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The Journal of Neuroscience, July 1, 1998, 18(13):4815-4824
Identification of the Amino Terminus of Neuronal
Ca2+ Channel  1 Subunits 1B and 1E as an
Essential Determinant of G-Protein Modulation
Karen M.
Page,
Carles
Cantí,
Gary J.
Stephens,
Nicholas S.
Berrow, and
Annette C.
Dolphin
Department of Pharmacology, University College London, London WC1E
6BT, United Kingdom
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ABSTRACT |
We have examined the basis for G-protein modulation of the neuronal
voltage-dependent calcium channels (VDCCs)  1E and 1B. A novel PCR
product of 1E was isolated from rat brain. This contained an
extended 5' DNA sequence and was subcloned onto the previously cloned
isoform rbEII, giving rise to 1Elong whose N terminus was extended by 50 amino acids. VDCC 1 subunit constructs were co-expressed with the accessory 2- and  2a subunits in
Xenopus oocytes and mammalian (COS-7) cells. The
1Elong showed biophysical properties similar to those of
rbEII; however, when G-protein modulation of expressed 1 subunits
was induced by activation of co-expressed dopamine (D2) receptors with
quinpirole (100 nM) in oocytes, or by co-transfection of
G1 2 subunits in COS-7 cells, 1Elong, unlike
1E(rbEII), was found to be G-protein-modulated, in terms of both a
slowing of activation kinetics and a reduction in current amplitude.
However, 1Elong showed less modulation than 1B, and
substitution of the 1E1-50 with the corresponding region of 1B1-55 produced a chimera 1bEEEE, with
G-protein modulation intermediate between 1Elong and
1B. Furthermore, deletion of the N-terminal 1-55 sequence from
1B produced 1B N1-55, which could not be
modulated, thus identifying the N-terminal domain as essential for
G-protein modulation. Taken together with previous studies, these
results indicate that the intracellular N terminus of
1E1-50 and 1B1-55 is likely to
contribute to a multicomponent site, together with the intracellular
I-II loop and/or the C-terminal tail, which are involved in G
binding and/or in subsequent modulation of channel gating.
Key words:
calcium channel; neuronal; G-protein; 1 subunit; G subunit; modulation
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INTRODUCTION |
G-protein inhibition of neuronal N
(1B) and P/Q type (1A) calcium currents is mediated by G
subunits (Herlitze et al., 1996 ; Ikeda, 1996). The extent of G-protein
modulation for the other non-L-type voltage-dependent calcium channel
(VDCC) subunit 1E is less well established (for review, see Dolphin,
1998). The human 1E subunit has recently been shown to be inhibited by overexpression of G subunits (Shekter et al., 1997) and by the
activation of G-protein-coupled receptors (Mehrke et al., 1997; Qin et
al., 1997). It is of interest that these effects are attenuated by the
presence of accessory VDCC subunits, suggesting functional
competition, as previously hypothesized (Campbell et al., 1995b). In
contrast, rat brain 1E(rbEII) (Soong et al., 1993) shows no
G-protein modulation (Bourinet et al., 1996; Page et al., 1997).
A number of recent studies have investigated the site(s) at which
G subunits bind to 1 subunits. Two such regions have been
identified on the non-L-type VDCC subunits. First, the intracellular loop that links transmembrane domains I and II has two binding sites:
one containing a QxxER amino acid consensus sequence common to many
G binding proteins, and one nearer the end of the I-II loop (De
Waard et al., 1997; Zamponi et al., 1997). Second, a C-terminal site
has recently been identified and proposed to be the unique region
responsible for G-protein inhibition of human 1E (Qin et al., 1997).
A 38 amino acid sequence in the center of the 1E C terminus has been
found to bind free G dimers (Qin et al., 1997).
Functionally, the site of G-protein action remains controversial.
Mutations within the I-II loop have been shown either to abolish
G binding and prevent the slowing of activation induced by
GTPS (De Waard et al., 1997) or to enhance modulation (Herlitze et
al., 1997), whereas conversion of the entire 1A consensus sequence
(QIEER) to that seen in 1C (QQLEE) did attenuate modulation (Herlitze et al., 1997). We observed that transfer of the IS6 and I-II
loop from 1B to 1E(rbEII) conferred minor aspects of G-protein
sensitivity to the resultant chimera, namely a slowing of activation
kinetics in the presence of GTPS, but did not result in modulation
of the calcium current amplitude, as seen in 1B (Page et al., 1997).
In contrast, the 1B subunit was reported to retain G-protein
modulation when its entire I-II loop was replaced by the corresponding
1C sequence (Zhang et al., 1996), which does not bind G (De
Waard et al., 1997). Their study implicated a role of domain I together
with the C terminus in G-protein modulation. In partial agreement with
this, the inhibition of human 1E by muscarinic agonists appears to
be caused by G binding solely at the C-terminal site (Qin et al.,
1997).
In the present study we have examined the major difference between rat
1E(rbEII) and the corresponding human clone, which is that the
latter contains an extended N-terminal sequence. We have isolated a
fragment of rat brain 1E containing an extended 5' DNA sequence and
have found that the 1Elong isoform so formed, unlike
rbEII, is subject to G-protein modulation. Furthermore, an 1B
construct in which the corresponding N-terminal region is deleted shows
no G-protein regulation. The data indicate that the N terminus of the
1B and 1E subunits is crucial for their G-protein modulation.
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MATERIALS AND METHODS |
Materials
The following cDNAs were used: rat 1E (rbEII, GenBank
accession number L15453), rabbit 1B (D14157), rat 2a (M80545), rat 2- (neuronal splice variant, M86621), rat D2long
receptor (X17458, N5 G), bovine G1 (M13236), bovine G2
(M37183), and mut-3 Green Fluorescent Protein (GFP, U73901). All cDNAs were subcloned into the expression vector pMT2 (Swick et al., 1992).
Production of VDCC 1 constructs
The constructs were produced by PCR methodology described
previously (Page et al., 1997). Individual constructs were produced as
follows.
1Elong. A 5' region of a longer isoform of
1E was isolated by RT-PCR from granule cells, prepared from rat
cerebella as described previously (Huston et al., 1993). Total RNA was
isolated using the RNeasy miniprep kit (Qiagen, Hilden, Germany).
Reverse transcription was performed using M-MLV reverse transcriptase (Promega, Madison, WI) in the presence of RNasin (Promega) and random
hexamer primers (Promega) at 37°C for 60 min. The forward primer
(primer 1) (see Fig. 1) for PCR (ATA GGT ACC ATG GCT CGC TTC GGG GAG
GC) is based on a region completely conserved at the N terminus of the
reported human (L27745), mouse (L29346), and rabbit (X67855) 1E cDNA
sequences, and also contains a 5' KpnI extension (GGTACC).
The reverse primer E899R (GCC GAT CCA GTC CTT ACA TTC A) is specific
for 1E(rbEII). PCR was performed using BIO-X-ACT DNA Polymerase
(Bioline), a high-fidelity enzyme mixture. The extended 1E 5' region
was subcloned between the KpnI site (pMT2 polylinker) and
the NotI site (bp 158 of rbEII) of 1E(rbEII) pMT2. The
DNA and protein sequences are shown in Figure 1. RT-PCR was also
performed to determine whether the short isoform of 1E(rbEII) (Soong
et al., 1993) could be detected in rat cerebellar granule neurons or
whole rat brain. Two separate forward primers, CAT GGT ACC TTG CAG ACC
CAG GAA (primer 2) (see Fig. 1) and AGC GGT ACC TGT TCT TCA TGG ATC
(primer 3) (see Fig. 1), both containing mutated KpnI sites
at the 5' end, were used together with the reverse primer E899R.
1bEEEE. The first 55 amino acids of the N terminus of
rabbit 1B was added onto the N terminus of rat 1E(rbEII) to give 1bEEEE. The forward primer (pMT2F) AGC TTG AGG TGT GGC AGG CTT and
the reverse primer TGG GGT TGT ACA GCG CCA TGG T were used with the
1B-pMT2 template to give a product of ~300 bp. This PCR product
was used as a forward primer, along with the reverse primer E899R, and
extended on 1E(rbEII) pMT2 to give a product of ~1 kb. Digestion
of the PCR product with KpnI and XbaI gave a
fragment of ~800 bp, and this was subcloned onto the 5' end of
1E(rbEII) in the pMT2 vector.
1B(N1-55). The 1B was
truncated at the 5' end using the forward primer CGC ACT AGT ACC ATG
GCG CTG TAC AA and the reverse primer GTC GCT TCT GCT CTT CTT GG. The
PCR product was digested with the enzymes SpeI and
KpnI and subcloned into 1B pMT2, which had also been
digested with SpeI (polylinker cloning site) and
KpnI (1285 bp position in 1B).
All PCR was performed using the proof-reading enzyme Pfu
(Stratagene, La Jolla, CA), except for 1Elong as
described above. The sequences of the subcloned PCR products were
verified by cycle-sequencing using SequiTherm EXCEL II
(Epicenter Technologies, Madison, WI). For
1Elong, a number of different RT-PCR reactions
were performed, and the products were sequenced. The sequences were
found to be the same for all PCR products tested, including the single
clone selected for expression studies.
Expression of constructs and electrophysiological recording
Xenopus oocytes. Oocytes were surgically removed
from adult Xenopus laevis females 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, 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 3:1:1:3 (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 sodium
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 40, TEA-OH 50, KOH
2, niflumic acid 0.4, HEPES 5, pH 7.4 with methanesulfonic acid. In
some experiments niflumic acid was omitted, and 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) 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 measured 100 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. 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 VDCC
expression were observed between 2 and 4 d post-transfection (Brice et al., 1997). Ca2+ currents were recorded
using the whole-cell patch technique. Borosilicate glass electrodes
(2-4 M) 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, 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, BaCl2 1, 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 after leak
and residual capacitance current subtraction (P/4 or P/8 protocol) and
series resistance compensation up to 85%.
All experiments were performed at room temperature (20-24°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 |
Isolation of a long N-terminal isoform of 1E
Amino acid alignment of the rat 1E(rbEII) and the rabbit 1B
shows that a high degree of conservation exists within these sequences
but that the 1E(rbEII) sequence is 55 amino acids shorter than that
of 1B. Alignment of the 1E N termini for mouse (L29346), human
(L27745), rabbit (X67855), and rat (L15453) shows that the mouse,
human, and rabbit sequences also contain ~50 additional amino acids
at the extreme N terminus. This region is homologous in these species
but is missing from the rat sequence. Furthermore, the proximal part of
the reported 5' untranslated region of rbEII shows extensive homology
with the mouse, human, and rabbit 1E cDNAs. The initial 5' DNA
sequences in these species are completely conserved, allowing the
design of a PCR primer (primer 1) (Fig. 1) that could anneal to a longer isoform
of 1E, including the ATG corresponding to the start codon in the
human, rabbit, and mouse 1E clones. RT-PCR was performed on RNA
isolated from rat cerebellar granule cells. The resulting product was
of the expected length, compared with the reported sequences of 1E
from mouse, human, and rabbit. This was subcloned onto the rat
1E(rbEII) construct to give 1Elong. DNA and protein
sequences are shown in Figure 1. The predicted N-terminal amino acid
sequence of the PCR-derived 1Elong clone was found to be
identical to that of the reported mouse 1E sequence (Williams et
al., 1994).
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Figure 1.
Sequence of 1Elong used in
this study. A, DNA alignment of the 5' sequences of
1E(rbEII) (L15453), rat 1Elong (AF057029), and mouse
1E (L29346). Shaded areas show translated
sequences. The vertical arrow shows the position of the
restriction site NotI, which was used to subclone the
extended 5' sequence onto 1E(rbEII). The boxed CGG
nucleotides before the ATG start site in the 1E(rbEII) were found
to be present in the rbEII clone but are absent from the L15453
sequence in the database. This triplet is also present in the published
mouse, human, and rabbit 1E sequences. The forward primers used (see
Materials and Methods) are shown as horizontal arrows,
below (primer 1) or above (primers 2 and
3) the corresponding sequence. Note that the extended
N-terminal sequence of 1Elong shows a high degree of
homology with part of the reported 5' untranslated sequence of the
rbEII cDNA. B, Amino acid alignment for the N termini of
rat 1Elong, rabbit 1B (published sequence),
and rat 1E(rbEII, published sequence). Conserved residues are
shaded. The rat 1Elong N-terminal amino
acid sequence was also identical to that of the published mouse 1E
sequence (L29346).
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To determine whether we could detect the shorter isoform of
1E(rbEII) in rat brain, RT-PCR was performed using two different forward primers (labeled 2 and 3 in Fig. 1),
located in the 5' noncoding region of rbEII, whose sequence is given in
the database, together with the same reverse primer as above. No
products were found, using mRNA from either whole rat brain or
cerebellar granule cells, with either forward primer, although we have
no positive control for the efficacy of the forward primers used,
because the rbEII clone that we have is truncated at the
NotI site in the 5'-untranslated region (Fig. 1).
Biophysical properties of 1Elong
We have compared the properties of 1Elong with
those of 1E(rbEII) and 1B. Current-voltage
relationships show no major differences between 1Elong
and 1E(rbEII), in terms of either expression levels or voltage
dependence of activation (Fig. 2, Table
1). Thus, the extended N terminus of
1Elong does not affect its ability to show functional
expression.
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Figure 2.
Properties and G-protein modulation of
1Elong: comparison with 1E(rbEII). A
shows the lack of modulation of 1E(rbEII) in the absence of
co-transfected VDCC subunits. 1E(rbEII) was expressed with
2- but without 2a subunits in Xenopus oocytes
(together with D2 dopamine receptors). Left panel,
Example currents, control (1), plus quinpirole
(2), and after a depolarizing prepulse to +100 mV
in the presence of quinpirole (3). The voltage
protocol is shown above the current traces. Middle
panel, Time course of IBa amplitude
during quinpirole application. Right panel,
I-V plot before ( ) and during ( )
quinpirole application (n = 6). The
I-V data were fitted with a modified
Boltzmann equation as described previously (Page et al., 1997).
B shows the modulation of 1Elong in the
presence of co-transfected VDCC subunits. 1Elong was
expressed with both 2- and 2a subunits in
Xenopus oocytes (together with D2 dopamine receptors).
Activation of dopamine D2 receptors by quinpirole (100 nM)
in oocytes caused a reversible inhibition of
IBa. Left panel, Example
currents, control (1), plus quinpirole
(2), and after a depolarizing prepulse in the
presence of quinpirole (3). Middle
panel, Time course of inhibition by quinpirole. Right
panel, I-V plot before () and
during () quinpirole application (n = 9). The
I-V data were fitted as described in
A. The boxed inset shows the
voltage-dependence of the inhibition by quinpirole from the
I-V data of 1Elong
(solid bars, n = 9). Data for 1B
(open bars, n = 8) are plotted for
comparison; * p < 0.01 (Student's
t test).
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G-protein modulation of rat brain 1Elong: comparison
with 1B
The calcium channel 2a subunit was co-expressed with parental
or chimeric 1 subunits because this auxiliary subunit markedly attenuates the voltage-dependent inactivation of all 1 subunits (Olcese et al., 1994). It therefore allows G-protein modulation of
activation and current amplitudes to be compared in 1E and other
constructs without the interference of differing intrinsic calcium
channel inactivation rates. Receptor-mediated calcium current
inhibition was reconstructed in Xenopus oocytes by
co-expressing the dopamine D2 receptor. Modulation was examined by
determining the effect of a saturating concentration of quinpirole (100 nM) on IBa and the reversibility of
the inhibition by a depolarizing prepulse. In parallel studies in COS-7
cells, G-protein modulation was studied by co-expression of G12
subunits and examination of the effect of a depolarizing prepulse on
activation kinetics and amplitude of IBa.
IBa resulting from 1B expression is strongly
modulated both by endogenous G-protein activation and by co-expressed
G12 in COS-7 cells (Page et al., 1997, 1998; Stephens et al.,
1998a). The inhibition induced after dopamine D2 receptor activation by 100 nM quinpirole was ~50%, associated with a 7.5 mV
depolarizing shift in the voltage for 50% activation
(V50) of the current-voltage (I-V) relationship (Table 1). The
activation rate of 1B IBa was also
significantly slowed by co-expression of G12 (Table 1). In
contrast, we observed no modulation of 1E(rbEII), co-expressed with
2- and 2a, either by activation of dopamine D2 receptors in
Xenopus oocytes or by co-expressed G12 in COS-7 cells
(Table 1). Because it has recently been observed that modulation of human 1E is only fully manifested in the absence of co-expressed subunits and is prevented by co-expression of 2a (Yassin et al.,
1996; Qin et al., 1997), we also examined whether there was any
modulation of 1E(rbEII) in the absence of co-expressed 2a. However, no modulation of 1E(rbEII) was observed by quinpirole in
the absence of exogenous subunits (n = 6) (Fig.
2A).
We next examined whether the longer 1E subunit
(1Elong) showed the ability to be
G-protein-modulated. When 1Elong was expressed in
oocytes (with 2- and 2a), quinpirole (100 nM)
caused an inhibition of IBa amplitude of ~26%
at 0 mV (Fig. 2B, Table 1). This inhibition was
associated with a significant depolarizing shift in the
V50 for activation of IBa
of 3.6 mV (Table 1) and was reversed by a depolarizing prepulse (Fig.
2B). However, the inhibition was significantly less
than the modulation observed for 1B (Fig. 2B,
inset box; Table 1). We then examined whether the smaller
quinpirole-induced inhibition of 1Elong, compared with 1B, was because of co-expression of 2a, but we observed 27.0 ± 2.6% (n = 7) inhibition by 100 nM quinpirole of 1Elong in the absence of
co-expressed Ca2+ channel subunits. Furthermore,
inhibition by quinpirole was not abolished when three times the normal
amount of 2a cDNA was injected but remained at 22.2 ± 1.9%
(n = 7).
Modulation of 1Elong by co-expressed
G subunits
When 1Elong was co-expressed with G12 in
COS-7 cells, there was a clear slowing of activation kinetics, compared
with IBa recorded in control cells in the
presence of GDPS (Fig. 3A,
Table 1), although again this was less than for 1B. In Figure
3B, the voltage-dependence of the activation kinetics of
1Elong are compared in the presence and absence of
G12. Data for 1E(rbEII), showing the lack of effect
of G co-expression, are also included for comparison. A
depolarizing prepulse to +120 mV, applied 10 msec before the test pulse
to activate the calcium channel current, is able to provide an estimate
of the amount of tonic G-protein modulation attributable to
co-expressed G (Ikeda, 1996). In the presence of co-expressed
G12, there was marked prepulse facilitation of the amplitude of
1Elong (Fig. 3C, Table 1), whereas this was
not seen in the absence of co-expressed G12 or for 1E(rbEII)
(Table 1). However, facilitation of 1Elong in the
presence of G was significantly less than that observed for 1B
(Table 1).
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Figure 3.
G-protein modulation of 1Elong
expressed in COS-7 cells. 1Elong was expressed with
accessory VDCC 2- and 2a subunits in the presence or absence
of co-expressed G12. A, Examples of current
density-voltage profiles for 1Elong in a control cell
in the presence of GDPS to limit any tonic G-protein modulation
(left), and a cell co-expressing G12
(right) (Vt = 40 to 10
mV, in 10 mV steps). B, Voltage-dependence of
 act for 1Elong with co-expressed
G12 (, n = 10), 1Elong in
the presence of GDPS ( , n = 7), and
1E(rbEII) with co-expressed G12 (, n = 5), * p < 0.01 compared with respective
control. C, Example of facilitation of 1Elong
IBa in the presence of co-expressed
G12 by a depolarizing prepulse to +120 mV, 10 msec before and
immediately after equivalent test pulses P1 and P2, to test potentials
(Vt) between 40 and 10 mV in 10 mV intervals. The voltage protocol is shown above the current
traces. Facilitation was then determined as the P2/P1 ratio of the
current amplitudes in P1 and P2 (Table 1).
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Role of the N terminus of the VDCC 1B subunit in
G-protein-mediated inhibition
The inhibition of 1B was significantly more extensive than that
of 1Elong, for all parameters measured relating
to the extent of modulation both by receptor activation and by
G12 co-expression (Table 1). Therefore, we next examined whether
substitution of the corresponding N-terminal sequence from 1B would
confer further G-protein modulation on 1E. There is a marked
divergence of sequence when 1B1-55 is compared with the
N-terminal sequence of 1Elong identified here, although
the remaining 40 amino acids of the N-terminal tail, proximal to the
first transmembrane domain, are highly conserved (Fig. 1). For this
reason, a cDNA sequence corresponding to the first 55 amino acids from
1B was added to 1E(rbEII) to give the 1bEEEE chimera (Fig.
4A). This construct exhibited a degree of G-protein modulation in oocytes that was similar,
although somewhat greater throughout the potential range, to that of
1Elong (Table 1; and compare boxed insets in
Figs. 2B, 4A). The extent of
inhibition by quinpirole (100 nM) was ~30% (Table 1),
and there was a 4.2 mV depolarizing shift in the
V50 for activation of IBa
compared with control (Fig. 4B, Table 1). Similarly,
in COS-7 cells, the slowing of activation kinetics with G12 was
less than that seen with 1B (Fig. 4C, Table 1), and the
facilitation of 1bEEEE IBa in the presence of
G12, by a depolarizing prepulse, was also less than that shown by
1B (Table 1).
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Figure 4.
G-protein modulation of an 1E construct
containing the N terminus of 1B. A, The 1 subunit
construct in which the 1B1-55 sequence was added to
1E(rbEII) to form 1bEEEE was expressed with accessory VDCC
2- and 2a subunits in Xenopus oocytes (together
with D2 receptors) or in COS-7 cells (together with G12
subunits). B, 1bEEEE currents expressed in oocytes.
Left panel, Example currents, control
(1), plus quinpirole (2),
and after a depolarizing prepulse in the presence of quinpirole
(3). The voltage protocol is the same as shown in
Figure 2A. Middle panel, Time
course of inhibition by quinpirole. Right panel,
I-V plot before () and during ()
quinpirole application (n = 9). The
I-V data were fitted according to the
legend to Figure 2. The boxed inset shows the
voltage-dependence of the inhibition by quinpirole from the
I-V data (open bars,
n = 9). Data for 1Elong
(solid bars, n = 9) are plotted for
comparison; * p < 0.05 (Student's
t test). C, 1bEEEE currents expressed
in COS-7 cells. Left panel, Example current
density-voltage profiles for control 1bEEEE
IBa in the presence of 2 mM
GDPS. Middle panel, 1bEEEE
IBa in the presence of G12
(Vt = 40 to 10 mV in 10 mV steps).
Right panel, Voltage-dependence of act
for 1bEEEE in the presence (, n = 5) or
absence (, n = 3) of co-expressed G12; *
p < 0.01 compared with respective control.
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Examination of the role of 1B1-55 in G-protein
modulation of 1B
Because G-protein modulation was observed only in
1Elong and 1bEEEE and not in the N-terminal truncated
isoform 1E(rbEII), although the expression levels and
biophysical properties of the currents were very similar (Table 1), we
next examined whether 1B1-55 also played an essential
role in the G-protein modulation of 1B. We therefore created an
1B construct in which this N-terminal sequence was deleted
(1BN1-55) (Fig.
5A). The expression level of
1BN1-55 was similar to that of 1B in both COS-7 cells and Xenopus oocytes (Table 1). However, this construct was no longer subject to modulation by 100 nM quinpirole in
oocytes co-expressing the dopamine D2 receptor, either in the presence of co-injected 2a cDNA (Fig. 5B, Table 1) or in its
absence (0.6 ± 1.6% inhibition; n = 7).
Similarly, there was no effect of G12 on the activation kinetics
of IBa in COS-7 cells, compared with controls
recorded in the presence of GDPS (Fig. 5C, Table 1).
Furthermore, there was no facilitation by a depolarizing prepulse of
the amplitude of IBa in the presence of
G12 (Table 1). These findings highlight the essential role of the
1B1-55 sequence in G-protein inhibition, in terms of
both slowed activation kinetics and inhibition of current
amplitude.
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Figure 5.
Lack of G-protein modulation of an N-terminally
truncated 1B construct. A, The 1 construct in
which the 1B1-55 sequence was deleted from 1B to
form 1BN1-55 was expressed with accessory VDCC
2- and 2a subunits in Xenopus oocytes (together
with D2 receptors) or in COS-7 cells (together with G12
subunits). B, 1BN1-55 currents
expressed in oocytes. Left panel, Example currents,
control (1), plus quinpirole
(2), and after a depolarizing prepulse in the
presence of quinpirole (3). The voltage protocol
is the same as shown in Figure 2A. Middle
panel, Time course of IBa amplitude
during quinpirole application. Right panel,
I-V plot before () and during ()
quinpirole application (n = 7). The
I-V data were fitted according to the
legend to Figure 2. C, 1BN1-55
currents expressed in COS-7 cells. Left panel, Example
current density-voltage profiles in the absence or presence of
G12 (Vt = 40 to 10 mV in 10 mV
steps). Right panel, Voltage-dependence of
act in the presence (, n = 10) or
absence (, n = 7) of co-expressed
G12.
|
|
Comparison of the reinhibition kinetics of 1E(long)
and 1B
A characteristic feature of voltage-dependent G-protein modulation
is that after a large depolarizing prepulse to remove modulation, the
G-protein effect may be reinstated in a time- and voltage-dependent manner. The time constant of this reinhibition
(reinhibition) can be determined from the exponential
increase of current amplitude, when the duration of the interpulse
interval (t) between the depolarizing prepulse and test
pulse is increased (Fig. 6). When this
analysis was performed for the quinpirole-induced inhibition of 1B
and 1Elong in oocytes, there was no difference in their reinhibition rates (measured at 100 mV, after a 50 msec depolarizing prepulse to +100 mV). The reinhibition was 96.6 ± 5.9 msec (n = 9) for 1B and 93.5 ± 5.4 msec
(n = 9) for 1Elong. This result suggests
that the binding site for G shows a similar affinity in these two
1 subunits.
View larger version (20K):
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|
Figure 6.
Reinhibition kinetics of 1Elong and
1B. Prepulses of 50 msec duration to +100 mV were applied, and the
time between prepulse and test pulse to 0 mV (interpulse interval
t at 100 mV) was increased, in 10 msec steps, up to
220 msec. There was no difference between the
reinhibition for 1Elong (,
n = 9) and 1B (, n = 9)
IBa.
|
|
| |
DISCUSSION |
The molecular determinants for the inhibition of neuronal VDCC
1 subunits by G have been the subject of intense
investigation. 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).
Existence of an extended N-terminal isoform of rat brain 1E
We have demonstrated the presence of a longer isoform of rat brain
1E (1Elong) in rat cerebellar granule cells.
This has an N-terminal sequence extended by 50 amino acids compared
with rbEII and shows extensive homology with the mouse, rabbit, and human 1E sequences. The 1Elong was the only isoform
detected in rat brain, although we have no positive control for the two different forward primers in the reported 5' untranslated sequence of
rbEII that were used (Fig. 1).
The rat 1Elong isoform is G-protein-modulated
Initially, both rat and human 1E were reported not to be
modulated by G-proteins (Bourinet et al., 1996; Toth et al., 1996; Page
et al., 1997). However, it then became clear that human 1E was
capable of being G-protein-modulated (Mehrke et al., 1997; Qin et al.,
1997) but showed high sensitivity to functional antagonism by VDCC subunits (Shekter et al., 1997), and particularly to 2a, which
occluded G-protein modulation (Qin et al., 1997). This would also be a
possible explanation for the lack of inhibition of 1E(rbEII) by
co-expressed G or by activation of dopamine D2 receptors.
However, a number of points argue against this explanation. First, the
novel rat 1Elong isoform identified here is clearly modulated despite the presence of 2a, and second, we also observed no receptor-mediated modulation of 1E(rbEII) expressed in
Xenopus oocytes in the absence of 2a. Thus, the presence
of 1E1-50 in 1Elong confers G-protein
sensitivity onto 1E(rbEII). The 1E clone has been suggested to be
the molecular counterpart of the resistant R-type calcium current in
cerebellar granule neurons, which makes up ~15-20% of the total
calcium current in these cells (Randall and Tsien, 1995); however, it
is not known whether R-type current shows G-protein modulation.
The 1B1-55 sequence contributes to G-protein
inhibition of 1B
Our initial studies have shown that transfer of a sequence
corresponding to 1B1-483 (representing the N terminus,
domain I, and the I-II loop of the 1B subunit) into 1E(rbEII)
conferred both slowing of activation kinetics and reduction in current
amplitude in response to either G overexpression or activation of
a G-protein-linked receptor (Stephens et al., 1998b), whereas a region
corresponding to the IS6/I-II loop of 1B conferred only partial
slowing of activation kinetics, with no modulation of current amplitude
(Page et al., 1997). The 1E(rbEII) N-terminal tail is 55 amino acids shorter than that of 1B, although the 40 amino acids that form the
1E(rbEII) N-terminal tail do have a highly (82%) conserved counterpart in 1B56-95 (Fig. 1). The present study
provides compelling evidence for the involvement of
1B1-55 in its G-protein modulation. Deletion of
1B1-55 (forming the 1BN1-55 construct) renders the 1B subunit, which exhibits the strongest degree of G-protein sensitivity of all the 1 subunits, completely refractory to receptor-mediated inhibition and to the direct effect of
G overexpression. For both 1E and 1B, the biophysical
properties of the truncated and N-terminal extended forms are very
similar, suggesting that the truncation does not produce global
structural changes. When the 1B1-55 sequence was
transferred to rbEII, the 1bEEEE construct showed slowed activation
kinetics and prepulse-induced facilitation in the presence of G
and receptor-mediated inhibition, but in these measures the G-protein
modulation was less than that shown by 1B itself. This suggests that
other elements of 1B are also important for its modulation. It is
also relevant to compare 1bEEEE with 1Elong,
which forms the backbone of the channel and was also less modulated
than 1B. In fact, 1bEEEE was inhibited to a slightly greater
extent than 1Elong in all parameters measured. Thus,
part of the basis for the greater intrinsic G-protein modulation of
1B than 1E is likely to be located within the first 55 amino
acids of the N terminus, and part is located elsewhere in the first
domain/I-II loop sequence of 1, because we have shown that the
1B-1E chimera containing 1B1-483 (to the end of
the I-II loop) is modulated by a similar extent as 1B itself
(Stephens et al., 1998b). Furthermore, 1Elong was not
further inhibited by quinpirole in the absence of exogenously expressed
subunits, whereas the difference in the extent of modulation
between 1A and 1B was attenuated in the absence of co-expressed
3 subunits (Roche and Treistman, 1998).
Having implicated the N-terminal domains of 1B and
1Elong in their G-protein modulation, it is of interest
to compare our results with those of a previous study of the
determinants of G-protein modulation that compared a series of chimeras
between 1B and 1A or 1C (Zhang et al., 1996). However, in this
paper, 1B and all the constructs containing the 1B first domain
were composed of approximately the first 70 amino acids of 1A
ligated onto a truncated 1B subunit, which was found to improve the
expression of rat 1B (Ellinor et al., 1994). In our study we report
receptor-mediated inhibition of 1B of ~50%, in line with most
other reported values (Bourinet et al., 1996; Currie and Fox, 1997),
all of which are higher than the inhibition of 1B (~20%) seen by
Zhang and co-workers (1996). Such an atypically small amount of
receptor-mediated inhibition of 1B might be explained by the
overexpression of G in their study, which will partially occlude
agonist effects (Herlitze et al., 1996; Ikeda, 1996). However, given
the role of 1B1-55, these differences may also be
attributable to the exchange of the 1B N-terminal sequence for that
of 1A, a subunit that has been widely reported to be more weakly
G-protein-modulated than 1B (Bourinet et al., 1996). Nevertheless, a
difference in modulation was still found between the 1B construct
used in their study and 1A, indicating that other regions in domain
I are of importance (Zhang et al., 1996).
Comparison of reinhibition kinetics of 1B
and 1Elong
Zhang et al. (1996) proposed that the weaker modulation of the
1A subunit relative to 1B is attributable to an increased rate of
dissociation of G from 1A than from 1B; however, differing results were obtained in another expression study (Roche and Treistman, 1998). Furthermore, when N and P/Q currents, which are their native counterparts, were compared in chromaffin cells, no difference in
reinhibition kinetics was observed (Currie and Fox, 1997). In the
present study, we found that although G-protein inhibition of
1Elong was significantly less than that of 1B, their
reinhibition kinetics were very similar. Thus, our findings may be more
consistent with intrinsic differences existing between these 1
subunits in terms of G efficacy. One important caveat is the
competitive role of accessory subunits, which have been shown to
differentially affect G-protein-1 subunit interactions (Roche and
Treistman, 1998). However, even in the absence of exogenous subunits, quinpirole inhibition of 1Elong remained
significantly less than that of 1B, although differential effects of
the endogenous oocyte 3 (Tareilus et al., 1997) cannot be
discounted.
Molecular mechanism of G-protein inhibition
Our findings implicating the N terminus of 1B and 1E
subunits in G-protein modulation prompt a reevaluation of the
composition of the G binding site. An unanswered question is
whether the N-terminal region comprises a G binding site or
whether it contributes an element to a multifaceted site, in which
high-affinity G binding occurs elsewhere, and the N-terminal
region contributes to the functional consequences of binding. Some
evidence against the former possibility comes from Qin et al. (1997),
who found no high-affinity binding of purified G subunits to a
fusion protein containing N-terminal amino acids 1-89 of human 1E,
which has a high degree of homology with the corresponding sequence of
rat brain 1Elong (Fig. 1). It is therefore unlikely,
although not impossible, that G binding would differ
significantly between such highly conserved sequences. G subunits
are capable of binding to the I-II loop of 1A, 1B, and 1E and
to the C terminus of 1E and possibly other 1 subunits; therefore,
it is likely that one (or both) of these elements contributes to a
multicomponent site. Recent evidence supports the hypothesis that
different elements may also contribute to VDCC 1- binding sites,
with the demonstration that some subunits (2a and 4) may bind
at two sites on the 1 subunit, one of high affinity (I-II loop) and
the other of much lower affinity (C-terminal tail) (Walker et al.,
1998). Any interaction between G or the VDCC subunit and the
1 N-terminal tail may be of a secondary, low-affinity nature, or the
N-terminal tail may be essential for subsequent inhibition of the
channel gating.
| |
FOOTNOTES |
Received Feb. 19, 1998; revised April 6, 1998; accepted April 10, 1998.
This work was supported by The Wellcome Trust and the European
Community (Marie Curie Fellowship to C.C.). We thank the following for
generous gifts of cDNAs: T. Snutch (University of British Columbia,
Vancouver, Canada), 1E(rbEII); H. Chin (National
Institutes of Health, Bethesda, MD), 2-; Y. Mori (Seriken,
Okazaki, Japan), 1B; E. Perez-Reyes (Loyola, New Orleans, LA),
2a; P. G. Strange (Reading, UK), rat D2 receptor; M. Simon
(CalTech, CA), G1 and G2; T. Hughes (Yale, New Haven, CT), mut-3
GFP; and Genetics Institute (CA), pMT2. We thank I. Tedder, M. Li, and
J. May for technical assistance, and J. Millar and A. G. Jones for
the cerebellar granule cells. This work benefited from the use of the
Seqnet facility (Daresbury, UK).
Correspondence should be addressed to Professor A. C. Dolphin,
Department of Pharmacology (Medawar Building), University College London, Gower Street, London WC1E 6BT, UK.
| |
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Top
Abstract
Introduction
