From the Interactions of G-protein A family of membrane-associated guanine nucleotide-binding
regulatory proteins
(G-proteins)1 is essential
for mediating signal transduction between cell-surface receptors and
intracellular effectors such as adenylate cyclase, phospholipase C,
phospholipase A2, and ion channels (1-4). G-proteins are
composed of three subunits termed High voltage-activated (HVA) Ca2+ channels are negatively
regulated by G-proteins in a membrane-delimited manner (2, 4). This
response is primarily mediated by pertussis toxin-sensitive G-proteins
(Go/Gi), in which Go To address these issues at the molecular level, we have functionally
expressed In Vitro Transcription
The 1.4-kb ApaI/ApaI and 6.8-kb
HindIII/HindIII fragment containing the entire
coding regions of DOR (17) and the Ca2+ channel
cRNAs specific for Construction of Mutant and Chimeric Ca2+ Channels
B3T B3TCD--
The 8.7-kb SrfI/SalI fragment
excised from pSPB3S was ligated with the 2.2-kb
ScaI/SalI fragment from pSPCDRS to obtain
pSPB3TCD. In this plasmid, the codon CGG for Arg-1911 of the
B3LCD and B3LCDT B3LCDTCD--
The 8.6-kb SrfI/SalI
fragment was excised from pSPB3LCD and ligated with the 2.2-kb
ScaI/SalI fragment from pSPCDRS to yield pSPB3LCDTCD.
CDT CDTB3--
The 5.2-kb HindIII/ScaI and the
3.0-kb HindIII/SalI fragments excised from
pSPCDRS were ligated with the 1.6-kb SrfI/SalI fragment from pSPB3 to obtain pSPCDTB3. In this plasmid, the segment encoding amino acid residues 1912-2339 of the CDLB3 and CDLB3TB3--
To delete an internal SacI
site, the plasmids pSPCDRS and pSPCDTB3 were partially digested with
SacI, blunted, and circularized to produce pSPCDRSS and
pSPCDTB3S. Another SacI site on pSPCDRSS was deleted by the
same procedure. The resulting plasmid and the plasmid pSPCDTB3S were
digested with SacI and StuI and blunted. The
9.5-kb SacI/StuI fragment from the former or the
8.9-kb SacI/StuI fragment from the latter was
ligated with the 890-bp XhoI/ApaI fragment that
was excised from pSPB3 and blunted with T4 DNA polymerase, in order to yield pSPCDLB3 or pSPCDLB3TB3. In these plasmids, the
segment encoding amino acid residues 242-537 of the B3L B3L B1T B1T CDB1--
The 640-bp BamHI/BstXI and
420-bp BstXI/XmnI fragments from pCARD3 (18), the
87-bp XmnI/HindIII fragment from pSPCBI-2 (20), and the HindIII/BamHI 2.4-kb fragment from pSP72
were ligated to yield pCB(Bm-Hd). The 1.5-kb
XhoI/BamHI fragment from pCARD3, the 1.1-kb
BamHI/HindIII fragment from pCB(Bm-Hd), and the
9.3-kb HindIII/SalI fragment from pSPCBI-2 were
ligated to yield pBC2. In the plasmid pBC2, the CDTB1--
The 7.2-kb XbaI/PflMI fragment
from pCARD3, the 3.1-kb SphI/XbaI fragment
from pSPCBI-2, and the annealed oligonucleotides CTGGATGAATACGTGCGGGTCTGGGCCGAGTACGACCCTGCTGCTTGGGGACGCATG and CGTCCCCAAGCAGCAGGGTCGTACTCGGCCCAGACCCGCACGTATTCATCCAGATG were ligated to yield pBC4. The plasmid pBC4 carries cDNA for the
Department of Neurochemistry and
¶¶ Department of Neurophysiology,
Department of Information Physiology,
Department of Molecular Genetics,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
(G
) and
subunits (G
) with N- (
1B) and P/Q-type
(
1A) Ca2+ channels were investigated using
the Xenopus oocyte expression system. Gi3
was found to inhibit both N- and P/Q-type channels by receptor
agonists, whereas G
1
2 was responsible for
prepulse facilitation of N-type channels. L-type channels
(
1C) were not regulated by G
or G
. For N-type,
prepulse facilitation mediated via G
was impaired when the
cytoplasmic I-II loop (loop 1) was deleted or replaced with the
1C loop 1. G
-mediated inhibitions were also impaired
by substitution of the
1C loop 1, but only when the C
terminus was deleted. For P/Q-type, by contrast, deletion of the C
terminus alone diminished G
-mediated inhibition. Moreover, a chimera
of L-type with the
1B loop 1 gained
G
-dependent facilitation, whereas an L-type chimera
with the N- or P/Q-type C terminus gained G
-mediated inhibition.
These findings provide evidence that loop 1 of N-type channels is a
regulatory site for G
and the C termini of P/Q- and N-types for
G
.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
,
, and
. The
subunit (G
) contains a binding site for guanine nucleotides and possesses GTPase activity. Upon receptor stimulation, heterotrimeric G-proteins disassociate into an
-GTP complex and
/
dimer. In most
systems, a GTP-bound G
activates or inhibits an effector system, and
the functional half-life is determined by the intrinsic GTPase activity of G
. Recently, it has been shown that the
dimer (G
) is significantly important in signal transduction as well (3).
has been
shown to inhibit current from HVA Ca2+ channels (5-7).
Additionally, it has been shown that G
also transduces an
inhibitory signal to HVA Ca2+ channels (8, 9). It remains
to be determined, however, which subunit arm of the G-protein complex
preferentially interacts with N- and P/Q-types of HVA Ca2+
channels. Recently, it has been determined that the intracellular loop
joining motif I and II (referred to as "loop 1" in the present study) is an interaction site on neuronal HVA Ca2+ channels
for G
(10-13). Nevertheless, mapping of region(s) on HVA
Ca2+ channels responsible for interactions with G
and/or
G
is still very incomplete (14).
1A,
1B, and
1C
of HVA Ca2+ channels in Xenopus oocytes. These
subunits were derived from rabbit brain N-type, P/Q-type, and cardiac
L-type Ca2+ channels, respectively. In addition, we have
co-expressed
-opioid receptor (DOR) together with G
or G
as
we did in determining a region of the muscarinic-gated K+
channel critical for activation by G
with the presumption that co-expression with G
or G
determines which kind of modulation takes place (15). In this paper, interactions of G
and G
with
Ca2+ channels were characterized using mutant and chimeric
N- (
1B) and P/Q-type (
1A)
Ca2+ channels. The results, together with evidence for a
direct binding provided by the companion paper (16), define the
interaction sites of Ca2+ channels for G
and
G
.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
1C subunit (18) were inserted into the
HindIII site of the pSPA2 vector (19), to yield pSPDOR and
pSPCDR, respectively. The plasmid pSPCDR was digested with
XbaI, blunted with T4 DNA polymerase, and ligated with a
SalI linker to yield pSPCDRS. The
1C subunit
cDNA was kindly provided by Drs. Atsushi Mikami and Tsutomu Tanabe.
The pSPA1, pSPA2, pSP72, pSP65, and pSP64 recombinant plasmids carrying
the entire protein-coding sequences of Gi3
, G
1, G
2, and Ca2+ channel
1B,
1A,
2, and
1a subunits were described previously (15, 18-21).
Nucleotide sequence analyses revealed that the deduced amino acid
sequence of Gi3
was the same as that reported (22) except that Ser-16, Asp-64, Ser-165, Glu-261, and Pro-282 were determined as Thr (ACG), Glu (GAA), Thr (ACC), Asp (GAC), and Ser
(TCA), respectively, in our clone, pG3
1 (15).
1A,
1B,
1C,
2, and
1a subunits of
the Ca2+ channel, 17 kinds of mutants and chimeric
1 subunits (see below), DOR, Gi3
,
G
1, and G
2 were synthesized in
vitro using a MEGAscript SP6 kit (Ambion).
1--
The plasmid pSPB3 carrying the entire
protein-coding sequences of
1B (21) was digested with
BamHI and circularized with T4 DNA ligase to
yield pSPB3BH. The 5.5-kb NotI/SrfI fragment excised from pSPB3BH was ligated with the 55-bp
NotI/BglI fragment from pSPB3 and the annealed
oligodeoxyribonucleotides, GGGCTGGCGGTCC and GGACCGCCAGCCCCGC. The
1.7-kb NotI/PflMI fragment from the resulting
plasmid was ligated with the 8.6-kb NotI/PflMI
fragment from pSPB3 to obtain pSPB3S. The plasmid pSPB3S was digested
with SrfI and SpeI, blunted with T4
DNA polymerase, and ligated with the annealed oligonucleotides,
GGGCTTAGCTGCGGAGAAGAGTTCTGAGACGTGCACCGGTT and
AACCGGTGCACGTCTCAGAACTCTTCTCCGCAGCTAAGCCC, to yield pSPB3T
1. In this plasmid, the codon TTC for Tyr-1913 was replaced with the codon
TAG for termination.
1B subunit was replaced with the codon CAC for (His),
and the segment encoding amino acid residues 1658-2171 of the
1C subunit was substituted for amino acid residues
1912-2339 of the
1B subunit.
1--
The plasmid pSPB3S was digested with
EcoRI, blunted, and circularized to delete the
EcoRI site. The resulting plasmid and the plasmid pSPB3T
1
were digested with BsmI, blunted, and ligated with the
EcoRI linker, dGGAATTCC, to produce pSPB3S.E. and
pSPB3T
1E. The plasmids pSPB3S.E. and pSPB3T
1E were digested with
PmlI and EcoRI, and the 9.3-kb
PmlI/EcoRI fragment from pSPB3S.E. or the 7.8-kb
PmlI/EcoRI fragment from pSPB3T
1E was ligated
with the 950-bp BamHI/EcoRI fragment from pSPCDR
and the annealed oligonucleotides, GTGGCCCTGGGTGTATTTTGTCAGTCTGGTCATCTTTG and
GATCCAAAGATGACCAGACTGACAAAATACACCCAGGGCCAC, to yield
pSPB3LCD or pSPB3LCDT
1. In these plasmids, the segment encoding amino acid residues 411-740 of the
1C subunit
was substituted for amino acid residues 332-668 of the
1B subunit.
1--
The plasmid pSPCDR was partially digested with
AvrII, blunted with T4 DNA polymerase, and
circularized with T4 DNA ligase to obtain pSPCDTD1. In this
plasmid, the codon AGG and CCC for Arg-1980 and Pro-1981 of the
1C subunit was replaced with the codon AGC (Ser) and TAG
for termination.
1B
subunit was substituted for amino acid residues of 1658-2171 of the
1C subunit, and the codon TAC for Tyr-1657 of the
1C subunit was replaced with the codon TGG (Trp).
1B
subunit were substituted for amino acid residues 318-610 of the
1C subunit, and the codon CTC for Leu-242 of the
1B subunit and CAG for Gln-611 of the
1C
subunit were replaced with the codon GTC for (Val) and GAG for (Glu),
respectively.
1--
The 9.6-kb PmlI/PflMI
fragment from pSPB3S was ligated with the 99-bp
PmlI/HhaI and 610-bp
KpnI/PflMI fragments excised from pSPB3 and the
annealed oligonucleotides, CGAGAGAGAGCTCAACGGGTAC and
CCGTTGAGCTCTCTCTCGCG, to yield pSPB3L
1. In this plasmid, the segment
encoding amino acid residues 366-383 of the
1B subunit were deleted.
2, B3L
3, and B3L
4--
The plasmid pSPB3 was
digested with SacI, blunted with T4 DNA
polymerase, and cleaved with NotI, PvuII,
XmnI, and/or PflMI. The 1.1-kb
NotI/SacI and 510-bp
PvuII/PflMI fragments, the 1.2-kb NotI/PvuII and 360-bp
XmnI/PflMI fragments, and the 1.1-kb
NotI/SacI and 360-bp
XmnI/PflMI fragments were ligated with the 8.6-kb
NotI/PflMI fragment from pSPB3S to produce
pSPB3L
2, pSPB3L
3, and pSPB3L
4, respectively. In the plasmid
pSPB3L
2, the segment encoding amino acid residues 384-420 of the
1B subunit was deleted. In the plasmid pSPB3L
3, the
segment encoding amino acid residues 421-470 of the
1B
subunit was deleted, and the codon ATG for Met-471 was replaced with
the codon GTG for (Val). In the plasmid pSPB3L
4, the segment
encoding amino acid residues 384-470 of the
1B subunit was deleted, and the codon ATG for Met-471 was replaced with the codon
GTG for (Val).
1--
pSPCBI-1 (20) was digested with HindIII
or SphI, blunted with T4 DNA polymerase, and digested with
SalI. The resulting 5.6-kb SalI/SphI
(blunted) and 3.6-kb HindIII (blunted)/SalI
fragments were ligated to yield pSPBIC
1 (originally pSPCBI
SH-1).
In the plasmid pSPBIC
1, the segment encoding amino acid residues
1856-2273 of the
1A (BI-1
1) subunit was
deleted, and the amino acid residues AFRLRAAERGR were attached.
2--
Oligonucleotides
GATCTATGCCGCCATGATGATCATGGAGTACTAC,
CGGCAGAGCAAAGCCAAAAAGCTGCAGGCCATGCGCGAGGAG,
CAGAACCGGACACCGCTCATGTTCCAGCGCATGGAGCCCCCG, and
CCGGATGAGGGGGGCGCCGGCCAGAACGCCCTGCCCTAGCGC were annealed with GGCCGCGCTAGGGCAGGGCGTTCTGGCCGGCGCCCCCCTC,
ATCCGGCGGGGGCTCCATGCGCTGGAACATGAGCGGTGTCCG, GTTCTGCTCCTCGCGCATGGCCTGCAGCTTTTTGGCTTTGCT, and
CTGCCGGTAGTACTCCATGATCATCATGGCGGCATA, respectively, ligated, and
cleaved with BglII and NotI. The resulting 170-bp
BglII/NotI fragment was ligated with the 5.8-kb
XbaI/NheI, 2.6-kb
NheI/BglII, and 1.1-kb
NotI/XbaI fragment from pSPCBI-1 to yield
pSPBIC
4. The plasmid carries cDNA encoding the
1A
(BI-1
1) subunit with a deletion of the C-terminal amino
acid residues 2015-2273.
1A (BI-2
1) subunit cDNA has a substitution of the nucleotide
sequence encoding residues 1-777 of the
1C subunit for
the sequence encoding the amino acid residues 1-707.
1C subunit which has the C-terminal tail residues
1524-2127 replaced with the tail residues 1838-2424 of the
1A (BI-2
1) subunit.
Functional Expression of Wild-type, Mutant, and Chimeric Ca2+ Channels in Xenopus Oocytes
After removal of the follicular cell layer (15),
Xenopus oocytes were injected either with 0.3 µg/µl
1 (
1B,
1A,
1C, mutant
1, or chimeric
1) cRNA in combination with 0.2 µg/µl
2 cRNA and 0.1 µg/µl
1a cRNA; 0.03 µg/µl DOR cRNA; 0.05 µg/µl Gi3
cRNA, or 0.05 µg/µl G
1 cRNA, and 0.025 µg/µl G
2
cRNA, unless otherwise specified. The average volume of injection was
~50 nl per oocyte. The injected oocytes were incubated for 3-5 days
and then subjected to electrophysiological measurements at 21 ± 2 °C.
In order to unmask the effect of endogenous G (16), a
deoxyoligonucleotide 20-mer (AGO) of the following sequence was used in
antisense experiments, CATGACTGCTCGGGGGGGGA. The AGO antisense oligonucleotide is complementary to nucleotides (
17 to 3) of the
Xenopus Go
mRNA (23). The endogenous
Xenopus Go
nucleotide sequence shows 40%
identity with the corresponding nucleotide sequence of
Go
cRNA injected. This antisense oligonucleotide (0.1 µg/µl, 50 nl) was injected 12-16 h prior to electrophysiological measurements.
The oocytes were positioned in a recording chamber (1.0 ml in volume)
and were perfused with a Ba2+ solution containing 40 mM Ba2+, 50 mM Na+, 2 mM K+, and 5 mM HEPES (pH 7.5 with
methanesulfonic acid). Membrane currents through the expressed
Ca2+ channels were measured with the two-microelectrode
voltage-clamp method as described previously (15). Also, the membrane
potential recorded by the potential electrode was monitored. The
membrane was held at 80 or
100 mV, and step depolarizations were
applied to activate the Ca2+ channels. Microelectrodes were
filled with 3 M KCl, and those showing resistances of
0.5-1.5 megohms were used.
We noticed slow tail currents upon repolarization as shown in Fig. 1. In these cases, the time resolution of clamping was within 4 ms and the potential error was within 3% of the command pulse, indicating no serious space-clamping problems in characterizing Ca2+ channel currents.
Unless otherwise stated, statistical data were represented by the mean and S.E.
![]() |
RESULTS |
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Functional Expression of the N-, P/Q-, and L-type Ca2+
Channels in Xenopus Oocytes--
To establish a recombinant expression
system, where current inhibition mediated by G-proteins can be
reconstituted individually, HVA N-, P/Q-, and L-type Ca2+
channels were co-expressed in Xenopus oocytes by injection
of cRNAs for three (1,
2, and
1) Ca2+ channel subunits and the
-opioid
receptor (DOR). Their responses to the opioid peptide, Leu-enkephalin
(Leu-EK), were examined by the two-microelectrode voltage-clamp
technique.
|
Effects of G and G
on the N- and P/Q-type Ca2+
Channels--
To determine which arm of the G-protein complex
contributes to regulation of N- and P/Q-type Ca2+ channels,
either Gi3
cRNA or G
1 plus
G
2 cRNAs were injected into oocytes in combination with
Ca2+ channel
1 (
1B or
1A),
2, and
1a subunits
cRNAs and DOR cRNA.
|
|
|
Combination of 1B,
2, and
1a Subunits Is Required for the Inhibitory Regulations
by Gi3
and G
--
The
1 subunit of
the Ca2+ channel forms the channel pore (4). As a result of
this, N-type Ca2+ channel currents were not detectable
without the injection of
1B subunit cRNA
(n = 13). However, when the
1B subunit
was expressed without the
2 and
1a
subunits, Leu-EK still produced channel inhibition and slowing of the
1B currents via Gi3
(n = 8). Moreover, the opioid-induced inhibition of
1B
currents was larger in the absence of
1a subunit
(n = 15) and did not change without the
2 subunit (n = 8) (27, 28). In addition,
the prepulse facilitation of
1B currents mediated via
G
1
2 (see Fig. 3) was also present without
the
2 and
1a subunits (n = 5). These results suggest that both G
and G
can interact
with the
1 subunit regardless of subunit composition and
are able to produce channel modulation.
Interaction of G and G
with Mutant and Chimeric
1B Channels--
By aiming at identifying the regions
on the
1B channel interacting with Gi3
and G
1
2, chimeric channels between
1B (Fig. 2A, B3) and
1C (Fig. 2A, CD) were constructed.
These constructs were generated (Fig. 2), taking advantage of the
inability of
1C channels to be inhibited by G-proteins
(Figs. 1F, 2A, and 3A). Neither a
deletion of the C-terminal region of
1B (amino acid
residues 1913-2339, see Fig. 5) nor a
replacement of the C-terminal region of
1B (amino acid
residues 1912-2339) by that of
1C (amino acid residues
1658-2171) affected the Leu-EK-induced inhibition of Ca2+
channels in oocytes co-expressed with Gi3
and
G
1
2 (Fig. 2, A and
B; B3T
1 and B3TCD, respectively).
Moreover, currents through the chimeric
1B channel,
B3LCD, in which a region of
1C (amino acid residues
411-740) containing the intracellular loop joining motif I and II
(loop 1) was substituted for that of
1B (amino acid
residues 332-668), were inhibited by Leu-EK in oocytes co-expressed with G
or G
(Fig. 2, A and B,
B3LCD). However, a deletion of the C-terminal region of
B3LCD (corresponding to amino acid residues 1913-2339 of
1B) produced a chimeric channel, B3LCDT
1, which was
insensitive to Leu-EK (Fig. 2, A and B,
B3LCDT
1).
|
Effects of Prepulse on the Inhibitions of Mutant and Chimeric
N-type Ca2+ Channels via Gi3 or
G
1
2--
The experiments described
above, in which the wild-type
1B and
1A
channels were used, demonstrated that G
plays a significant role in
G-protein-mediated inhibition of neuronal Ca2+ channels.
However, there is a possibility that G
exerts its effect indirectly
upon Ca2+ channels through G
. To exclude this
possibility, it was necessary to investigate further molecularly and
structurally the dependence of G
on G
when interacting with
Ca2+ channel
1 subunits.
Interaction Site on the P/Q-type Ca2+ Channel for
G-protein--
In order to determine the interaction site on the
P/Q-type Ca2+ channel for G and G
, procedures
similar to those for
1B channels (Figs. 2 and 3) were
applied to
1A channels (Fig. 4). In oocytes co-expressed
with Gi3
or G
1
2 together
with DOR and Ca2+ channel (
1,
2 and
1a subunits), deletion of the C
terminus of
1A (amino acid residues 2015-2273) reduced
the sensitivity to Leu-EK (Fig. 4A, B1T
2) as
compared with the wild-type
1A (B1). This
stands clearly in contrast to the mutant
1B channel (B3T
1), in which deletion of the C terminus alone did not influence the sensitivity to Leu-EK (Fig. 2A). In addition, the
chimeric
1C channel (CDTB1), in which the C terminus of
1C (amino acid residues 1524-2127) was replaced by that
of
1A (amino acid residues 1838-2424), acquired
sensitivity to Leu-EK (Fig. 4A, CDTB1), whereas the wild-type
1C channel was not affected by Leu-EK
(Figs. 2A and 3A). Another chimeric
1C/
1A channel (CDB1), in which the N
terminus of
1C (amino acid residues 1-777) substituted
for that of
1A (amino acid residues 1-707), still
exerted sensitivities to Leu-EK (Fig. 4A, CDB1).
As shown in Fig. 4B, currents through the B1T
2 and CDTB1
channels were comparable to those through the wild-type
1A and
1C channels (Fig. 1), and the
CDTB1 currents were blocked by nifedipine.
![]() |
DISCUSSION |
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---|
In the present study, the -CTx-sensitive N-type
(
1B) and
-Aga-sensitive P/Q-type (
1A)
Ca2+ channels were functionally expressed in
Xenopus oocytes, an in vivo expression system. As
described, we found that Gi3
co-expressed in oocytes
mediated receptor agonist-induced inhibition of N-type
1B and P/Q-type
1A channels. On the other
hand, a depolarizing prepulse relieved current inhibition caused by the
G
1
2 complex, and the facilitatory effects
were more pronounced in
1B than in
1A.
Because responsiveness of the
1B and
1A
channels to the inhibition mediated by Gi3
and
G
1
2 was maintained even in the absence of
the Ca2+ channel auxiliary subunits
2 and
1, the
1 subunit should bear the
interaction sites for both the G
subunit and the G
dimer. Finally, we defined loop 1 of
1B as an interaction site
for G
and the C termini of
1B and
1A for G
, based on the responses of mutant and
chimeric channels to G
and G
.
The Native Type 1B,
1A, and
1C Channels Expressed in Xenopus Oocytes--
The
electrophysiological and pharmacological properties of the
1B,
1A, and
1C channels
determined were identical to those of the N-, P/Q-, and L-type
Ca2+ channels described previously (18, 20, 33). This
indicates that
1B (N-type),
1A
(P/Q-type), and
1C (L-type) Ca2+ channels
were functionally expressed with the Ca2+ channel
2 and
1 subunits in Xenopus
oocytes. When DOR was further co-expressed,
1B and
1A channel currents, but not
1C channel currents, were inhibited within seconds when stimulated by Leu-EK. It
is likely that agonist-induced inhibitions of
1B and
1A channels are mediated by endogenous oocyte G-proteins
that are coupled to the receptor, because the inhibitions of
1B and
1A channels were reduced when the
antisense oligonucleotide AGO against Xenopus Go
was injected (16).
The Loop 1 of 1B Channel as an Interaction Site for
G
--
When G
1
2 was co-expressed,
the Leu-EK-induced inhibition was not potentiated in either
1B or
1A channels. In the case of N-type
1B, however, a depolarizing prepulse to +80 mV
facilitated the currents in the absence of the receptor agonist,
suggesting that the exogenous G
inhibits the
1B
channel by itself (8, 9). Thus, the difference between the current
traces before and after the prepulse should correspond to an
1B current component that is mainly inhibited by
exogenous G
1
2.
The C Termini of 1B and
1A Channels
as an Interaction Site for G
--
Receptor stimulation by agonist
is known to catalyze activation of G
and lead to dissociation of the
G
heterotrimer (1). In fact, application of a prepulse did not
facilitate N-type
1B channels when co-expressed with
Gi3
and DOR, unless DOR was stimulated by Leu-EK. The
potentiating action of Gi3
on the agonist-induced inhibition of
1B channels was abolished by application
of a large conditioning prepulse. This suggests that exogenous G
,
unlike G
, does not influence the
1B channel by
itself and stays in its inactive form. It appears, therefore, that
potentiation of agonist-induced inhibition via exogenous G
results
from the interaction of the channel with activated exogenous G
and,
probably, with an endogenous G
dissociated from the G
. This
idea is further evidenced by the observation for mutant (B3T
1) and
chimera (B3TCD)
1B channels, in which loss of the C
terminus (a possible interaction site for Gi3
, see
below) did not affect the potentiation of inhibition via
Gi3
. However, a further loss of the loop 1, an
interaction site for G
1
2, of the mutant
B3T
1
1B channel (B3LCDT
1) eliminated the
potentiation of inhibition via Gi3
as well as the
prepulse facilitation via G
1
2. Moreover,
an
1B channel chimerized with the
1C loop
1 (B3LCD), in which the interaction site exclusively for
G
1
2 was lost, retained the potentiation
of inhibition via Gi3
but no longer the prepulse
facilitation via G
1
2. These results
indicate that there are two distinct interaction sites, namely loop 1 and the C terminus, for G
and G
on N-type Ca2+
channels and that the two sites regulate the channel activity independently when they receive inhibitory signals from G
and G
. This independence of loop 1 and the C terminus in the
1B channel modulation is supported by the observation
that the Gi3
-dependent potentiation of
inhibition was not affected by single application of the loop 1 peptide
(PL1) or a C-terminal peptide (PB3T4) inside the oocyte but abolished
by simultaneous application of both of them (16).
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ACKNOWLEDGEMENTS |
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We are grateful to Drs. Atsushi Mikami and
Tsutomu Tanabe for providing us with 1C cDNA. We
also thank Dr. Mark Strobeck for the critical reading of the
manuscript.
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FOOTNOTES |
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* This investigation was supported in part by Ministry of Education, Science and Culture of Japan Research Grants 08770519 (to T. F.), 02557013, 06264101, 08680855 (to T. N.), and 04807013 (to M. Y.) and by National Institutes of Health Grant P01 HL22619-20 (to Y. M. and M. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Neurochemistry, Tokyo Institute of Psychiatry, 2-1-8 Kamikitazawa, Setagaya-ku, Tokyo 156, Japan. Tel.: 81-3-3304-5701; Fax: 81-3-3329-8035.
1
The abbreviations used are: G-proteins, guanine
nucleotide-binding regulatory proteins; G, G-protein
subunit;
G
, G-protein
subunit; HVA, high voltage-activated; loop 1, intracellular loop joining the segments I and II; DOR,
-opioid
receptor; Leu-EK, Leu-enkephalin;
-CTx,
-conotoxin GVIA;
-Aga,
-agatoxin IVA; DHP, dihydropyridine; bp, base pair; kb, kilobase
pair.
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REFERENCES |
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