see Fig. 5) when defined in this way
and 47 ± 6.4% (n = 13) when defined as
[I(baseline) I(R*)]/I(baseline),
suggesting a nearly 50% reduction in the SEM of the
estimated effect size. Consistent with the involvement of
heterotrimeric G-proteins (Marchetti et al., 1986; Ikeda, 1991; Pollo
et al., 1992), this current reduction was greater with respect to
currents evoked before a voltage prepulse than for currents evoked
after a prepulse (total modulation, 38.6 ± 3.7% after prepulse;
n = 13) (Fig. 1). Kinetic slowing was evident in the
majority of cells (Fig. 1) but was not examined in detail for the
purpose of this study.
To further explore the specificity of agonist-independent opioid
receptor effects on Ca currents, we transfected cells with 1-B1, 1B,
2B, CD8, and the mouse -opioid receptor
(R). This receptor has well characterized agonist-independent
activity; ICI174,864 is known to act as an inverse agonist, and DPDPE
(enkephalin, [D-Pen2,5]) is known to
act as an agonist (Chiu et al., 1996; Mullaney et al., 1996; Merkouris
et al., 1997). These cells showed inhibition and prepulse facilitation
qualitatively similar to 1R-expressing cells before drug
exposure (Fig. 1D). When norBNI was added to the
bath, R-expressing cells showed no alteration in their
1B-1 currents (Fig. 1D;
n = 3). However, when ICI174,864 was added to the bath,
an increase in current amplitudes was noted (28.0 ± 14.0%;
n = 3), and facilitation decreased (current ratio,
1.4 ± 0.2 at baseline; current ratio, 1.0 ± 0.1 in the
presence of ICI174,864). When DPDPE was subsequently added to the bath,
current amplitudes rapidly decreased and facilitation increased
(current ratio, 1.5 ± 0.4), and the DPDPE-mediated inhibition
could be reversed by reapplication of ICI174,864 over a time frame of
minutes (total modulation, 37.0 ± 13.0%; n = 3).
The response of these cells to agonist and inverse agonist was
therefore qualitatively similar to cells expressing 1R
but selective for R-specific agents.
When 1R- and 1E-3-expressing cells were
exposed to U69593 and norBNI, much less current inhibition (25.3 ± 1.9%; n = 11) (Fig.
2; see Fig. 5) was observed than for
1B-1-expressing cells (p < 0.001), consistent with previous findings (Toth et al., 1996; Yassin et
al., 1996). 1E-3-expressing cells also showed partial voltage-dependent relief of inhibition such that inhibition after a
prepulse was less than before a prepulse (total modulation, 21.9 ± 1.6% after prepulse), but cells fell far short of the ~50% relief seen in 1B-1-expressing cells. Prepulse ratios in
the presence of U69593 corrected for inactivation (see Materials and Methods) were 1.052 ± 0.02 for 1E-3-expressing
cells but 2.184 ± 0.02 for 1B-1-expressing cells
(p < 0.001; see Fig. 5).
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Figure 2.
Opioid agonist and inverse agonist effects on Ba
currents for 1E-3- and 1R-expressing
tsA-201 cells. Currents were taken from the points indicated.
Calibration: 250 pA, 10 msec. See Figure 1 for details.
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Structural determinants of G-protein regulation
To elucidate the structural basis for differences in the degree of
G-protein regulation of 1B-1 and 1E-3 Ca
channels, we constructed Ca channels in which portions of
1B-1 were inserted into an 1E-3
background. When the entire N-terminal half of 1B-1 was
transferred into the 1E-3 background to create the
bBbBbEeEe chimera (where lowercase letters indicate intracellular
sequence, and uppercase letters indicate transmembrane domains, written in the sequence N terminus, domain I, I/II loop, etc.), currents were
markedly reduced by U69593 and markedly enhanced by norBNI (Fig.
3; see Fig. 5) (n = 7).
Overall modulation was 69.4 ± 4.3% and not significantly
different from modulation seen with 1B-1. Inhibition was
partially relieved by a voltage prepulse (to 46 ± 7.9%), and
somewhat less relief was seen than for 1B-1 (corrected prepulse ratio, 1.78 ± 0.20; p > 0.05; see Fig.
5).
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Figure 3.
Representative currents from various
1B-1 and 1E-3 chimeric Ca channels with
sensitivity to modulation approaching that of native
1B-1 channels (Fig. 1) before prepulse
(green) and after a 30 msec depolarizing prepulse
(black). Maximal effects of U69593 and norBNI are
illustrated. Calibration: 250 pA, 10 msec.
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To determine the minimal sequence responsible for these observations,
we transferred two of the intracellular loops contained in the
bBbBbEeEe chimera into the 1E-3 background, individually and in combination. The I/II intracellular loop has been shown by GST
fusion studies to bind G-protein subunits (De Waard et al.,
1997) and to posses a consensus sequence QXXER that it shares with
adenyl cyclase and a number of other G-protein effectors (Pragnell et
al., 1994). Somewhat consistent with the results of Page et al. (1997),
the eEbEeEeEe chimera (Figs.
4-6)
showed current inhibition that was slightly greater but not
significantly different from 1E-3 (33.8 ± 2.3%;
n = 9) and much less than that of 1B-1
(p < 0.001) or the bBbBeEeE chimera
(p < 0.001). When the II/III intracellular loop
of 1B-1 was transferred into the 1E-3
background (Figs. 4, 5, eEeEbEeEe chimera), inhibition
similar to that of 1E-3 was seen (23.1 ± 4.8%;
n = 7). When the II/III loop was transferred into the
1E-3 background, in combination with the I/II loop
(Figs. 4, 5, eEbEbEeEe chimera), the resulting construct
also showed inhibition similar to that of 1E-3
(24.6 ± 1.1%; n = 8). All three constructs
showed prepulse facilitation similar to 1E-3
(p > 0.05 in each case) and significantly less facilitation than 1B-1 (p < 0.001 for each).
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Figure 4.
Representative currents from various
1B-1 and 1E-3 chimeric Ca channels with
sensitivity to modulation approaching that of native
1E-3 channels (Fig. 2) before prepulse
(green) and after a 30 msec depolarizing prepulse
(black). Maximal effects of U69593 and norBNI are
illustrated. Calibration: 250 pA, 10 msec.
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Figure 5.
Summary of inhibition and facilitation seen
in native 1B-1- and 1E-3-expressing cells
as well as for various chimeric Ca channels. A, Summary
of 1R inhibition obtained for Ba currents evoked before
a depolarizing prepulse from native 1B-1- and
1E-3-expressing cells, as well as cells expressing
various 1B-1 and 1E-3 chimeras. Values
are plotted as mean ± SEM. Sample sizes for each construct were
 6 as detailed in Results. Asterisks indicate
modulation significantly different from native 1E-3
(p < 0.05). B, Summary of
corrected 1R-mediated prepulse facilitation ratios from
native 1B-1- and 1E-3-expressing cells,
as well as cells expressing various 1B-1 and
1E-3 chimeras. Values are plotted as mean ± SEM.
Ratios were calculated by multiplying prepulse ratios in the presence
of U69593 [I(+pp)/I(pp)] by an index
of inactivation [I(pp)/I(+pp)]
calculated from currents in the presence of norBNI to adjust for
differences in inactivation between the various constructs. Sample
sizes for each construct were 6 as detailed in Results.
Asterisks indicate facilitation significantly different
from native 1E-3 (p < 0.05).
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Figure 6.
Current-voltage relations for selected Ca channel
constructs in the presence of U69593 (circles) and
norBNI (triangles) before (green)
and after (black) a depolarizing prepulse
(pp). Sample sizes were 5 as detailed in
Results.
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Because the I/II loop alone failed to significantly enhance modulation
but may play some role in mediating the binding of subunits to
Ca channels (De Waard et al., 1997), a series of additional constructs
was created to assess the role of sequences near the I/II loop in
determining effector sensitivity. When domain II, the I/II loop, and
the II/III loop from 1B-1 were transferred into the
1E-3 background (eEbBbEeEe chimera), modulation and facilitation similar to that of 1E-3 was obtained (total
modulation, 24.6 ± 1.1%; p > 0.05; corrected
ratio, 1.03 ± 0.02; p > 0.05; n = 8). The addition of 1B-1 sequence from the N terminus
to the C-terminal end of the I/II loop into the 1E-3
background (Figs. 3, 5, 6, bBbEeEeEe chimera) resulted in a
channel showing inhibition not significantly different from
1B-1 (66.6 ± 4.1%; n = 8).
However, as in the case of the bBbBbEeEe chimera, this inhibition was
less completely relieved by a prepulse (to 46.0 ± 7.9%;
corrected prepulse ratio, 1.55 ± 0.11; p < 0.001) than in the case of 1B-1, although
facilitation was greater than that seen in the case of
1E-3 (p < 0.05). When domain I
and the I/II loop from 1B-1 were transferred alone into
the 1E-3 background (Figs. 3, 5, eBbEeEeEe
chimera) modulation that was not significantly different from
1B-1 was also observed (56.8 ± 4.4%;
n = 7; p > 0.05). These channels
showed less facilitation (corrected prepulse ratio, 1.27 ± 0.05)
than 1B-1-expressing cells (p < 0.001) and not significantly more facilitation than
1E-3-expressing cells (p > 0.05). We also attempted to examine the contribution of the N terminus
in isolation by the chimera bEeEeEeEe but were unable to obtain
adequate current expression from this chimera. When domain I of
1B-1 was transferred alone into the 1E-3
background (Figs. 3, 5, eBeEeEeEe chimera), modulation
significantly greater than 1E-3 was observed (44.6 ± 0.03%; n = 10; p < 0.001), but this was significantly less than that observed for 1B-1
(p < 0.05). Facilitation (corrected ratio,
1.3 ± 0.02) was not significantly different from
1E-3 (p > 0.05).
Domain I of Ca channels is thought to be partly responsible for
determining the voltage dependence of Ca channel inactivation (Zhang et
al., 1994), although the C terminus (Klockner et al., 1995; Soldatov et
al., 1997) and I/II loop (Herlitze et al., 1997) are also thought to
play a role. The regions in domain I responsible for determining
inactivation are thought to be a C-terminal portion of the Is5
sec6 linker as well as Is6 (Zhang et al., 1994).
When this sequence alone was added along with the I/II loop (Figs. 4,
5, eE(90-309)B(315-355)bEeEeEe
chimera), the resulting construct showed modulation (30.0 ± 3.9%; n = 7) and facilitation (corrected ratio,
1.04 ± 0.01) similar to 1E-3 and the eEbEeEeEe chimera (p > 0.05 in both cases), although
inactivation was reduced compared with the eEbEeEeEe chimera
(p < 0.05).
Recently Qin et al. (1997) have provided biochemical and functional
evidence that the C terminus of non-DHP-sensitive Ca channels is
involved in binding and current inhibition. To assess the role
of the C terminus in our system, we exchanged the C terminus of
1B-1 into the bBbBbEeEe chimera to produce the bBbBbEeEb
chimera (Figs. 3, 5). These channels showed inhibition (65.6 ± 4.2%; n = 7) not significantly different from
1B-1 or the bBbBbEeEe chimera (p > 0.05 in both cases). However,
facilitation more closely approximated that of 1B-1 than
was seen in the case of the bBbBbEeEe chimera (corrected ratio,
2.04 ± 0.12).
Because Ca channel modulation by G-proteins is voltage-dependent, we
collected current-voltage data in the presence of norBNI and U69593
for a number of the constructs (Fig. 6). The bBbBbEeEb chimera
(n = 7) closely resembled wild-type 1B-1
(n = 5) both in terms of inhibition and facilitation
(Fig. 6). As we observed using a single test potential of 0 mV, the
bBbBbEeEe construct (n = 5) showed less facilitation
than bBbBbEeEb across a range of activating potentials (Fig. 6). These
results suggest that the C terminus may be partly responsible for the
differences in facilitation between 1B-1 and
1E-3, but it does not appear to mediate the
differences in inhibition between the two channels. The bBbEeEeEe
chimera (n = 5) showed strong inhibition, similar in
magnitude to 1B-1, but showed little facilitation
at any activating potential. The eEbEeEeEe chimera (Fig. 6;
n = 8) showed relatively little facilitation or
inhibition at any activating potential. For example, at a potential of
10 mV, the current ratio in the presence of U69593 for
1B-1 (2.28 ± 0.34) was not significantly different
from the bBbBbEeEb chimera (2.00 ± 0.20; p > 0.05) but was significantly greater than the ratios for the bBbEeEeEe
(1.22 ± 0.11), eEbEeEeEe (0.92 ± 0.04), and bBbBbEeEe
(1.40 ± 0.11) chimeras (all p < 0.05). These
data further reinforce the conclusion that facilitation and inhibition
are mediated by different structural elements.
We defined agonist-independent modulation as the difference in the
current amplitude in the presence of norBNI compared with that at
baseline. The degree of such modulation would be expected to correlate
with the index of total modulation for a particular construct if both
indices reflect sensitivity to inhibition by G-proteins. We computed
this correlation across all of our observations and found that the
correlation was large and highly significant (r = 0.74;
p < 0.00001). In addition, the magnitude of the
agonist-independent modulation seen for each construct was well
correlated with the magnitude of the total modulation seen for the
construct (0.03 ± 0.01%, 1E-3; 0.02 ± 0.01%, eEbEeEeEe; 0.22 ± 0.03%, eBeEeEeEe; 0.38 ± 0.06%, 1B-1) This provides additional support
for the notion that the index of total modulation is indeed a valid
index of sensitivity to current inhibition by G-proteins.
We were concerned that the observed differences in facilitation between
the constructs were attributable to differences in the kinetics of
G-protein association with the channels. We measured the G-protein
reassociation rates for a number of our constructs using a variable
interval between the prepulse and the second test pulse. Facilitation
decayed as a single exponential with time constants between the limits
of 15.9 ± 2.3 msec (eEbEeEeEe; n = 4) (data not
shown) and 41.6 ± 2.4 msec (1B-1;
n = 6) (data not shown). Facilitation will have decayed
to 73-89% of its maximum after a 5 msec interval for our constructs.
Therefore, the large differences in facilitation observed (Figs. 5, 6)
cannot be accounted for by differences in reassociation kinetics.
As noted above, current ratios (Fig. 5) were corrected for inactivation
to facilitate comparisons between constructs that differed markedly in
terms of inactivation. An adequate kinetic model that can account for
modulation, activation, and inactivation of Ca channels has not yet
been elaborated, and the relationship between inactivation and
modulation therefore remains unclear. However, with respect to the most
important constructs used in the present study, it is clear that
modulation and inactivation are relatively independent. For example,
the constructs bBbBbEeEb, bBbBbEeEe, bBbEeEeEe, eBbEeEeEe, eBeEeEeEe,
and e(E90-309B315-355)bEeEeEe did not differ
in terms of inactivation compared with bBbBbBbBb (all p > 0.9). However, these constructs differed markedly from one another
in terms of facilitation. The chimeras bBbBbEeEb and bBbBbEeEe did not
differ significantly from 1B-1 in terms of facilitation
(p > 0.1), but bBbEeEeEe, eBbEeEeEe, eBeEeEeEe,
and e(EB)bEeEeEe did differ significantly (all p < 0.0001). Because these constructs differ greatly in terms of
facilitation but not in terms of inactivation, it seems clear that the
two are not highly correlated.
| |
DISCUSSION |
We have identified a domain in the family of DHP-insensitive Ca
channels that appears to largely dictate the efficacy of
G-protein-induced channel inhibition. The results of the present study
also suggest that the 1R, like the R (Chiu et al.,
1996; Mullaney et al., 1996; Merkouris et al., 1997), has
agonist-independent activity that can be suppressed by drugs with
inverse-agonist properties. Therefore, it appears that these
pharmacological properties may be general characteristics of the opioid
receptor family. Agonist-independent activity of the 1R
has not been previously reported. It is likely that we observed such
activity in our experiments because of the high transient expression
levels obtainable in tsA-201 cells or because of some other
cell-dependent variables. The observation of this agonist-independent
activity allowed us to identify norBNI as an inverse agonist at the
1R. The use of inverse agonists in combination with
agonists appears to have important utility in that receptor states near
maximal and minimal activity can be readily generated, allowing for
more precise estimates of receptor effects on G-protein regulated
effectors. This consideration would theoretically be of greatest
importance for systems with the highest levels of receptor expression,
that would be expected to exhibit significant agonist-independent
receptor activity. We achieved substantial improvements in the
precision of our estimates of G-protein modulation by using this
approach.
With regard to the structural basis for the differences in G-protein
sensitivity between 1B-1 and 1E-3 Ca
channels, the results of the present study suggest that domain I of
1-B1 is the single most important structural feature
required for determining the higher sensitivity to opioid receptor
modulation of 1B-1 compared with 1E-3.
Whereas 1R-mediated modulation of 1E-3 was only 37% of that obtained for native 1B-1 channels,
exchange of domain I alone yielded a chimeric channel with 65% of the
modulation of 1B-1. The further addition of the I/II
linker increased modulation to 83% of native 1B-1
channels, and the further addition of the N terminus increased
modulation to 100% of 1B-1 (Fig. 5). These same regions
appear largely responsible for facilitation, although the determinants
of facilitation appear to be complex and may also include the C
terminus (Figs. 5, 6). The lack of significant differences in
modulation between the eEbEeEeEe chimera and native 1E-3
(Fig. 5) suggests that the I/II loop is not sufficient to determine the
differential sensitivity to modulation between 1B-1 and
1E-3. Likewise, the lack of significant differences in
inhibition between the bBbBbEeEe and bBbBbEeEb chimeras (Fig. 5)
suggests that the C terminus is not involved in determining differences in sensitivity to modulation. Therefore, the two structural elements shown to be involved in G-protein binding to Ca channels (De Waard et al., 1997; Qin et al., 1997; Zamponi et al., 1997) do not
appear to account for differences in sensitivity to inhibition between
1B-1 and 1E-3.
The role of the I/II loop in G-protein modulation of Ca channels is
controversial. A number of other studies have also suggested that the
I/II loop is not sufficient to account for high sensitivity to
G-protein modulation. Page et al. (1997) showed that transfer of the
I/II loop from 1B to 1E or from
1B to 1A conferred kinetic
slowing but not inhibition on the recipient channel. Qin et al. (1997)
showed that transfer of the I/II loop from 1C to 1E yielded a chimeric channel that was still modulated
like 1E. Zhang et al. (1996) showed that transfer of the
I/II loop from 1A or 1C into
1B yielded a channel that was still modulated like
1B. In contrast, Zamponi et al. (1997) showed that
transfer of the I/II loop from 1B into 1A
yielded a chimeric channel with significantly more modulation than
native 1A. Herlitze et al. (1997) and De Waard et al.
(1997) showed that mutations in the sequence QXXER of 1A
can disrupt modulation, and peptides made according to sequences in the
I/II loop can block modulation of both channels (Zamponi et al., 1997).
In addition, a number of groups have demonstrated that the I/II loop
can indeed bind subunits (De Waard et al., 1997; Qin et al.,
1997; Zamponi et al., 1997), and this may be important in mediating the
effects of subunits in some general way. The results of Qin et
al. (1997) and Zhang et al. (1996) also suggest that the C terminus plays a critical role in mediating modulation, and the latter study
suggests a role of the N terminal portion of the Ca channel that is
N-terminal to the I/II loop. Overall, the results of the present study
are most consistent with those of Zhang et al. (1996) in terms of the
involvement of the N terminal portion of the channel, although we find
that the C terminus plays little if any role in determining
inhibition.
There are at least three models that could account for the findings of
the present study. First, differences in the kinetics of the two
channels, probably determined by multiple and complex structural
features, could account for at least some of the differences in
modulation. For example, we numerically integrated the kinetic model of
G-protein modulation proposed by Patil et al. (1996) while
systematically varying the rate constants of the model a small amount
near their fitted values and have observed alterations in predicted
inhibition for each of the rate constants in the model (data not
shown). Apart from rate constants directly introduced to model
modulation (e.g., "willing" to "reluctant" transitions), predicted inhibition is particularly sensitive to the values of the
rate constants for exit from the inactivated state and for transitions
between "willing" closed states.
A second model that could account for these data is an allosteric model
in which the I/II loop and/or C terminus binds G-protein
subunits and changes conformation but then must induce conformational changes in the gating apparatus of the channel. The C terminus and I/II
loop may interact directly with the gating apparatus or may be coupled
to these domains through other structures. According to this view, it
is possible that domain I of 1B-1, which may itself contain sequence involved in voltage-dependent channel gating,
is more susceptible to structural alteration by binding to the
I/II loop and/or C terminus than is domain I of
1E-3, and that the N terminus somehow facilitates
such interactions. Alternatively, domain I may play some role in
linking binding to the C terminus and/or I/II linker to regions of the
channel involved in gating to generate modulation.
Finally, it is possible that Ca channel regions other than the I/II
loop and C terminus play a role in binding. Biochemical binding
studies performed to date (Qin et al., 1997; De Waard et al., 1997)
have demonstrated that the I/II loop and C terminus have affinities
that are by themselves sufficient for tight binding but have not shown
that other sequences do not play an important role in binding to
the intact channel. Although domain I (and the N terminus) may not be
competent to bind subunits in isolation, it is possible that it
does contribute to the affinity of subunit binding to the intact
channel and that domain I of 1B-1 is differentially capable of stabilizing binding in comparison to domain I of 1E-3.
The notion that different structural elements account for facilitation
and inhibition is similar to the results of Zhang et al. (1996), who
showed that both the first domain and the C terminus of
1B were required within an 1A background
to confer facilitation and inhibition, but N- or C-terminal halves of
1B in an 1A background were sufficient
for inhibition alone. A physical interaction of the C terminus of
1B-1 with G subunits may play some role in the
functional uncoupling of G and Ca channels after a depolarizing pulse.
Whatever the precise mechanism by which domain I and the N terminus
contribute to Ca channel modulation, the findings of the present study
suggest that Ca channel sensitivity to G-protein modulation not only is
determined by sequences with high-affinity interactions with
subunits but also requires another sequence that is perhaps not
directly involved in high-affinity binding. Domain I and the N
terminus of the channel appear largely responsible for these
differences. The N-terminal tails of the two channels are fairly
dissimilar (60% identity), especially in their N terminal halves. The
only significant regions of dissimilarity between 1B-1
and 1E-3 in domain I fall within the extracellular loops (s1-s2, s3-s4, and s5-s6). One or both of these regions of
dissimilarity may therefore account for the observed effects of
exchanging domain I between 1B-1 and
1E-3.
| |
FOOTNOTES |
Received Nov. 12, 1997; revised March 2, 1998; accepted March 4, 1998.
This work was supported by Public Service Grants DA02121, DA02575,
MH40165, NS33502, DK42086, and DK44840. A.A.S. was supported by Public
Service Grants HD07009 and DA02575. We are grateful to Dr. R. Harpold
(Sibia Neurosciences) for the Ca channel subunits used in these studies
and to Dr. G. I. Bell (Howard Hughes Medical Institute, University
of Chicago) for helpful discussions regarding the molecular techniques
used in these studies, for the use of his laboratory facilities, and
for the opioid receptor clones used in this work.
Correspondence should be addressed to Dr. Richard J. Miller, Department
of Pharmacological and Physiological Sciences, University of Chicago,
947 East 58th Street, Chicago IL 60637.
| |
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Top
Abstract
Introduction
