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The Journal of Neuroscience, August 15, 1999, 19(16):6855-6864
Identification of Residues in the N Terminus of  1B Critical
for Inhibition of the Voltage-Dependent Calcium Channel by G 
Carles
Cantí,
Karen M.
Page,
Gary J.
Stephens, and
Annette C.
Dolphin
Department of Pharmacology, University College London, London WC1E
6BT, United Kingdom
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ABSTRACT |
To examine the role of the intracellular N terminus in the
G-protein modulation of the neuronal voltage-dependent calcium channel
(VDCC)  1B, we have pursued two routes of investigation. First, we
made chimeric channels between 1B and 1C, the latter not being
modulated by G  subunits. VDCC 1 subunit constructs were
coexpressed with accessory 2 and 2a subunits in
Xenopus oocytes and mammalian (COS-7) cells. G-protein
modulation of expressed 1 subunits was induced by activation of
coexpressed dopamine (D2) receptors with quinpirole in oocytes, or by
cotransfection of G12 subunits in COS-7 cells. For the chimeric
channels, only those with the N terminus of 1B showed any G-protein
modulation; further addition of the first transmembrane domain and I-II
intracellular linker of 1B increased the degree of modulation. To
determine the amino acids within the 1B N terminus, essential for
G-protein modulation, we made mutations of this sequence and identified three amino acids (S48, R52, and R54) within an 11 amino acid sequence
as being critical for G-protein modulation, with I49 being involved to
a lesser extent. This sequence may comprise an essential part of a
complex G-binding site or be involved in its subsequent action.
Key words:
calcium channel; neuronal; G-protein; 1 subunit; G subunit; modulation
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INTRODUCTION |
The inhibition of N- (1B) and
P/Q-type (1A) calcium currents by receptors, usually acting through
pertussis toxin-sensitive G-proteins, appears to be mediated by G
subunits (Herlitze et al., 1996 ; Ikeda, 1996). There has been some
controversy concerning whether the 1E calcium channel is
G-protein-modulated (Page et al., 1998). We have now established that,
whereas an N-terminally truncated isoform of rat 1E is not subject
to modulation, an isoform with a full-length N terminus is
G-protein-modulated, either by coexpression of G subunits or by
activation of a G-protein-coupled receptor (Page et al., 1998), which
would agree with results obtained previously for full-length human
1E (Qin et al., 1997).
A number of recent studies have established the importance of the
intracellular loop that links transmembrane domains I and II, both in
binding G and in mediating its effects to produce inhibition of
the channel (Herlitze et al., 1997; Zamponi et al., 1997). However,
this result is controversial, and several studies have suggested either
that the I-II loop plays no role in G-protein modulation of 1B
(Zhang et al., 1996) or 1E (Qin et al., 1997), or that alone it
cannot mediate the effects of the G subunits (Page et al., 1997,
1998; Simen and Miller, 1998). Nevertheless it is not disputed that the
I-II loops of 1A, B, and E comprise a major binding site or sites
for G and contain a QxxER amino acid consensus sequence common to
many G-binding sites (De Waard et al., 1997; Herlitze et al.,
1997; Zamponi et al., 1997; Dolphin et al., 1999). Secondly, a
C-terminal G-binding site has recently been identified and
proposed to be a region responsible for G-protein inhibition of human
1E (Qin et al., 1997). However, it is clear that there are also a
number of other sites in the 1 subunit of G-protein-modulated
calcium channels that are involved in expression of the inhibition by
G. First, we have found that part of the intracellular N terminus
of 1B and 1E is essential for their G-protein modulation (Page et
al., 1998). Second, the transmembrane domain I has been found to have
an important role (Zhang et al., 1996; Stephens et al., 1998b).
In the present study we have examined the critical nature of the
intracellular N terminus of 1B, by making chimeric channels between
1B, which is strongly G-protein-modulated and 1C, which is not
G-protein-modulated by this mechanism, and has a completely different
N-terminal sequence. We have shown an absolute requirement for the
1B N terminus for observation of G-protein modulation in all the
chimeric constructs. Second, we have made specific deletions and point
mutations to identify the sequence in the N terminus of 1B that is
responsible for conferring G-protein modulation.
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MATERIALS AND METHODS |
Materials
The following cDNAs were used: rat 1C (isoform CII, GenBank
accession number M67515), rabbit 1B (D14157), rat 2a (M80545),
rat 2-1 (neuronal splice variant, M86621), rat D2long receptor (X17458, N5 G), bovine G1
(M13236), bovine G2 (M37183), the C-terminal minigene of -ARK
(M34019), and mut-3 green fluorescent protein (GFP; U73901). All cDNAs were subcloned into the expression vector pMT2 (Swick et al., 1992).
Construction of chimeras
Chimeras were created using PCR following the methods described
previously (Page et al., 1998; Stephens et al., 1998b). All constructs
were subcloned into the pMT2 vector, and the sequences of the PCR
products were confirmed using cycle-sequencing. The constructs were
assembled as follows: bCCCC, amino acid residues 1-95 of 1B,
125-2143 of 1C; bBcCCC 1-359 1B, 409-2143 1C; bBbCCC 1-483
1B, 525-2143 1C; cBcCCC 1-124 1C, 96-359 1B, 409-2143
1C; cBbCCC 1-124 1C, 96-483 1B, 525-2143 1C; cCbCCC 1-408 1C, 360-483 1B, 525-2143 1C;
and bCbCCC 1-95 1B, 125-408 1C, 360-483 1B, 525-2143
1C. Chimeric primers were used with the reverse primer CCA CCA GCA
GGT CCA GGA TAT TGA (R1). The resulting PCR product was extended
against a template using a forward primer (pMT2F2) directed against the
vector TCT CCA CAG GTG TCC ACT. The following chimeric primers were
used: GTG CTG GGT GTG CTG AGC GGA GAG TTT for bBcCCC; CAG CCA GTA GAA
GAC CTG TGC CTT CAC CAT (reverse primer R2) for bBbCCC; CAC CGA GTG GCC
TCC ATT TGA AAT AAT T for bCCCC. These chimeras were used as templates
to make others. The primers TTT GAG CGG AGA GTT TGC TAA GG and R2 were
used to make the first PCR product, which was then extended on bCCCC to
give bCbCCC. The chimeras cBbCCC and cBcCCC were made using bBbCCC and
bBcCCC as templates. In each case, the PCR product made using the
primers TGT TGA ATG GAA ACC GTT CGA GTA CAT G and R1 was extended on
1CpMT2 template to add the N terminus of 1C. For cCbCCC,
restriction digestion of an MfeI site in domain I was used
to substitute the N terminus of bCbCCC with that of 1C.
Construction of N-terminal deletion and point mutations
The 1B N terminus was truncated at the 5' end by introducing
a start codon before amino acid E7 to make 1B  2-6, Y45 (1B 2-44), and Q51 (1B 2-50). The following primers were used; CGC ACT AGT ATG GAG CTG GGC GGC CGC TAT (2-6), CAG ACT AGT ATG TAC
AAA CAG TCG ATC GCG (2-44), and CAG ACT AGT ATG CAG CGC GCG CGG ACC
AT (2-50). The 1B 45-55 construct was made by using the
primer GGC CAG CGG GTC CTC ATG GCG CTG TAC AAC to delete the 11 amino
acids, YKQSIAQRART. For all of the 1B point mutations, primers were
designed so that single residues were mutated to alanines or so that a
number of residues were mutated within the same primer. The following
primers were used; R52A-R54A, TCG ATC GCG CAG GCC GCG GCG ACC ATG GCG
CT; Y45A, CAG CGG GTC CTC GCC AAA CAG TCG ATC; K46A, CGG GTC CTC TAC
GCA CAG TCG ATC GCG; Q47A, GTC CTC TAC AAA GCG TCG ATC GCG CAG; S48A,
CTC TAC AAA CAG GCG ATC GCG CAG C; I49A, TAC AAA CAG TCG GCC GCG CAG
CGC GCG; Q51A, CAG TCG ATC GCG GCG CGC GCG CGG ACC; R52A, TCG ATC GCG
CAG GCC GCG CGG ACC ATG; R54A, GCG CAG CGC GCG GCG ACC ATG GCG CTG;
45YKQSIAAAAAA, GCC GCA GCA GCT GCC GCG CAG CGC GCG CGG (forward) and
GGC AGC TGC TGC GGC GAG GAC CCG CTG (reverse); and 45YKQAAA, CGG GTC CTC GCC GCA GCG TCG ATC GCG CAG. The reverse primer used in each case
was GTC GCT TCT GCT CTT CTT GG. For the PCR extension reactions, the
forward primer used was AGC ACT AGT ATG GTC CGC TTC GGG GAC. The
sequences of all constructs were verified.
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Expression of constructs and electrophysiological recording |
Xenopus oocytes. Adult female Xenopus
laevis were killed by anesthetic overdose in a 0.25% solution of
tricaine, decapitated, and pithed. Oocytes were removed and
defolliculated by treatment with 2 mg/ml collagenase type Ia in a
Ca2+-free ND96 saline containing (in
mM): NaCl, 96; KCl, 2;
MgCl2, 1; and HEPES, 5, pH-adjusted to 7.4 with
NaOH for 2 hr at 21°C. Plasmid cDNAs for the different 1 subunits,
plus accessory 2a and 2 subunits and rat D2 receptors, were
mixed in a ratio of 3:4:1:3 (except where stated), and ~10 nl was
injected into the nuclei of stage V or VI oocytes. Injected oocytes
were incubated at 18°C for 3-7 d in ND96 saline (as above plus 1.8 mM CaCl2) supplemented with
100 µg/ml penicillin, 100 IU/ml streptomycin (Life Technologies,
Gaithersburg, MD), and 2.5 mM Na pyruvate. Whole-cell recordings from oocytes were made in the two-electrode voltage-clamp configuration with a chloride-free solution containing (in mM): Ba(OH)2, 5;
TEA-OH, 80; NaOH, 25; CsOH, 2; and HEPES, 5 (pH 7.4 with
methanesulfonic acid). In all experiments, oocytes were injected with
30-40 nl of a 100 mM solution of
K3-1,2-bis (aminophenoxy)
ethane-N,N,N',N'-tetra-acetic
acid (BAPTA) in order to suppress endogenous
Ca2+-activated
Cl currents. Electrodes contained 3 M KCl and had resistances of 0.3-2 M . The
holding potential (VH) was  100 mV,
and the test potential (Vt) used for
time course studies was 0 mV. All illustrated traces are at this
potential, and the current amplitude was always measured 20 msec after
the start of the test pulse. Membrane currents were recorded every 15 sec, amplified, and low-pass filtered at 1 KHz using a Geneclamp 500 amplifier and digitized through a Digidata 1200 interface (Axon
Instruments, Foster City, CA). In all cases currents were leak
subtracted on-line by a P/4 protocol.
COS-7 cells. Cells were cultured and transfected, using the
electroporation technique, essentially as described previously (Campbell et al., 1995a). The 1, 2, 2a, and GFP cDNAs were used at 15, 5, 5, and 1 µg, respectively. When used, G1 and G2 were included at 2.5 µg each, or -ARK was included at 5 µg.
Blank pMT2 vector was included where necessary to maintain the total cDNA at 31 µg/transfection. Cells were replated using nonenzymatic cell dissociation medium (Sigma, St. Louis, MO), and then maintained at
25°C for between 1 and 16 hr before electrophysiological recording. Maximum GFP fluorescence and voltage-dependent calcium channel (VDCC)
expression were observed between 2 and 4 d after transfection (Brice et al., 1997). Ca2+ channel
currents were recorded using the whole-cell patch technique. Borosilicate glass 2-5 M electrodes were used. The internal
(electrode) and external solutions were similar to those described
previously (Campbell et al., 1995b). The patch pipette solution
contained in mM: Cs aspartate, 140; EGTA, 5;
MgCl2, 2; CaCl2, 0.1;
K2ATP, 2; and HEPES, 10; pH 7.2, 310 mOsm with
sucrose. GDPS (2 mM) was included where stated. The
external solution contained in mM: tetraethylammonium (TEA)
bromide, 160; KCl, 3; NaHCO3, 1.0; MgCl2, 1.0; HEPES, 10; glucose, 4; and
BaCl2, 1 or 10, pH 7.4, 320 mOsm with sucrose.
Whole-cell currents were elicited from VH of 100 mV and recorded using an
Axopatch 1D amplifier. Data were filtered at 2 kHz and digitized at
5-10 kHz. The junction potential between external and internal
solutions was 6 mV, the values given in the figures and text have not
been corrected for this. Current records are shown following leak and
residual capacitance current subtraction (P/4 or P/8 protocol) and
series resistance compensation up to 85%. Current amplitudes were
measured 50 msec after the start of depolarization.
All experiments were performed at room temperature (18-20°C).
Analysis was performed using pClamp6 and Origin software. Data are
expressed as mean ± SEM. Statistical analysis was performed using
paired or unpaired Student's t test, as appropriate.
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RESULTS |
In a previous study we made chimeras between the rat brain 1E
(rbEII) clone, which is not G-protein-modulated, and the strongly modulated 1B. The results of this study showed that rbEII was not
modulated because it was N terminally truncated, and a full-length rat
1E isoform showed clear G-protein modulation, although not to such a
great extent as 1B. We further showed the importance of the first
domain of 1B in increasing the extent of G-protein modulation of
1B/1E chimeras (Page et al., 1998; Stephens et al., 1998b), as
has another recent study (Simen and Miller, 1998). In the present
study, we wished to examine the distinct role of the N terminus of
1B in G-protein modulation. To do this we have taken two approaches.
First, we have made chimeras between 1B and the 1C channel, which
is not modulated by a G-mediated pathway under any conditions.
Second, we have produced selective deletions and mutations of the 1B
N-terminal sequence. With such constructs we can determine the domains
necessary for the expression of G-protein modulation.
G-protein modulation of 1B/1C chimeras by activation of the
dopamine D2 receptor
In this part of the study, all channels were expressed with the
accessory subunits 2 and 2a (unless stated) in
Xenopus oocytes, where they were coexpressed with the
dopamine D2 receptor. A series of chimeras were made, in which the N
terminus, first transmembrane domain, and I-II loop of 1B were
systematically substituted for those in 1C, in different
permutations. Figure 1A
shows the chimeras that were made, and the nomenclature employed, which
uses capital letters for the transmembrane domains and small letters
for the intracellular N-terminal and I-II loop. All chimeras contained
the last three domains and C-terminal tail of 1C (denoted CCC), and
all showed good expression levels with one exception (Table
1). However, because 1B, 1C, and
the chimeras between them showed differences in their voltage
dependence of activation (Table 1), we could not compare G-protein
modulation at a single step potential (Fig.
2). Therefore, we have estimated the
amount of G-protein modulation in two ways in Xenopus
oocytes, first by determining the ability of the D2 agonist quinpirole
to cause a depolarizing shift in the voltage dependence of activation, determined from current-voltage plots (Fig. 1B).
Second, we have determined the percentage inhibition by quinpirole of
the current activated at all potentials between 20 and +30 mV (Fig.
2). In all cases, the modulation by quinpirole occurred within 30-60 sec of its application and was fully reversible.
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Figure 1.
G-protein modulation of chimeras between 1B and
1C. A, Chimeras made between 1B
(white) and 1C (black), together with
the nomenclature used. B, Chimeras and parental
constructs were expressed in Xenopus oocytes together
with 2 and the dopamine D2 receptor. The
V50 in the absence and presence of the D2
agonist quinpirole (100 nM) was determined from
current-voltage relationships performed before and during its
application, as described in the legend to Table 1, and the
V50 was calculated (mean ± SEM).
The number of experiments is given for each histogram bar. The
statistical significance of V50 was
determined by paired t test; **p < 0.01.
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Table 1.
Biophysical properties and G-protein modulation of calcium
channel 1 subunit chimeras in Xenopus oocytes
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Figure 2.
Voltage dependence of modulation of the chimeras
between 1B and 1C by activation of the dopamine D2 receptor. The
percentage inhibition by quinpirole (100 nM) was determined
at voltages between 20 and +30 mV, from current-voltage
relationships performed in the absence and presence of quinpirole.
Measurements were made isochronally, 20 msec after the start of the
voltage step. A, 1B; B, 1bBbCCC;
C, 1bBcCCC; D, 1bCbCCC; and
E, 1bCcCCC. Experiments were performed both in the
presence (white bars) and the absence (black
bars) of overexpressed 2a, except for 1bCcCCC, where no
expression was seen in the absence of 2a. The numbers of experiments
(with, without 2a) are 8, 6 (A); 12, 6 (B); 7, 6 (C); 10, 6 (D); and 9 (E). The statistical significance of
the differences at each potential between inhibition in the presence
and absence of 2a is indicated by *p < 0.05;
**p < 0.01. Example currents in the presence of
2a are given as insets to parts A-C
for 1B and for all the chimeras shown. They were expressed as
described in the legend to Figure 1. These traces were evoked by a
pulse from 100 to 0 mV, and therefore do not show the maximum
inhibition. Traces are shown before (con) and during
quinpirole (100 nM) application (quin).
F, Example traces showing the lack of effect of
quinpirole on 1C, 1cBbCCC, 1cBcCCC, and 1cCbCCC, all
expressed with 2a. The calibration bars are all 50 msec and 500 nA,
unless otherwise stated.
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The modulation of 1B by activation of the dopamine D2 receptor with
100 nM quinpirole was voltage-dependent, as we have shown previously (Page et al., 1998; Stephens et al., 1998b). This is manifested by a depolarizing shift in the voltage for 50% activation of the current (V50) (Fig.
1B), and also by a reduction in the percentage
inhibition at increasing test potentials (Fig. 2A). Maximum inhibition was usually seen at a test potential ~10 mV below
the peak of the current-voltage relationship (47% at 10 mV for
1B; Fig. 2A). The transfer of the entire N
terminus, first transmembrane domain, and I-II loop sequence of 1B
into 1C gave a chimera showing G-protein modulation that was smaller
at all potentials than the 1B parent (Figs. 1B,
2B). The depolarizing shift in the
V50 for 1bBbCCC was less than for
1B (Fig. 1B), and the maximum modulation was 24%
at 20 mV (Fig. 2B). With respect to both
measurements, a similar degree of modulation by quinpirole was seen for
1bBcCCC (Figs. 1B, 2C), providing
strong evidence that the I-II linker from a modulatable channel such as
1B is not essential for exhibition of G-protein modulation.
Modulation by quinpirole was also still present in the chimera
1bCbCCC (18% at 10 mV; Figs. 1B,
2D). Furthermore, there was still a significant degree of modulation of the minimal chimera 1bCcCCC (13% at 10 mV; Figs. 1B, 2E), again indicating
that the I-II linker from a modulatable channel is not essential for
the observation of G-protein modulation.
In contrast, none of the chimeras containing the N terminus of 1C
instead of 1B showed any inhibition by quinpirole at any potential
from 30 to +40 mV under these conditions (inhibition by quinpirole at
0 mV: 0.66 ± 1.0% for 1cBbCCC, -0.4 ± 0.3% for 1cBcCCC, and -0.86 ± 0.92% for 1cCbCCC; n
values given in Table 1) (Fig. 2F). There was also no
quinpirole-induced depolarizing shift in the
V50 for activation (Fig.
1B). This was also the case for 1C (0.25 ± 0.21% inhibition by quinpirole at 0 mV; Figs. 1B,
2F). Thus, the N terminus of 1B is essential and
sufficient for the expression of any G-protein modulation, whereas the
first transmembrane domain and I-II linker of 1B can be substituted by that of 1C, and significant, although reduced, G-protein
modulation is still observed.
Antagonism by 2a of G-protein modulation of the
1B/1C chimeras
It has previously been shown that the G-protein modulation of
1E currents is antagonized by 2a (Qin et al., 1998). To study the
interaction between the presence of overexpressed VDCC 2a and the
extent of G-protein modulation, we also examined the degree of
G-protein modulation by dopamine D2 receptor activation in the absence
of exogenously coexpressed VDCC subunit in Xenopus oocytes. Nevertheless, it should be stressed that Xenopus
oocytes contain an endogenous 3-like subunit, and when this was
depleted with an antisense construct, no functional currents were seen (Tareilus et al., 1997). The G-protein modulation of 1B and the chimera 1bBbCCC was found to be significantly greater in the absence
of coexpressed 2a than in its presence (Fig.
2A,B). In contrast, the extent of
quinpirole-induced modulation of the 1bBcCCC and 1bCbCCC chimeras
was not significantly increased in the absence of exogenous 2a (Fig.
2C,D). Furthermore, the absence of 2a did not
uncover G-protein modulation in any of the chimeras lacking the N
terminus of 1B that were not modulated in the presence of 2a
(results not shown). We were unable to examine 1bCcCCC currents in
the absence of 2a, because no expression was observed (n = 3 experiments). These results suggest that the
presence of the I-II linker and first transmembrane domain of 1B,
although not being essential for G-protein modulation, are together
required for the reduction of G-protein modulation in the presence of
the exogenously expressed VDCC 2a subunit, seen under these conditions.
Coexpression of subunits with 1 subunits in Xenopus
oocytes and other systems results in a hyperpolarizing shift in current activation (for review, see Walker and De Waard, 1998), and it is of
interest that this is greatest for 1B, 1C and those chimeras in
which all the transmembrane domains are identical (1bCbCCC and
1cCbCCC; Table 1). However, despite the reduced 2a-induced hyperpolarizing shift in the activation of the 1bBbCCC chimera, compared to 1B, there was still a clear 2a-induced reduction in
the amount of G-protein inhibition at all potentials (Fig. 2B), indicating that 2a was influencing this channel.
G-protein modulation of 1B/1C chimeras by coexpression of
G subunits
The role of G in mediating the inhibition observed was
confirmed by coexpression of the chimeric 1 channels with 2,
2a, and G12 in COS-7 cells. A prepulse protocol was used (Fig.
3, left panels), giving steps
to potentials between 40 and +40 mV, before (P1) and 10 msec after
(P2) a large depolarizing step to +120 mV (Page et al., 1998). The
prepulse reverses G-mediated modulation, and hence P2 acts as an
internal control. The G-mediated modulation was determined from
the hyperpolarizing shift in the V50
of the current-voltage relationship in P2 compared to that in P1 (Fig.
3, right panels). For 1B, this shift was almost 10 mV
(Figs. 3A, 4), and it was not
significantly smaller for the chimeras 1bBbCCC and 1bCbCCC (Figs.
3B, 4). It was reduced, but still significantly different
from 1C for the 1bBcCCC and 1bCcCCC chimeras (Figs.
3C, 4). Of the other chimeras, all of which had the N
terminus of 1C, none showed any greater shift in
V50 than 1C itself (Figs.
3D, 4). In control experiments recorded in the absence of
coexpressed G12 and in the presence of intracellular GDPS, the
shift in V50 caused by a depolarizing
prepulse was approximately 1.8 mV for 1C (n = 10),
very similar to the value for 1C coexpressed with G12 (2.1
mV; Fig. 4). A similar level of control facilitation was observed for
1B (n = 10) (Fig. 4). Similar control results were
obtained when the -ARK1 G-binding domain was coexpressed, to
act as a sink for endogenous G and prevent tonic modulation
(Stephens et al., 1998a,b) (results not shown). This control
prepulse potentiation is therefore likely to be caused by a mechanism
other than G-protein modulation (Dolphin, 1996). The main discrepancy
between the results examining direct G modulation and those
examining receptor-mediated modulation involve the 1bCbCCC chimera,
which is strongly modulated by overexpression of G12 (Figs.
3B, 4), and more weakly modulated by receptor-mediated inhibition (Figs. 1B, 2D). The
reason for this may relate to differences in G subtype or
concentration between the two systems.
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Figure 3.
Examples of direct modulation by G12 of the
chimeras between 1B and 1C. The 1 subunits shown were
coexpressed with 2, 2a, G1, and G2 in COS-7 cells.
Left panel, Traces obtained before and after a
depolarizing prepulse (+120 mV, 100 msec). The prepulse protocol is
above the top trace. Right panel, Current-voltage
relationships (steps from 40 to +50 mV in 10 mV intervals, from a
holding potential of 100 mV), measured 50 msec after the start of the
step, for the currents in P1 (open circle) and P2
(filled circle). The current-voltage
relationships were fitted (solid lines) with a modified
Boltzmann equation as given in the legend to Table 1. The mean
depolarizing shifts in V50 resulting from
the depolarizing prepulse are given in Figure 4. A,
Currents resulting from 1B expression (currents shown resulting from
steps 40 to 0 mV, and recorded in 1 mM
Ba2+). B, Currents resulting from
1bCbCCC expression (steps 40 to +20 mV shown, recorded in 10 mM Ba2+). C, Currents
resulting from 1bCcCCC expression (steps 40 to +20 mV shown,
recorded in 10 mM Ba2+).
D, Currents resulting from 1C expression (steps 40
to 10 mV shown, recorded in 1 mM
Ba2+). In this example the depolarizing prepulse was
not preceded by a 10 msec step to the holding potential, but this had
no effect on the subsequent results.
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Figure 4.
Modulation by G12 of the chimeras between
1B and 1C. Histogram giving the mean ± SEM of the
hyperpolarizing shifts in V50 after a
depolarizing prepulse for the same chimeras as in Figure 1.
*p < 0.05; **p < 0.001 compared to 1C/G12. All 1B currents were recorded with 1 mM Ba2+ and all chimeras with 10 mM Ba2+ as charge carrier. It was
checked for parental 1B that the use of 1 or 10 mM
Ba2+ did not affect the
V50 caused by a depolarizing prepulse.
For the bars marked control, the parental constructs
were expressed without G subunits, in the presence of GDPS (1 mM), and a small prepulse-induced hyperpolarizing shift in
V50 was observed for 1B and 1C. A
similar control shift was also observed for all the chimeras tested
[for example for 1bCbCCC the control
V50 was 2.7 ± 0.8 mV
(n = 8)]. This shift was not significantly
different from that for 1C coexpressed with G12. The number of
experiments performed is given at the base of each bar.
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Isolation of the amino acid residues of the N terminus essential
for G-protein modulation
We have made a number of deletions to determine the amino acid
sequences that are essential for G-protein modulation. From our
previous study (Page et al., 1998), we found that the truncated 1-55 1B construct was not G-protein modulated, in agreement with
the N-terminally truncated 1E (rbEII) isoform, that is also not
G-protein-modulated. In the present series of experiments, the effect
of quinpirole (100 nM) was determined during steps to 0 mV,
because none of the constructs showed major shifts in voltage
dependence of current activation, compared to 1B. Inhibition of
wild-type 1B was 35.3 ± 2.2% at 0 mV under these conditions (n = 8). We made a number of truncations: 1B2-6
and 2-44, in line with regions of homology between the N-terminal
sequences of all the G-protein modulated 1 subunits (Fig.
5A). These two constructs were
as strongly G-protein-modulated as 1B itself [respectively,
35.8 ± 2.5% (n = 5), and 36.1 ± 5.3% (n = 6) inhibition by quinpirole; Fig.
5B,C]. This identifies the 11 amino acid sequence of 1B 45-55 (YKQSIAQRART) (Fig.
5A) as being required for the G-protein modulation of 1B.
To confirm this finding, deletion of only this sequence created a
construct, 1B 45-55, in which G-protein modulation was
completely abolished [0.7 ± 2.1% inhibition by quinpirole
(n = 5); Fig.
5B,C].
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Figure 5.
The effect of various deletions and point
mutations of the N terminus of 1B on inhibition of
IBa by the D2 agonist quinpirole. The
sequence of the N terminus of 1B, with the 11 amino acid sequence
identified as being involved in G-protein modulation in
bold, and the points at which deletions were made shown
by arrows beneath the sequence. Example traces, showing
the effect of quinpirole (100 nM) on
IBa in the 1B 2-44 mutant
(left), the 1B 45-55 mutant (center
left), the 1B I49A mutant (center right), and
the 1B R54A mutant (right). Traces (100 msec
duration) were obtained at a test potential of 0 mV, from a holding
potential of 100 mV. Con, Control traces;
quin, after perfusion of quinpirole. Histogram of the
percentage inhibition by 100 nM quinpirole (mean ± SEM) of IBa in the various deletion and
point mutants of the N terminus of 1B. The currents were activated
at 0 mV, and the degree of inhibition was determined from the currents
activated every 15 sec. The number of experiments for each condition is
given in parentheses, and the significance of the
differences compared to the inhibition of 1B are given by
*p < 0.005.
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Point mutations to alanine (A) were then carried out to identify the
specific amino acids in this 11 amino acid sequence that are essential
for G-protein modulation. Mutation of both arginines to alanine (R52A,
R54A) produced a construct that showed no G-protein modulation
(2.5 ± 2.5% inhibition by quinpirole; n = 8).
Point mutations of the individual amino acids in the QRART sequence (Q51A, R52A, and R54A) subsequently identified both arginines as being
critical for G-protein modulation, because either mutation produced a
construct that showed almost complete loss of inhibition by quinpirole
(Fig. 5B,C).
The N-terminal part of this 11 amino acid sequence also contains
residues that are critical for G-protein modulation. When YKQSI was
mutated to AAAAA (Fig. 5A), the channel was not
G-protein-modulated [0.3 ± 2.1% inhibition by quinpirole
(n = 4); Fig. 5C]. To confirm the
importance of the amino acids 45-50 (YKQSIA), an intermediate deletion
1B N2-50 was made, to give a construct starting with methionine
followed by Q51. This was also found not to be G-protein-modulated (Fig. 5C). Subsequent point mutations were made of the
individual amino acids in the YKQSIA sequence to A (Y45A, K46A, Q47A,
S48A, and I49A). This identified only the serine and, to a lesser
extent, isoleucine in the sequence as being involved in G-protein
modulation. These mutations resulted in reduced quinpirole-induced
inhibition of IBa to 4.5 ± 1.0%
(n = 6) for S48A and 17.4 ± 2.1%
(n = 11) for I49A, respectively (Fig. 5C).
Although the individual point mutants Y45A, K46A, and Q47A were all
strongly G-protein-modulated by quinpirole, the modulation of the
construct containing the triple mutation YKQAAA was reduced
(18.8 ± 3.9% inhibition by quinpirole; n = 5;
Fig. 5C), indicating an influence of these amino acids.
Modulation of N-terminal mutants of 1B by G
We have confirmed that the identified amino acids are similarly
involved in direct G-induced modulation of 1B by performing experiments with coexpressed G12 in COS-7 cells. Examples of results obtained are shown in Figure 6.
For the Q47A mutation, G-protein modulation was still observed, with
slowly activating currents in P1 and a clear hyperpolarizing shift in
the V50 for current activation
resulting from the depolarizing prepulse (Fig. 6A).
In contrast, for the R52A mutation, no G-protein modulation was
observed (Fig. 6B). The mean results for all the
constructs are given in Figure 7,
expressed as P2/P1 facilitation ratio at 10 mV (Fig. 7A).
The V50 for the
IBa current-voltage relationship was
also plotted, because this shows a depolarizing shift in
G-protein-modulated channels, compared to the control 1B expressed
in the absence of G (Fig. 7B). These two sets of
measurements are strongly correlated (r = 0.76, data
not shown), and the depolarizing shift in activation
V50 is also highly correlated to the
percentage inhibition by quinpirole observed for the same
constructs in the Xenopus oocyte experiments (Fig.
7C), suggesting that direct G modulation and
quinpirole-induced modulation of these constructs are using the same
mechanism. The I49A mutation stands out in both these systems as
producing a reduction, but not a complete inhibition of modulation
(Fig. 7C).
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Figure 6.
Examples of the effect of 1B N-terminal
mutations on G modulation in COS-7 cells. Coexpression of two
1B N-terminal mutations with 2, 2a, and G12, recorded
with 1 mM Ba2+ charge carrier.
Left panel, Current traces are shown, evoked by the same
protocol given in Figure 3. Right panel,
Current-voltage relationships are given, from 40 to +50 mV, in 10 mV
intervals, before (open circles) and after
(filled circles) the depolarizing prepulse,
fitted (solid lines) with the modified Boltzmann
equation given in the legend to Table 1. A, The 1B
Q47A mutation (traces from 40 to 0 mV are shown). B,
The 1B R52A mutation (traces from 40 to +10 mV are shown).
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Figure 7.
Mean effect of 1B deletions and mutations on
G modulation in COS-7 cells. A, The P2/P1 ratio
was determined in COS-7 cells overexpressing G12, from current
amplitudes during steps to 10 mV before and after a depolarizing
prepulse (+120 mV, 100 msec), for the same N-terminal deletions and
point mutations shown in Figure 5. Comparison is made with 1B in the
absence of G, recorded with 1 mM GDPS in the patch
pipette (1). The value [(P2/P1) 1] is plotted,
which will be 0 if there is no facilitation. B, The
activation V50 was determined for the same
constructs coexpressed with G12, and compared to the value for
1B in the absence of G, recorded with 1 mM GDPS
in the patch pipette (1). The dashed
lines are 1 SEM more positive than the mean value for 1B
(1), and 1 SEM more negative than the mean value
for 1B/G (2). C,
Correlation between activation V50 (the
data given in B, after subtraction of the
V50 for 1B) on the y-axis,
and the data from Figure 5C (percentage inhibition of
IBa by 100 nM quinpirole), on
the x-axis. The numbers identifying the constructs refer
to the bars in A and B. Regression
analysis (dotted line) gives a coefficient,
r of 0.92 (p < 0.001). The
data divide into a group of modulated and a group of nonmodulated
constructs, as identified, except for constructs 14 (I49A) and 9 (YKQAAA).
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Basis for the reduction in receptor-mediated modulation of the
N-terminal point mutation I49A
G-protein modulation of calcium channels is strongly
voltage-dependent, in that more inhibition is observed at low than at high depolarizations (Bean, 1989). To examine the basis for the reduced
modulation of the partially modulated mutant (1B I49A) compared to
1B, we first examined, in Xenopus oocytes, the voltage dependence of the removal of inhibition by quinpirole, during a
depolarizing prepulse (see Fig.
8A for voltage
protocol). There was no significant effect of the I49A mutation on the
voltage dependence of the prepulse-induced facilitation in the presence of quinpirole (Fig. 8B), or on the time course of
removal of quinpirole-induced inhibition during a 100 mV depolarizing
prepulse. Single exponential fits gave  values for removal of
inhibition (possibly representing dissociation of G at this
depolarized potential) of ~20 msec for both constructs (Fig.
8C). The only clear difference between I49A 1B and
wild-type 1B was in the more rapid time course of reinstatement of
G-protein modulation after its removal by a 100 msec prepulse to +100
mV (Fig. 8D). This could be fit to a single exponential with reinhibition of 187 msec for
1B and 85 msec for the I49A mutant (Fig. 8D,
inset).
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Figure 8.
Voltage dependence of inhibition, rate of loss of
inhibition, and reinhibition rate for 1B and 1B I49A in
Xenopus oocytes. A, Voltage protocol,
showing variation of the prepulse voltage
(V), the prepulse duration
(tdep), and the interpulse interval
(tinter) between the prepulse and the test pulse.
The prepulse potential was 100 mV and 50 msec duration, and the
interpulse interval was 20 msec, unless these parameters were varied.
B, Effect of increasing the 50 msec prepulse voltage
(V) on prepulse facilitation in the
presence of quinpirole. Facilitation was measured as
(IBa in P2) (IBa in P1) and normalized to the maximum
facilitation observed (normalized I). 1B (open
circles), 1B I49A (filled circles).
The inset histogram gives the
V50 values (mean ± SEM, determined by
fitting Boltzmann functions to the data from the number of individual
experiments given above each bar) for 1B (white bar)
and 1B I49A (black bar). C, Effect of
increasing the duration of the 100 mV prepulse
(tdep) on prepulse facilitation in the presence
of quinpirole. Facilitation was measured as described in
B. 1B (open circles), 1B I49A
(filled circles). The inset
histogram gives the dissociation values (mean ± SEM, determined by fitting a single exponential to the data from the
number of experiments given above each bar) for 1B (white
bar) and 1B I49A (black bar).
D, Effect of increasing the interval between the 100 mV,
50 msec prepulse and the subsequent test pulse P2 on the facilitation
in the presence of quinpirole. Facilitation was measured as described
in B: 1B (open circles), 1B I49A
(filled circles). The inset
histogram gives the reinhibition values (mean ± SEM, determined by fitting a single exponential to the data from the
number of experiments given above each bar) for 1B (white
bar) and 1B I49A (black bar).
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If we consider G-protein modulation as a simple bimolecular reaction,
as has been done previously (Zhang et al., 1996; Stephens et al.,
1998a):
where C is one of the closed states of the calcium
channel 1B subunit, k1 is the
association rate constant, and k1 is the dissociation rate constant for G. At equilibrium, from the
law of mass action:
at
the holding potential,
1/reinhibition=k1[G]+k1 (1)
![<UP>and steady state inhibition</UP>=k<SUB>1</SUB>[G&bgr;&ggr;]/(k<SUB>1</SUB>[G&bgr;&ggr;]+k<SUB><UP>−</UP>1</SUB>)](/content/vol19/issue16/fulltext/6855/img003.gif)
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(2)
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From our previous study (Stephens et al., 1998a), we estimated the
G concentration to reach ~300 nM, when G was
overexpressed. Taking an approximate value of 100 nM for
the concentration of G resulting from quinpirole-induced receptor
activation in the present study [a value of 130 nM can be
calculated making the assumptions described in Stephens et al.
(1998a)], we can obtain estimates by substitution of steady-state
inhibition and reinhibition values into
Equations 1 and 2, of k1 and
k1. For 1B,
k1 is 25.2 µM1sec1
, and k1 is 2.8 sec1, whereas for the I49A mutant
k1 is 21.0 µM1sec1,
and k1 is 9.6 sec1. Clearly, the major difference is
an apparent 3.4-fold increase in the off-rate for G in the I49A
mutant. However, assuming that reassociation of G is very slow at
+100 mV, the dissociation of G during the prepulse to +100 mV,
found from Figure 8C, is 53.2 sec1 for 1B and 46.7 sec1 for 1B I49A, indicating that the
apparent off-rate is more rapid for both constructs at this depolarized
potential [as previously observed in Stephens et al. (1998a)], and
the difference between the parental 1B and the I49A mutant is lost.
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DISCUSSION |
The molecular determinants for the inhibition of neuronal
VDCC 1 subunits by G have been the subject of several studies. However, there remains no consensus of opinion concerning the functional importance of biochemically identified G-binding sites
on the I-II loop and C terminus (De Waard et al., 1997; Page et al.,
1997; Qin et al., 1997; Zamponi et al., 1997) (for review, see Dolphin,
1998). Furthermore, there has been little agreement on the extent of
modulation of the E-type VDCCs (Bourinet et al., 1996; Toth et al.,
1996; Yassin et al., 1996; Mehrke et al., 1997; Page et al., 1997; Qin
et al., 1997). However, following our recent study (Page et al., 1998),
it now seems clear that all 1E orthologues are G-protein-modulated
when the long N terminus is present.
Requirement for the N terminus of 1B for
G-protein modulation
The present study was performed to further our understanding of
the involvement of the N terminus of the VDCC 1B in G-protein modulation, first identified by Page et al. (1998). We therefore made a
series of chimeras between 1B, which is strongly
G-protein-modulated, and 1C, which is not modulated by G, in
the systems studied. Our conclusions are that the N terminus of 1B
is absolutely essential for its G-protein modulation. No modulation was
observed of any channel that contained the N terminus from 1C. The
sequences of the intracellular N termini of 1B and 1C show little
homology, and it is thus clear that the N terminus of 1B plays a
role in G-protein modulation that cannot be substituted by that of
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TOP
ABSTRACT
INTRODUCTION
![k<SUB>1</SUB>[G&bgr;&ggr;][C]=k<SUB><UP>−</UP>1</SUB>[C·G&bgr;&ggr;]](/content/vol19/issue16/fulltext/6855/img002.gif)
