Supplemental Material can be found at:
http://www.jbc.org/cgi/content/full/M306645200/DC1
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2003 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 278, No. 49, Issue of December 5, pp. 49386 –49400, 2003
Printed in U.S.A.
Competitive and Synergistic Interactions of G Protein ␤2 and Ca2ⴙ
Channel ␤1b Subunits with CaV2.1 Channels, Revealed by
Mammalian Two-hybrid and Fluorescence Resonance Energy
S
Transfer Measurements*□
Received for publication, June 23, 2003, and in revised form, September 22, 2003
Published, JBC Papers in Press, September 24, 2003, DOI 10.1074/jbc.M306645200
Alexander Hümmer, Oliver Delzeith, Shannon R. Gomez, Rosa L. Moreno, Melanie D. Mark,
and Stefan Herlitze‡
From the Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4975
Voltage-gated Ca2⫹ channels of the N-, P/Q-, and R-type are
inhibited by G protein-coupled receptors. The voltage-dependent inhibition is mediated by G protein ␤␥ subunits (1, 2) but is
also induced by coexpression of the G protein ␤ subunit alone
(1). Ca2⫹ channels consist of at least three subunits: the poreforming ␣1 and several auxiliary subunits such as the intracellulary located ␤ subunit and the transmembrane subunit ␣2␦.
The ␣1 subunit consists of four channel domains, which are
connected via intracellular peptide loops (for review see Refs.
* This work was supported by National Institutes of Health Grant
R01 NS42623-01A1 (to S. H.). The costs of publication of this article
were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
□
S The on-line version of this article (available at http://www.jbc.org)
contains 2 tables.
‡ To whom correspondence should be addressed: Dept. of Neurosciences, Case Western Reserve University, School of Medicine, Rm.
E604, 10900 Euclid Ave., Cleveland, OH 44106-4975. Tel.: 216-3681804; Fax: 216-368-4650; E-mail: [email protected].
3–5). G protein ␤␥ subunits modulate the channel via interaction with the intracellular peptide domain (loop I–II) and the C
terminus of the ␣1 subunit (6 –12). Interestingly, the G␤␥ subunit-binding sites overlap with the Ca2⫹ channel ␤ subunitbinding sites on the ␣1 channel subunit (5, 7, 9, 10). In addition
to the overlapping binding sites, G protein ␤␥ and Ca2⫹ channel ␤ subunits induce antagonistic effects on defined biophysical properties of the channel. For example, Ca2⫹ channel ␤
subunits (with the exception of ␤2 subunits) shift the voltage
dependence of activation to more hyperpolarized potentials,
whereas G␤␥ subunits have the opposite effects, i.e. a depolarizing shift (e.g. Refs. 13–18). The overlapping binding sites on
the channel as well as their antagonistic effects on the voltage
dependence of channel activation suggest that Ca2⫹ channel ␤
and G protein ␤␥ subunits may compete for binding sites on the
␣1 subunits during G protein modulation. Early biophysical
analysis of the Ca2⫹ channel G protein modulation suggested
that Ca2⫹ channels are stabilized in a certain conformational
state during G protein modulation from which channel opening
is more difficult to achieve (19). According to this model, G
protein ␤␥ subunits may induce and stabilize this reluctant
state of the channel (20 –24). In addition, one splice variant of
the N-type channel mimics a G protein-modulated channel
in the absence of activated G proteins, supporting the idea that
the G protein binding to the channel induces and stabilizes an
intrinsic state of the Ca2⫹ channel (25).
Green fluorescent protein (GFP)1 has become an important
fluorescent tag to study the localization, targeting, and interaction of proteins (26). Visualization of GFP does not require
any cofactor or enzymatic reaction and is therefore suitable as
a reporter gene for an immediate read out in a two-hybrid
interaction assay, for example. Because of the existence of
several spectrally distinguishable variants of GFP, two reporter genes can be used to record and compare the expression
and interaction of two independent proteins. Dual-color imaging and fluorescence resonance energy transfers (FRET) were
performed in various studies with promising results by using
CFP and YFP as fluorescent pairs (27–30).
By using a modified mammalian two-hybrid system (MTH)
and FRET, we asked how Ca2⫹ channel ␤ and G protein ␤
subunits interact at the binding sites on the ␣1 subunit and
1
The abbreviations used are: GFP, green fluorescent protein; aa,
amino acid(s); AD, activation domain; AID, ␣1 interaction domain; BD,
binding domain; CFP, cyan fluorescent protein; FRET, fluorescence
resonance energy transfer; HEK, human embryonic kidney; MLR, multilinear regression; MTH, mammalian two-hybrid system; NLS, nuclear
localization signal; YFP, yellow fluorescent protein; OK, opossum kidney; GTP␥S, guanosine 5⬘-3-O-(thio)triphosphate.
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This paper is available on line at http://www.jbc.org
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Presynaptic Ca2ⴙ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein ␤␥ subunits modulate the binding
of the Ca2ⴙ channel ␤ subunit on the Ca2ⴙ channel complex and induce a conformational state from which
channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca2ⴙ channel ␤ and G
protein ␤ subunits on the two separate binding sites, i.e.
the loopI–II and the C terminus, and on the full-length
P/Q-type ␣12.1 subunit by using a modified mammalian
two-hybrid system and fluorescence resonance energy
transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the
Ca2ⴙ channel ␤1b subunit induces a strong fluorescent
signal when interacting with the loopI–II but not with
the C terminus. In contrast, the G protein ␤ subunit
induces FRET signals on both the C terminus and loopI–
II. Analysis of the interactions on the full-length channel
indicates that Ca2ⴙ channel ␤1b and G protein ␤ subunits
bind to the ␣1 subunit at the same time. Coexpression of
the G protein increases the FRET signal between ␣1/␤1b
FRET pairs but not for ␣1/␤1b FRET pairs where the C
terminus was deleted from the ␣1 subunit. The results
suggest that the G protein alters the orientation and/or
association between the Ca2ⴙ channel ␤ and ␣12.1 subunits, which involves the C terminus of the ␣1 subunit
and may corresponds to a new conformational state of
the channel.
G Protein Modulation of P/Q-type Ca2⫹ Channels
whether the G protein induces a new conformational state of
the channel. Our results suggest that Ca2⫹ channel ␤1b and G
protein ␤ subunits differentially interact with the two isolated
binding sites of the ␣1 subunit (i.e. loopI–II and C terminus).
On the functional full-length channel the Ca2⫹ channel ␤1b
subunits may interact more strongly than G protein ␤ subunits,
because the FRET signals were larger for ␣1/␤1b FRET pairs.
Interestingly, when G protein ␤ subunits were coexpressed
with Ca2⫹ channel ␣1 and ␤ subunits, there was an increase in
the fluorescence signal between the Ca2⫹ channel subunits.
This increase in FRET was abolished when the C terminus was
deleted from the ␣1 subunit or overexpressed in the untagged
form. The results suggest that the G protein induces an altered
conformational state of the P/Q-type channel, which probably
involves the binding of the G protein to the C terminus of the ␣1
subunit.
Construction of DNA-BD and AD Fusion Proteins and pHASH-3—
The following cloning and reporter vectors of the mammalian Matchmaker two-hybrid assay kit (Clontech) were used: pM as cloning vector
to construct the GAL4 DNA-BD fusion constructs; pVP16 as cloning
vector for AD fusion constructs; pM3-VP16, pM-53, and pVP16-T as
positive control vectors; pVP16 and pM without insert as negative
control vectors. G␤2, G␥3, and the Ca2⫹ channel constructs ␤1b, ␣12.1loop I–II, and ␣11.2-loop I–II were amplified by a single PCR, and
constructs were subcloned in-frame into pM and pVP16. All amplified
products were verified by sequencing. For construction of pHASH-3,
five consensus GAL4-binding sites (UASG17-mer (x5)) and an adenovirus E1b minimal promoter were amplified by a single PCR and were
subcloned into pEYFP-C1 (Clontech). This vector was called pHASH-1.
Then two nuclear localization signals were added 3⬘ in-frame into the
YFP gene. This vector was called pHASH-2. For creating pHASH-3, two
nuclear localization signals were also added 3⬘ to the CFP gene in
pECFP (pECFP-NLS). The inducible YFP gene was cut out from
pHASH2 using BspTI and subsequently subcloned into the BspTI site of
pECFP-NLS. The 5⬘ BspTI site was introduced into pHASH-2 with the
inducible promoter via PCR.
Cell Culture and Immunohistochemistry—Opossum kidney (OK),
Chinese hamster ovary, and human embryonic kidney (HEK) 293 cells
were transfected with the indicated DNAs in each set of experiments
with EffecteneTM Transfection Reagent (Qiagen GmbH, Germany). 0.25
␮g of each DNA in combination with 0.5 ␮g of the reporter plasmids
(pHASH-3) were used for transfection. Equal amounts of total DNA
within one set of experiments were used by adding unrelated DNA
(plasmid pBF-1) to the transfection mixture when necessary. For example in Fig. 1 a maximum of 5 different DNAs were transfected in a
2:1:1:1:1 (with pHASH-3 added at twice the molar ratio than the other
DNAs). When only 3 or 4 different DNAs were transfected, the missing
DNA was replaced by pBF-1 DNA at the same ratio.
Western blot analysis of transfected OK cells were performed with a
polyclonal anti-Gal4 binding domain antibody and a monoclonal antiVP-16 activation domain antibody (Santa Cruz Biotechnology, Santa
Cruz, CA) according to standard procedures as described by Mark et al.
(31).
Immunocytochemistry and Quantification of YFP and CFP Signals—
Cells were embedded in Fluoromount (133 mM Tris/HCl, 30% glycerol;
11% Mowiol, 2% diazabicyclo[2.2.2]octane). Fluorescence was detected
with a conventional fluorescence microscope (Axioskop; Carl Zeiss,
Oberkochen, Germany). For CFP and YFP detection, the following filter
sets were used: CFP, excitation, short-pass D436/10; beamsplitter
460DCLP and emission, bandpass filter 480/30; YFP, excitation, shortpass HQ 500/20; beamsplitter Q515LP and emission, bandpass filter
535/30. All filters were obtained from AHF Analysentechnik AG,
Germany.
Intensity ratios between nuclear YFP and CFP fluorescence were
calculated by dividing the mean intensity values for YFP by the mean
intensity values for CFP. Mean intensity values of YFP and CFP fluorescence were calculated by subtracting the intensity values measured
from the extracellular background from the intensity values measured
from the fluorescence in the nucleus of the individual cells. Intensity
values are defined as the sum of the gray scale values for all pixels
contained in a defined object area. For every fluorophore in each set of
experiments the optimal exposure time for the YFP and the CFP fluo-
rescence signals was determined for the strongest signal of the positive
control (i.e. pM53/pVP16-T/pHASH-1–3). Fluorescence intensities were
compared with the fluorescence signal at the defined exposition time. In
addition, CFP fluorescence background after YFP excitation was measured by expressing pHASH-3 alone, and values were subtracted from
all experiments performed for the same transfection. Images were captured with a CCD camera (RTE/CCD-1300-Y/HS Princeton Instruments; Tucson, AZ), and pictures were analyzed with MetaMorph 4.01
(Visitron Systems GmbH, Puchheim, Germany). All experiments described were performed at least in triplicate, and data were presented
as means ⫾ S.E. Colors used for YFP, CFP, and DAPI in Fig. 1 are
computer-generated colors (Adobe Photoshop 5.5).
Fluorescence Resonance Energy Transfer
Constructs—Constructs ␣12.1-loopI–II (residues 369 – 418), ␣12.1loopI–II-Y/S, ␣11.2-loopI–II (residues 406 –520), ␣12.1-C terminus (residues 1766 –2212), ␣12.1 full-length, ␣12.1 full-length-⌬C terminus (residues 1–1857), ␤1b, ␤4, G␤2, G␥3 were either PCR-amplified or if
restriction sites were suitable cloned into either pECFP-C1, pEYFP-C1
(Clontech), pECFP-C2, and pEYFP-C2 (derived from pEGFP-C2) or as
non-tagged versions into pcDNA1, -3, or pcDNA3.1. All PCR-amplified
products were verified by sequencing.
FRET Measurements—For the calculation of FRET values and
FRET-derived values, a two-step approach was used, which is based on
the formalism and procedures of Erickson et al. (32). In the first step,
the constants RD1 and RA1 were determined by a multilinear regression (MLR) of the type FRETfl ⫽ ␣ ⫻ CFPfl ⫹ ␤ ⫻ YFPfl ⫹ ␥ [MLR]
(where fl indicates fluorescence). A simple manipulation of (FRET ⫽
(FRETfl ⫺ RD1 ⫻ CFPfl)/(RA1 ⫻ (YFPfl ⫺ RD2 ⫻ CFPfl))) yields the
relations RD1 ⫽ ␣ ⫹ FRET ⫻ RA1 ⫻ RD2 and RA1 ⫽ ␤/FRET. Because
the term FRET ⫻ RA1 ⫻ RD2 turns out to be exceedingly small in
comparison to RD1 (see Table in the Supplemental Material), the constant RD1 was estimated by ␣ to a good degree of approximation. This
regression method has the advantage of producing the results completely independent of any additive adjustments of the basic input data
usually necessary because of background variation. Furthermore, the
correlation coefficient r of [MLR] can be calculated. In case r is close to
1 or ⫺1, it indicates the appropriateness of a linear relation between the
variables, as predicted by the theory given in Erickson et al. (32).
Considering the data of cells expressing donor (X-CFP) only, the
constant RD1 was set to be ␣ in [MLR]. By using the data of the cells
expressing acceptor protein (X-YFP) only, RA1 was determined as ␤ of
the [MLR], because in this case the FRET ratio (FRET) is equal to 1 by
definition (FRET ⫽ (FRETfl (from FRET pair) ⫹ FRETfl (from YFP))/
FRETfl (from YFP)) (32).
In the second step the constants ⌬FRmax and FRETmax were calculated by an ordinary linear fit of the data of cells expressing both donor
(X-CFP) and acceptor protein (X-YFP). As suggested by equation
FRET ⫽ ⌬FRmax ⫻ Ab ⫹ 1 (32), the data were linearly fitted according
to the free type FRET ⫽ m ⫻ Ab ⫹ c (y ⫽ mx ⫹ q). The predicted value
FRET of the FRET ratio was given according to FRET ⫽ (FRETfl ⫺
RD1 ⫻ CFPfl)/(RA1 ⫻ (YFPfl ⫺ RD2 ⫻ CFPfl)) (see above) (32) using the
original data and the R constants calculated in the first step. The
percentage Ab of bound acceptors was calculated according to Ab ⫽
(CFPest ⫹ YFPest ⫹ Kd(Eff) ⫺ ((CFPest ⫹ YFPest ⫹ Kd(EFF))2 ⫺ 4 ⫻ CFPest ⫻
YFPest)1/2]/(2 ⫻ YFPest) [A34] (with CFPest ⫽ CFPfl/M_D; YFPest ⫽
YFPfl/M_A; M_A and M_D set as in Erickson et al. (32), KD(EFF) set 0),
where Eff indicates efficiency and est indicates estimate. The quality of
the linear fit was measured by the correlation coefficient r shown in the
figures.
Throughout the experiments statistical significance (p) was determined with a two-tailed Student’s t test with p ⬍ 0.05 (*) and p ⬍ 0.01
(**). Standard errors are the mean ⫾ S.E.
Electrophysiology—CFP- and YFP-tagged Ca2⫹ channel subunits
(␣12.1, ␤1b) and the G protein subunit (G␤2) were coexpressed in tsA201
cells, and Ca2⫹ channel-mediated Ba2⫹ currents were measured and
analyzed as described previously (1, 33, 34).
RESULTS
A Modified Mammalian Two-hybrid System to Detect Protein-Protein Interactions—The yeast two-hybrid system is
based on the finding by Fields and Song (35) that eukaryotic
trans-acting transcription factors like GAL4 can be divided into
two physically separated but still functional independent domains. Both domains are normally part of a nuclear protein,
which binds to a specific activation sequence of the target genes
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EXPERIMENTAL PROCEDURES
Mammalian Two-hybrid System
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G Protein Modulation of P/Q-type Ca2⫹ Channels
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FIG. 1. A YFP/CFP-based mammalian two-hybrid system to detect protein-protein interactions. a, schematic representation of the
YFP/CFP-based mammalian two-hybrid system. Upper, transfection of the vectors pM (expressing the interacting protein X fused to the GAL4
DNA-BD), pVP16 (expressing the interacting protein Y fused to the VP16-AD), and the reporter plasmid pHASH-3 (encoding for constitutively
expressed CFP and induced YFP) are cotransfected into a mammalian cell line. Middle, if protein X interacts with protein Y, transcription of YFP
is induced via the GAL4 DNA-BD and VP16-AD. The YFP gene carries two nuclear localization signals at the 3⬘ end and is cloned 3⬘ to four
GAL4-binding sites and an adenovirus E1b minimal promoter. CFP is constitutively expressed and is under cytomegalovirus promoter control. The
CFP gene also carries two nuclear localization signals (NLS) at the 3⬘ end. Both YFP and CFP genes are located on one vector (pHASH-3). Lower,
interaction of protein X with protein Y leads to the induction of YFP expression and targeting of YFP to the nucleus. Because CFP is constitutively
expressed and also localized to the nucleus, CFP fluorescence can be compared with the YFP fluorescence. The intensity values for CFP and YFP
fluorescence were determined by imaging techniques in combination with analysis programs. Intensity values were defined as the sum of the gray
scale values for all pixels in a defined object area. The intensity values from the nucleus were subtracted from the background intensity values,
and the ratio between the corrected intensity values between YFP and CFP was determined. b– d, time dependence of YFP/CFP ratios. OK cells
G Protein Modulation of P/Q-type Ca2⫹ Channels
incubation time after transfection. YFP fluorescence was first
detected 12 h after transfection in 20 –50% of constitutively
CFP-expressing cells for the positive control constructs, i.e.
pM3-VP16 (fused GAL-4-DNA-BD and VP16-AD), interacting
p53 protein/SV40 large T-antigen (pM53/pVP16-T), and G␤2/
G␥3 interaction (Fig. 1, b– e). The relative cell number in which
YFP fluorescence was detected in CFP-positive cells increased
to 75–100% 24 h after transfection (Fig. 1b). In contrast, for the
negative controls (non-interacting pairs pM/pVP16, pM53/
pVP16, and pHASH-3 alone), YFP fluorescence was only detected after 18 h in 15% of the CFP-positive cells, and the cell
number with YFP fluorescence further increased to a saturating level after 36 – 48 h of expression (Fig. 1c). Forty-eight h
after transfection 60 –70% of the CFP-positive cells revealed
YFP fluorescence due to the leakage of the GAL4/E1b promoter
(Fig. 1c). As shown in Fig. 1d, the optimal signal to noise ratio
for the detection of YFP fluorescence and quantification of
protein interactions occurred after 18 –24 h of expression for
OK cells. To demonstrate further that the signal to noise ratio
decreased for incubation times longer than 24 h, we compared
the YFP/CFP fluorescence ratios from cells incubated for 18
and 36 h after transfection. As shown in Fig. 1, e and f, the
YFP/CFP fluorescence intensity ratios decline significantly for
G␤2/G␥3 interaction from 0.52 ⫾ 0.08 (n ⫽ 21) to 0.29 ⫾ 0.05
(n ⫽ 54) (p ⬍ 0.05, Student’s t test). Thus, optimal YFP/CFP
fluorescence ratios are obtained between 18 and 24 h following
transfection, which is the time where background fluorescence
is minimized.
Interaction of the Ca2⫹ Channel ␤ Subunit and the G Protein
␤ Subunit with the Intracellular Loop I–II of the Ca2⫹ Channel
␣1 Subunits—The binding sites of the Ca2⫹ channel ␤ and G
protein ␤␥ subunits are localized at the intracellular domain
connecting domain I and II and the C terminus of the Ca2⫹
channel ␣1 subunit (6 –12). We first analyzed the interaction of
both proteins (Ca2⫹ channel ␤ and G protein ␤ subunit) on the
␣1-loopI–II of the P/Q-type channel with the MTH system (Fig.
1h). P/Q-type channel loopI–II and G␤2 (0.24 ⫾ 0.04 (n ⫽ 35))
induced a YFP fluorescent signal, which was significantly
weaker than the signal induced with ␤1b (0.45 ⫾ 0.03 (n ⫽ 53)).
In contrast, no YFP fluorescent signal was detected for coexpression of L-type channel ␣11.2-loopI–II and G protein ␤2
subunits. This result was expected, because G protein ␤␥ subunits do not interact with the L-type channel ␣1 subunits.
were transfected with pHASH-3 and positive control constructs (interacting proteins or fusion proteins that induce YFP expression) in b with
pM3-VP16 (●), pM53/pVP16-T (u), and pM-G␤2, pVP16-G␥3 (shaded triangle), or negative controls (no induction of YFP) in c with pHASH-3 alone
(⽧), pM and VP16 separated (shaded diamond) and pM53/pVP16-CP () both transfected with pHASH-3. The numbers of cells with YFP
fluorescence was divided by the number of cells with CFP fluorescence at the indicated time point. d shows the averaged relative cell number of
detectable YFP fluorescence relative to CFP fluorescence for positive controls in b (●) and negative controls in c (shaded triangle). Optimal
transfection/signal efficiencies were reached between 18 and 24 h incubation time (gray bar). Standard errors are mean ⫾ S.E. e and f, ratio of
YFP/CFP intensity ratio for 18 h (e) and 36 h (f) incubation time. OK cells were transfected with pM53/pVP16-T (white bar) (18 h, 1 ⫾ 0.11 (n ⫽
20); 36 h, 1 ⫾ 0.1 (n ⫽ 30)) and pM-G␤2, pVP16-G␥3 (gray bar) (18 h, 0.52 ⫾ 0.08 (n ⫽ 21); 36 h, 0.29 ⫾ 0.05 (n ⫽ 54); *, p ⬍ 0.05) together with
pHASH-3; for negative controls with pM and VP16 separated (18 h, 0.04 ⫾ 0.01 (n ⫽ 17); 36 h, 0.04 ⫾ 0.005 (n ⫽ 48)) and pM53/pVP16-CP (18
h, 0.03 ⫾ 0.01 (n ⫽ 27); 36 h, 0.01 ⫾ 0.004 (n ⫽ 41)) both transfected with pHASH-3. Intensity values at 18 and 36 h are normalized to the positive
control (pM-53/pVP16-T). The results indicate that the YFP/CFP intensity ratio after 18 h is higher for the pM-G␤2 and pVP16-G␥3 interaction than
after 36 h. Number of cells analyzed are indicated in parentheses. *, p ⬍ 0.05, Student’s t test for G␤2␥3-induced intensities after 18 and 36 h. g,
Western blot and induction of YFP/CFP fluorescence of fusion proteins expressed in OK cells from studies in b–f. Proteins were detected with a
monoclonal antibody against p53 and VP16, respectively. All fusion proteins used produced proteins of the correct size: pM53 (16.17 kDa), G␥3
(51.81 kDa), VP16-T (82.76 –90.89 kDa), G␤2 (46.94 kDa). Positive control combinations pM-53/pVP16-T and G␥3/G␤2 induced YFP fluorescence,
whereas negative control pM/pVP16 did not. h, detection and relative quantification of the interaction of Ca2⫹ channel ␣1-loopI–II with Ca2⫹
channel ␤ and G protein ␤ subunits. OK cells were transfected with the indicated vector combinations including pHASH-3, and fluorescence
intensity values were analyzed after 24 h of transfection. Left, YFP/CFP intensity ratios were calculated for the following cotransfections: pM53
and pVP16-T (0.99 ⫾ 0.06 (n ⫽ 47)); pM-␣12.1-loopI–II (aa 369 – 418), pVP16⫺␤1b (0.45 ⫾ 0.025 (n ⫽ 53)); pM-␣12.1-loopI–II (aa 369 – 418),
pVP16-G␤2 (0.24 ⫾ 0.04 (n ⫽ 35)); pM-␣12.1-loopI–II (aa 369 – 418), pVP16-G␤2 and pcDNA3-␥3 (aa 1–73) (0.22 ⫾ 0.062 (n ⫽ 31)); pM-␣11.2.loopI–II (aa 408 –520), pVP16-G␤2 (0.013 ⫾ 0.009 (n ⫽ 66)) and pM/pVP16 (0.018 ⫾ 0.005 (n ⫽ 55)). Right, Western blot of fusion proteins expressed
in OK cells from studies in h. Proteins were detected as described in g. All fusion proteins used produced proteins of the correct size: ␣12.1-loopI–II
(aa 369 – 418) (21.56 kDa), ␣11.2.-loopI–II (29.52 kDa), ␤1b (74.94 kDa), and G␤2 (46.94 kDa). Western blots are from the same cell preparation.
Number of cells analyzed are indicated in parentheses. Note: for G␥3 subunit expression in the MTH system a C-terminal truncation of G␥3 was
used. The C terminus of G␥ contains a CAAX motif that directs prenylation of the molecule and is responsible for anchoring the G␤␥ complex to
the plasma membrane (54). It has been shown that C-terminal truncations of G␥ do not interfere with its ability to bind G␤ subunits (55). *, p ⬍
0.05, Student’s t test.
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and regulates their transcription. Therefore, the DNA binding
domain (DNA-BD) binds to certain upstream activating sequences (UAS) in close proximity to the promoter of the gene.
One or several activation domains (AD) increase the transcription rate by directing the RNA polymerase II complex for downstream action. The AD and DNA-BD have to be in close physical proximity for efficient gene transcription. Separating AD
from DNA-BD results in loss of gene transcription, whereas
tethering AD to the DNA-BD by fusion of interacting proteins
restores the function of the transcription factor. The possibility
to separate the two domains and fuse them to putative interacting proteins allows one to monitor the interaction via expression of a certain reporter gene (36) (Clontech and Stratagene). Recently, GFP has been used for monitoring protein
interactions in an MTH system, which is based on the same
principles as the yeast system (37).
One problem concerning the detection of especially weak
protein interactions in the MTH system is the identification of
positively transfected cells and the detection of a signal relative
to background. In addition, the induced fluorescence intensity
among several cells within a single assay may vary because of
unequal numbers of reporter plasmids within the cell, different
expression times after transfection, or because of the cell type.
To overcome these problems, we introduced a second constitutively expressed reporter, the cyan fluorescence protein (CFP),
into a vector where YFP is under the control of the GAL4inducible promoter. The CFP is under CMV promoter control
and is also transported to the nucleus. CFP-mediated fluorescence therefore indicates a positively transfected cell. The induction of YFP fluorescence can now be compared with the
fluorescence signals of CFP, which are both expressed in the
same restricted area, i.e. the nucleus (Fig. 1a). Thus, induced
YFP signals can be detected and monitored relative to the CFP
signals within single cells.
As shown in Fig. 1, b– d, we first determined the optimal
expression time for detection of protein-protein interactions in
the MTH system. This was necessary because the YFP reporter
plasmid gene reveals low expression over time in the absence of
interacting proteins. We therefore analyzed the signal ratio
between YFP-induced fluorescence and the constitutive CFP
fluorescence for positive and negative controls at various expression times after transfection. As shown in Fig. 1, b– d, the
signal ratio between YFP and CFP fluorescence depends on the
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G Protein Modulation of P/Q-type Ca2⫹ Channels
To verify the results observed with the MTH system,
we compared and analyzed the direct interaction of the
␣1-loop-I–II with the Ca2⫹ channel ␤ and G protein ␤ subunits
using the three cube FRET method between CFP-tagged donor
proteins (loopI–II) and YFP-tagged acceptor proteins (␤1b/G␤2)
(Fig. 2). We calculated independently two FRET-based values
according to a modified version of Erickson et al. (32). First, we
determined the average FRET value (Fig. 2b); second, we determined the maximal FRET (FRETmax) value for the interacting protein pairs (Fig. 2, c ⫹ d) to qualitatively compare the
protein interaction with other interactions examined. Differences in the FRETmax values correspond to a difference in the
affinity of the interaction or the distance and/or orientation
between the donor relative to the acceptor protein.
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
FIG. 2. Interaction of Ca2ⴙ channel ␤1b and G protein ␤2 subunits with the intracellular loopI–II of P/Q-type ␣1 subunit. a, schematic
diagram of the interaction of N-terminal YFP-tagged ␤1b or G␤2 subunit with N-terminal CFP-tagged intracellular loopI–II of the ␣1 subunit. b,
average FRET measurements for the following FRET pairs: CFP-␣12.1-loopI–II ⫹ YFP-␤1b; CFP␣12.1-loopI–II ⫹ YFP-G␤2; ␣11.2-loopI–II ⫹
YFP-G␤2. The following controls were also analyzed but are not shown in the diagram: CFP-␣12.1-loopI–II ⫹ YFP; CFP-␣11.2-loopI–II ⫹ YFP;
CFP ⫹ YFP-␤1b; CFP ⫹ YFP-G␤2; CFP ⫹YFP; “chameleon” Ca2⫹ indicators localized to the cytoplasm and localized to the nucleus. c, FRET
measurements and calculation of FRETmax of a single transfection of CFP-␣12.1-loopI–II ⫹ YFP-␤1b (upper) CFP-␣12.1-loopI—II ⫹ YFP-G␤2
(lower). Values were fitted to y ⫽ mx ⫹ q with no constraints and given in the figure. d, FRETmax values calculated from the fits from the single
experiments as shown in c for the interaction of CFP-␣12.1-loopI–II ⫹ YFP-␤1b and CFP-␣12.1-loopI–II ⫹ YFP-G␤2. Data were fitted according to
y ⫽ mx ⫹ q with no constraints. Only experiments with r ⬎0.65 were considered. e, competition of acceptors (YFP-␤1b) for donor (␣12.1-loopI–II)
binding. Average FRET measurements for the following FRET pairs: CFP-␣12.1-loopI–II ⫹ YFP-␤1b; CFP-␣12.1-loopI–II ⫹ YFP-␤1b ⫹ ␤1b;
CFP-␣12.1-loopI–II ⫹ YFP-␤1b ⫹ G␤2. f, competition of acceptors (YFP-G␤2) for donor (␣12.1-loopI–II) binding. Average FRET measurements for
the following FRET pairs: CFP-␣12.1-loopI–II ⫹ YFP-G␤2; CFP-␣12.1-loopI–II ⫹ YFP-G␤2 ⫹ ␤1b; CFP-␣12.1-loopI–II ⫹ YFP-G␤2 ⫹ G␤2. For all
FRET experiments shown in Figs. 2– 8 and described throughout the text, the following control experiments were performed. The acceptor protein
(protein or protein domain fused to YFP) was expressed with untagged CFP, and the donor protein (protein or protein domain fused to CFP) was
expressed with untagged YFP, and the average FRET values were determined. The average FRET values were around 1 and are given in the
Supplemental Material, Table b. *, p ⬍ 0.05; **, p ⬍ 0.01, two-tailed Student’s t test.
G Protein Modulation of P/Q-type Ca2⫹ Channels
higher affinity on a second, more C-terminally located binding
site of loopI–II.
As described in our earlier work (1, 8, 33), G protein ␤
subunits when coexpressed alone with P/Q-type channels in
HEK293 cells induce G protein modulation. To rule out the
possibility that G protein ␥ subunits alter the interaction with
G␤ interacting proteins, we cotransfected G protein ␥3 subunits
in equal concentrations and 5 times higher concentrations. At
equal concentration the G protein ␥3 subunit did not alter
the fluorescence intensity ratios in the MTH (Fig. 1h;
␣12.1-loopI–II ⫹ G␤2, 0.24 ⫾ 0.04 (n ⫽ 35), and ␣12.1-loopI–II
⫹ G␤2 ⫹ G␥3, 0.22 ⫾ 0.06 (n ⫽ 31)) and FRET measurements
for G␤2 interaction with ␣12.1-loopI–II (␣12.1-loopI–II-CFP ⫹
G␤2-YFP, 3.31 ⫾ 0.24 (n ⫽ 39) and (␣12.1-loopI–II-CFP ⫹
G␤2-YFP ⫹ G␥3, 3.76 ⫾ 0.25 (n ⫽ 33)), and in FRET measurements for interaction with the full-length (fl) ␣1 subunit (␣12.1fl-CFP ⫹ G␤2-YFP, FRET 1.35 ⫾ 0.09 (n ⫽ 94) ␣12.1-fl-CFP ⫹
G␤2-YFP ⫹ G␥3, 1.33 ⫾ 0.03 (n ⫽ 102)). Higher concentrations
of G protein ␥3 subunits reduced the FRET and also the Ab
signals (␣12.1-fl-CFP ⫹ G␤2-YFP, FRET 1.35 ⫾ 0.09; Ab
0.149 ⫾ 0.01 (n ⫽ 94) ␣12.1-fl-CFP ⫹ G␤2-YFP ⫹5 times
G␥3, 1.16 ⫾ 0.02; Ab 0.02 ⫾ 0.003 (n ⫽ 50)). The reduction in
FRET and Ab signals further support the idea that G protein ␥
subunits are expressed and act as a G protein ␤ subunit sink as
already observed when G protein subunits were coexpressed
with P/Q-type channels. Expression of the G␥3 subunit abolished GTP␥S-mediated modulation of P/Q-type channels (1).
Because we did not see a significant change in the interaction
between G␤2 and ␣12.1-loopI–II or ␣12.1-full-length subunit in
the presence of equal molar concentrations of G␥3, we did not
cotransfect G␥3 in the other experiments.
To verify further that the FRET3 method is capable of detecting affinity changes between interacting proteins, we introduced a point mutation (Tyr to Ser exchange) into the AIDbinding site of the loopI–II. The mutation within the loopI–II
has been described in biochemical assays to reduce binding
between loopI–II and the Ca2⫹ channel ␤ subunits as well as G
protein ␤␥ subunits (7, 38). As expected, the loopI–II Tyr-Ser
mutation reduced the FRET signal significantly for the interaction with ␤1b and G protein ␤ subunit in comparison to wild
type loopI–II (␣12.1-loopI–II ⫹ ␤1b, 3.18 ⫾ 0.13 (n ⫽ 97); ␣12.1loopI–II-Y/S ⫹ ␤1b, 1.95 ⫾ 0.13 (n ⫽ 85); ␣12.1-loopI—II ⫹ G␤2,
2.25 ⫾ 0.06 (n ⫽ 98); ␣12.1-loopI–II-Y/S ⫹ G␤2, 1.55 ⫾ 0.05 (n ⫽
94)), indicating that FRET3 can detect affinity changes within
protein-protein interactions (Fig. 3).
In summary, the FRET data confirm the quantified protein
interactions of the MTH system. Specifically, the interactions
between P/Q-type channel ␣12.1-loop-I–II and Ca2⫹ channel
␤1b subunit induced a stronger fluorescent signal in comparison to the interaction between the P/Q-type channel
␣12.1-loop-I–II and the G protein. According to the competition
experiments, ␤1b subunits can probably compete the binding of
G␤2 on the AID-binding site of the loopI–II. Because the FRET
measurements are more sensitive and direct than the MTH
measurements and FRET measurements are capable of detecting affinity changes within a protein as demonstrated with the
Tyr/Ser loopI–II mutations, we decided to analyze the following
protein interactions with the more sensitive FRET system.
Interaction of the Ca2⫹ Channel ␤ Subunit and the G Protein
␤ Subunit with the C Terminus of the ␣12.1 Subunit—The
second binding site of the Ca2⫹ channel ␤ subunit and the G
protein ␤ subunit on the P/Q-type channel is located within the
C terminus of the ␣1 subunit (6, 9, 39, 40). However, this
binding site seems to be ␤ subunit type-specific. It has been
described for the P/Q-type channel C terminus (rabbit) that ␤2a
and ␤4 subunits but not ␤1b and ␤3 subunits interact with the
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
As shown in Fig. 2b, the interaction between P/Q-type channel ␣12.1-loopI–II with ␤1b subunits (FRET 3.45 ⫾ 0.11 (n ⫽
328)) induced a significantly stronger FRET signal than the
interaction between ␣12.1-loopI–II with the G␤2 subunit (FRET
2.49 ⫾ 0.08 (n ⫽ 335)). In contrast, cotransfection of L-type
channel ␣11.2-loopI–II-CFP and G␤2-YFP did not result in an
average FRET signal larger than 1, indicating that these two
fusion proteins do not interact (Fig. 2b). Furthermore, cotransfection of CFP and YFP and cotransfection of the donor constructs with YFP or the acceptor constructs with CFP also did
not result in a FRET signal larger than 1 (see the Supplemental Material), indicating that the FRET signals measured for
the interacting proteins are due to the interaction between
donor and acceptor protein.
The average FRET value depends on the percentage of acceptor protein interacting with donor protein within one cell
and is maximal (FRETmax) when all acceptors within one cell
interact with one donor. Because this is not the case for nonfused proteins, the actual FRET value is related to the amount
of acceptor to donor association, which was calculated by Erickson et al. (32) and is given by the Ab value (32). Thus, the Ab
value corresponds to the fraction of YFP-tagged proteins that
are preassociated with CFP-tagged proteins. For non-fused
donor and acceptor proteins, Ab values between 0 and 1 are
expected by definition. The distribution may vary according to
the incubation time, the protein pair analyzed, the cell type,
the amount of binding sites, and other factors. The distribution
should be fitted with a straight line (32). Fig. 2c shows the
distribution of a single experiment for the interaction between
␣12.1-loopI–II-CFP cotransfected with ␤1b-YFP (upper panel)
or G␤2-YFP (lower panel). The results indicate that the data
can be fitted with a straight line. The interaction between
P/Q-type channel ␣12.1-loopI–II with ␤1b induced a stronger
FRETmax value (3.63 ⫾ 0.37 (n ⫽ 6)) than the interaction
between P/Q-type channel ␣12.1-loopI–II and G␤2 (2.6 ⫾ 0.14
(n ⫽ 10)) (Fig. 2d).
As suggested by the antagonistic function and the overlapping binding sites on the ␣1 subunit between the Ca2⫹ channel
␤ subunit and G protein ␤ subunit, we asked whether Ca2⫹
channel ␤ subunits compete with G protein ␤ subunits for
binding on the ␣12.1-loopI–II. To analyze whether competition
occurs between G␤2 or ␤1b on the ␣12.1-loopI–II, we coexpressed first equal DNA amounts of CFP-tagged ␣12.1-loop-I-II
and YFP-tagged ␤1b or G␤2 with untagged ␤1b and/or G␤2
subunits (Fig. 2, e and f). For competition of acceptor to donor
binding, a reduction in the interacting pairs should result in a
reduction in the average FRET value. In our experiments a
significant reduction in the fluorescence intensity ratio for the
average FRET values was detected when the acceptor molecule
was coexpressed with its untagged version, i.e. tagged ␣12.1loop-I–II/␤1b coexpressed with untagged ␤1b (Fig. 2e) and
tagged ␣12.1-loop-I-II/G␤2 coexpressed with untagged G␤2 (Fig.
2f). Thus, the corresponding untagged protein reduces the
number of available acceptor proteins for FRET. In addition, a
significant decrease was observed for the FRET signal of ␣12.1loopI–II/G␤2 coexpressed with untagged ␤1b (Fig. 2f), whereas
even a 5-fold excess of untagged G␤2 could not significantly
reduce the FRET signal in the ␣12.1-loopI–II/␤1b interaction
(Fig. 2e). A further increase in the G␤2 concentration resulted
in loss of viable cells necessary for FRET measurements probably due to toxic cell effects of G␤2. The results suggest that the
Ca2⫹ channel ␤1b subunit interacts differently than the G protein ␤2 subunit on the ␣12.1-loopI–II. This is consistent with
the fact that ␤ subunits bind to the AID on the loopI–II,
whereas G protein ␤␥ subunits interact with the AID and with
49391
49392
G Protein Modulation of P/Q-type Ca2⫹ Channels
C-terminal binding domain (9, 40). Therefore, we wanted to
analyze how Ca2⫹ channel ␤ and G protein ␤ subunits interact
with the C terminus of the rat ␣12.1 subunit (Fig. 4). Coexpression of the C terminus and the G protein ␤ subunit induced a
consistent, average FRET signal (FRET 2.63 ⫾ 0.05 (n ⫽ 1041))
with a FRETmax value of 3.48 ⫾ 0.5 (n ⫽ 7). In contrast,
coexpression of the C terminus and ␤1b subunit did induce a
small average FRET signal (FRET 1.14 ⫾ 0.02 (n ⫽ 767)),
which could not be fitted with a straight line, indicating that
the ␤1b/C-terminal signal is unspecific. As a positive control of
Ca2⫹ channel ␤ subunit interaction with the C terminus, we
analyzed the interaction between the Ca2⫹ channel ␤4 subunit
and the C terminus. Coexpression of YFP-␤4 with the CFPtagged C terminus induced an average FRET signal of 1.97 ⫾
0.02 (n ⫽ 146) and a FRETmax signal of 3.15 ⫾ 0.62 (n ⫽ 12),
which was not significantly different from the FRETmax signal
observed for the interaction between the C terminus and the G
protein. Thus, the FRET measurements correlate with the
described biochemical data for the interaction of G␤2, ␤4, and
␤1b subunits on the ␣12.1 P/Q-type channel and support the
view that ␤1b subunits bind to the loopI–II (AID), whereas G␤2
bind to both the C terminus and the loopI–II of the P/Q-type
channel.
Protein Interactions at the Full-length ␣12.1 Subunit—By
having analyzed the protein interactions of Ca2⫹ channel ␤1b
and G protein ␤2 subunits with the isolated binding domains of
the ␣1 subunit, we were next interested in how these proteins
may interact with the full-length P/Q-type ␣12.1 subunit. Originally, the FRET3 method was developed and described for
Ca2⫹ channel ␣1 subunits as fluorescent acceptor proteins in
the FRET experiments. In our experiments we tagged Ca2⫹
channel ␣1 subunits with CFP or YFP (using the ␣1 subunit as
donor or acceptor) and analyzed the protein interactions with
the YFP- or CFP-tagged Ca2⫹ channel ␤1b or G protein ␤2
subunits. We first verified that our tagged proteins were functional by coexpressing the tagged subunits in HEK293 cells
and analyzing the underlying Ba2⫹ currents. As indicated in
Fig. 5a, coexpression of CFP-tagged ␣12.1 subunits with YFPtagged ␤1b and G␤2 subunits resulted in Ca2⫹ channel-mediated Ba2⫹ currents, which were G protein-modulated (Fig. 5a,
lower part). Application of a high positive prepulse to ⫹100 mV
released G protein modulation from the channel. Therefore, the
current elicited by the second test pulse to ⫹10 mV is larger
than the current elicited by the first test pulse to the same
potential. Comparable modulation was observed for the YFPtagged ␣12.1 subunits with CFP-tagged ␤1b and G␤2 subunits
and for the untagged constructs as described in our previous
studies when untagged G␤2 was coexpressed with untagged
␣12.1 and ␤1b subunits (1, 8, 33) or when GFP-tagged ␤4 subunits were coexpressed with ␣12.1 (34). On the full-length ␣1
subunit, ␤1b-YFP (paired with ␣12.1-fl-CFP) as well as ␤1b-CFP
(paired with ␣12.1-fl-YFP) subunits induced a significantly
stronger average FRET (␤1b-YFP/␣12.1-fl-CFP FRET 1.55 ⫾
0.02 (n ⫽ 587); ␤1b-CFP/␣12.1-fl-YFP 2.67 ⫾ 0.06 (n ⫽ 425)) and
FRETmax signal (␤1b-YFP/␣12.1-fl-CFP FRETmax 6.48 ⫾ 0.84
(n ⫽ 8); ␤1b-CFP/␣12.1-fl-YFP 5.36 ⫾ 0.38 (n ⫽ 7)) than G
protein ␤2 subunits (G␤2-YFP/␣12.1-fl-CFP FRET 1.1 ⫾ 0.09
(n ⫽ 93); FRETmax 2.93 ⫾ 0.34 (n ⫽ 9); G␤2-CFP/␣12.1-fl-YFP
FRET 1.68 ⫾ 0.06 (n ⫽ 98); FRETmax 4.17 ⫾ 0.18 (n ⫽ 7)) (Fig.
6d). Because full-length ␣1 subunits need the Ca2⫹ channel ␤
subunit for transport to the plasma membrane (18, 41), untagged ␤1b subunits were also cotransfected for studying the
interaction with G␤2 on the full-length channel. The data indicate that the spatial orientation and the proximity of fluorophores is more optimal for energy transfer between the interacting proteins ␣1/␤1b in comparison to ␣1/G␤2. This result may
imply that the interaction between ␣1/␤1b is stronger than the
␣1/G␤2 interaction.
The FRET3 method allows us to correlate the percentage of
acceptor proteins (YFP-tagged protein) associated with the donor proteins (CFP-tagged proteins) in a given FRET experiment via the calculation of the Ab value (see above). We determined the percentage of ␣1 subunits associated with Ca2⫹
channel ␤1b or G protein ␤2 subunits and compared these
values to the percentage of ␤1b and G␤2 associated with ␣12.1fl. Although 26% of ␤1b subunits were associated with Ca2⫹
channel ␣1 subunits (␣12.1-fl-CFP/␤1b-YFP Ab 0.26 ⫾ 0.01 (n ⫽
587)), 67% of ␣1 subunits were associated with ␤1b subunits
(␤1b-CFP/␣12.1-fl-YFP Ab 0.67 ⫾ 0.01 (n ⫽ 438)) (Fig. 6c). No
significant change in the association percentage was observed
for the G␤2 subunits interacting with Ca2⫹ channel ␣1 subunits. For both fluorophore orientations the percentage of association between G␤2 with ␣12.1 (␣12.1-fl-CFP/G␤2-YFP Ab
0.375 ⫾ 0.03 (n ⫽ 93)) or ␣12.1 with G␤2 (G␤2-CFP/␣12.1-fl-YFP
Ab 0.41 ⫾ 0.02 (n ⫽ 98)) was around 40%. The experiments
suggest that Ca2⫹ channel ␤ subunits are closely associated
with ␣1 subunits at the plasma membrane, supporting the
results of Bichet et al. (41) for the role of ␤ subunits for ␣1
subunit trafficking.
Interaction of Ca2⫹ Channel ␣1 and ␤1b Subunits in the
Presence of G Protein ␤2 Subunits—Activation of a G proteincoupled receptor leads to the dissociation of G protein ␣ and ␤␥
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FIG. 3. Interaction of Ca2ⴙ channel ␤1b and G protein ␤2 subunits with wild type and mutated intracellular loopI–II of the P/Q-type
␣1 subunit. a, schematic diagram of the AID within the loopI–II and the location of the point mutation (Tyr/Ser) which reduces the binding affinity
of Ca2⫹ channel ␤ and G protein ␤␥ subunits. b, average FRET measurements for the following FRET pairs: CFP-␣12.1-loopI–II ⫹ YFP-␤1b;
CFP-␣12.1-loopI–II-Y/S ⫹ YFP-␤1b; CFP-␣12.1-loopI–II ⫹ YFP-G␤2; CFP-␣12.1-loopI–II-Y/S ⫹ YFP-G␤2. The following control was also analyzed
but is not shown in the diagram: CFP-␣12.1-loopI–II-Y/S ⫹ YFP. Values for shown experiments and controls are given in the Supplemental
Material, Table b. *, p ⬍ 0.05; **, p ⬍ 0.01, two-tailed Student’s t test.
G Protein Modulation of P/Q-type Ca2⫹ Channels
49393
subunits, the active components of the G protein. It is assumed
that G␤␥ after its dissociation from G␣ interacts with the Ca2⫹
channel to induce modulation. The binding of G␤␥ to the channel complex could either result in the release of the Ca2⫹
channel ␤ subunit from the channel (which is not supported by
our interaction studies (Fig. 2)) or could induce a conformational change in the channel complex. To mimic the G protein
modulation of the channel complex in the heterologous expression system, we coexpressed untagged G␤2 together with the
FRET Ca2⫹ channel pair ␣1/␤1b. If the G protein competes the
binding of Ca2⫹ channel ␤1b subunit to ␣1, we should observe a
decreased FRET value, whereas rearranging the ␣1/␤1b subunits could either result in an increase or decrease in the FRET
signal depending on the reorientation of the fluorophores. We
observed a significant increase in the average FRET value from
1.45 ⫾ 0.03 (n ⫽ 200) to 1.62 ⫾ 0.04 (n ⫽ 151) for the interac-
tion between ␣12.1-fl-CFP and the ␤1b-YFP when coexpressed
with untagged G␤2. This indicates a change of the interaction
between Ca2⫹ channel ␣12.1-fl and ␤1b subunits in the presence
of G protein ␤2 subunits. The result was confirmed by determination of the FRETmax values. In the presence of G␤2 subunits, the FRETmax value was significantly increased for ␣12.1fl/␤1b interaction from FRETmax 4.81 ⫾ 0.85 (n ⫽ 3) to FRETmax
9.26 ⫾ 1.07 (n ⫽ 3) (Fig. 7, a– c). We next analyzed the effect of
G␤2 on the ␣1/␤1b FRET pair, when ␣1 was the acceptor in
FRET. Again we observed a significant increase in the average
FRET value from 2.29 ⫾ 0.07 (n ⫽ 240) to 2.54 ⫾ 0.09 (n ⫽ 255)
and FRETmax value from 5.79 ⫾ 0.3 (n ⫽ 7) to 7.48 ⫾ 0.46 (n ⫽
7) (Fig. 7, d–f), indicating that G protein ␤ subunits change the
orientation between Ca2⫹ channel ␣1 and ␤1b subunits rather
than competing for binding with the Ca2⫹ channel ␤1b subunits
on the channel complex.
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
FIG. 4. Interaction of Ca2ⴙ channel ␤1b and G protein ␤2 subunits with the intracellular C terminus of the P/Q-type ␣1 subunit. a,
schematic diagram of the interaction of N-terminal-YFP tagged ␤1b and G␤2 subunit with N-terminal CFP-tagged intracellular C terminus of the
␣12.1 subunit. b, average FRET measurements for the following FRET pairs: CFP-␣12.1-C terminus ⫹ YFP-␤1b; CFP-␣12.1-C terminus ⫹ YFP-G␤2;
CFP-␣12.1-C terminus ⫹ YFP-␤4. The following controls were also analyzed but are not shown in the diagram: CFP-␣12.1-C terminus ⫹ YFP and
CFP ⫹ YFP-␤4. c, FRET measurements and calculation of FRETmax of a single transfection of CFP-␣12.1-C terminus ⫹ YFP-G␤2 and CFP-␣12.1-C
terminus ⫹ YFP-␤4. Values were fitted to y ⫽ mx ⫹ q with no constraints and given in the figure. d, FRETmax values calculated from the fits from
the single experiments as shown in c for the interaction of CFP-␣12.1-C terminus ⫹ YFP-G␤2 and CFP-␣12.1-C terminus ⫹ YFP-␤4. Data were
fitted according to y ⫽ mx ⫹ q. Only experiments with r ⬎ 0.65 were considered. Values for shown experiments and controls are given in the
Supplemental Material, Table b. *, p ⬍ 0.05; **, p ⬍ 0.01, two-tailed Student’s t test.
49394
G Protein Modulation of P/Q-type Ca2⫹ Channels
Because G protein ␤ but not Ca2⫹ channel ␤1b subunits
interact with the ␣12.1-C terminus, binding of the G protein to
the C terminus of the P/Q-type channel may be involved in
increasing the FRET signal between the Ca2⫹ channel subunits. To verify this idea we first overexpressed the untagged C
terminus to compete the binding of G␤ subunits to the channel
complex, and second we truncated the C terminus of the fulllength ␣12.1 subunit by 355 amino acids, which includes the G
protein-binding site, to demonstrate that the C terminus is
involved in the G protein-mediated FRET increase of the Ca2⫹
channel ␤1b to ␣12.1 interaction (Fig. 8). For analyzing the
involvement of the C terminus in G protein-mediated FRET
increase, we studied the interaction between the CFP-tagged
␣1 subunit and the YFP-tagged ␤1b subunit. Again coexpression
of CFP-tagged ␣1 and YFP-tagged ␤1b subunits with untagged
G␤2 subunits increased the average FRET (2.61 ⫾ 0.09 (n ⫽
174)) and FRETmax (4.91 ⫾ 0.27 (n ⫽ 6)) values in comparison
to FRET pairs expressed without the G protein (average FRET
1.9 ⫾ 0.03 (n ⫽ 194) and FRETmax 2.36 ⫾ 0.44 (n ⫽ 3)).
Coexpression of the C terminus did not change the average
FRET (1.93 ⫾ 0.03 (n ⫽ 196)) and FRETmax (3.09 ⫾ 0.31 (n ⫽
5)) values for ␣1/␤1b interactions but abolished the G protein-
mediated FRET increase (average FRET 2.18 ⫾ 0.03 (n ⫽ 294)
and FRETmax 2.79 ⫾ 0.18 (n ⫽ 6)). Coexpression of the Cterminally deleted CFP-tagged ␣12.1 subunit (␣12.1-fl-⌬C terminus) with the YFP-tagged ␤1b subunit resulted in FRET
signals, which were comparable with the interaction between
the full-length ␣1 subunit and ␤1b subunit (average FRET
2.16 ⫾ 0.04 (n ⫽ 196) and FRETmax 3.27 ⫾ 0.39 (n ⫽ 6)).
However, coexpression of G␤2 with the C-terminally deleted
channel (␣12.1-fl-⌬C terminus/␤1b FRET pair) did not result in
an increase in the average FRET (2.11 ⫾ 0.04 (n ⫽ 195)) and
the FRETmax values (2.87 ⫾ 0.15 (n ⫽ 6)). These results indicate that the isolated C terminus probably acts as a sink for G
protein ␤ binding and is most likely involved in the G proteinmediated FRET increase and/or the reorientation of the Ca2⫹
channel ␣1 and ␤ subunits.
Interaction of Ca2⫹ Channel ␤1b Subunits and G Protein ␤2
Subunits on the Full-length P/Q-type Channel—The observations that ␤1b subunits and G protein ␤2 subunits possess at
least partially separated binding sites on the full-length channel and that G␤2 subunits alter the FRET signal between Ca2⫹
channel ␣1 and ␤1b subunits suggest that both G protein ␤2 and
Ca2⫹ channel ␤1b subunits can bind to the channel at the same
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
FIG. 5. Interaction of Ca2ⴙ channel ␤1b and G protein ␤2 subunits with the full-length P/Q-type channel ␣12.1 subunit. a, P/Q-type
channel-mediated Ba2⫹ currents measured in tsA-201 cells. CFP-tagged ␣12.1 subunits were coexpressed with YFP-tagged ␤1b and YFP-tagged G␤2
subunits in tsA-201 cells, and currents were measured after 48 –72 h after transfection with patch clamp measurements. Upper trace, a 500-ms
voltage ramp from a holding potential of ⫺70 to ⫹ 100 mV elicit a whole cell Ba2⫹ current. Lower trace, prepulse facilitation of P/Q-type channels
was determined by comparing two 5-ms test pulses to the indicated test potential separated by a 1-s interval at the holding potential of ⫺70 mV.
2 ms before the second test pulse a 10-ms conditioning prepulse to ⫹100 mV was applied which resulted in an increase in the peak and tail current.
b, distribution and FRET signal of N-terminal CFP-tagged full-length ␣12.1 subunits coexpressed with YFP-tagged ␤1b or YFP-tagged G␤2 subunits
and untagged ␤1b subunits. Top, detected CFP fluorescence; middle, detected FRET fluorescence; bottom, detected YFP fluorescence for the
cotransfected FRET protein pairs. c, schematic diagrams for the interactions of N-terminal YFP-tagged ␤1b and G␤2 subunits with N-terminal
CFP-tagged full-length ␣12.1 subunit (upper) and N-terminal CFP-tagged ␤1b and G␤2 subunit with N-terminal YFP-tagged full-length ␣12.1
subunit (lower). d, average FRET measurements for the following FRET pairs: CFP-␣12.1-fl ⫹ YFP-␤1b; CFP-␣12.1-fl ⫹ YFP-G␤2 ⫹ ␤1b; YFP-␣12.1-fl ⫹
CFP-␤1b; YFP-␣12.1-fl ⫹ CFP-G␤2 ⫹ ␤1b. The following controls were also analyzed but are not shown in the diagram: CFP-␣12.1-fl ⫹ YFP ⫹ ␤1b and
YFP-␣12.1-fl ⫹ CFP ⫹ ␤1b. Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p ⬍ 0.05; **, p ⬍ 0.01, two-tailed
Student’s t test.
G Protein Modulation of P/Q-type Ca2⫹ Channels
49395
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FIG. 6. Interaction of Ca2ⴙ channel ␤1b and G protein ␤2 subunits with the full-length P/Q-type channel ␣12.1 subunit. a, schematic
diagram for the interactions of N-terminal YFP-tagged ␤1b and G␤2 subunits with N-terminal CFP-tagged full-length ␣12.1 subunit (upper). FRET
measurements and calculation of FRETmax of a single transfection of CFP-␣12.1-fl ⫹ YFP-␤1b (middle) and CFP-␣12.1-fl ⫹ YFP-G␤2 ⫹ ␤1b (lower).
Values were fitted to y ⫽ mx ⫹ q with no constraints and are given in the figure. b, schematic diagram for the interactions of N-terminal
CFP-tagged ␤1b and G␤2 subunit with N-terminal YFP-tagged full-length ␣12.1 subunit (upper). FRET measurements and calculation of FRETmax
of a single transfection of YFP-␣12.1-fl ⫹ CFP-␤1b (middle) and YFP-␣12.1-fl ⫹ CFP-G␤2 ⫹ ␤1b (lower). Values were fitted to y ⫽ mx ⫹ q with no
constraints and are given in the figure. c, average Ab values calculated according to Erickson et al. (32) for the following FRET pairs: CFP-␣12.1-fl ⫹ YFP-␤1b;
CFP-␣12.1-fl ⫹ YFP-G␤2 ⫹ ␤1b; YFP-␣12.1-fl ⫹ CFP-␤1b; YFP-␣12.1-fl ⫹ CFP-G␤2 ⫹ ␤1b. d, FRETmax values calculated from the fits from the single
experiments as shown in a and b for the interaction of CFP-␣12.1-fl ⫹ YFP-␤1b; CFP-␣12.1-fl ⫹ YFP-G␤2 ⫹ ␤1b; YFP-␣12.1-fl ⫹ CFP-␤1b; YFP-␣12.1-fl ⫹
CFP-G␤2 ⫹ ␤1b. Data were fitted according to y ⫽ mx ⫹ q. Only experiments with r ⬎ 0.65 were considered except for YFP-␣12.1-fl ⫹ CFP-␤1b. Due to the
fact that FRET values were clustered at Ab values of 1 for this particular FRET pair (see also Erickson et al. (32)), the correlation for this FRET pair was
not as high as for the other FRET pairs. We therefore considered experiments with r ⬎ 0.5. Values for shown experiments and controls are given in the
Supplemental Material, Table b. *, p ⬍ 0.05; **, p ⬍ 0.01, two-tailed Student’s t test.
time. We therefore analyzed the average FRET signal between
CFP-tagged Ca2⫹ channel ␤1b subunits and YFP-tagged G protein ␤2 subunits either coexpressed with untagged ␣1 subunits
or in the absence of ␣1 (Fig. 9). A FRET signal over background
level could only be observed for ␤1b-CFP/G␤2-YFP pairs when
coexpressed with the ␣1 subunit (FRET 1.33 ⫾ 0.06 (n ⫽ 98)).
The data fitted a straight line and gave rise to a FRETmax value
of 1.84 ⫾ 0.22 (n ⫽ 4) (Fig. 9, c and d), indicating that Ca2⫹
channel ␤1b subunits and G protein ␤2 subunits interact at the
same time on the ␣12.1 subunit in COS7 cells.
49396
G Protein Modulation of P/Q-type Ca2⫹ Channels
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FIG. 7. Coexpression of G protein ␤2 subunits with the Ca2ⴙ channel ␣12.1/␤1b FRET pairs. a, schematic diagram of the interaction
between N-terminal YFP-tagged ␤1b subunit with N-terminal CFP-tagged ␣12.1-fl in the presence of untagged G␤2 subunits. b, FRET measurements and calculation of FRETmax of a single transfection of CFP-␣12.1-fl ⫹ YFP-␤1b (upper) and of CFP-␣12.1-fl ⫹ YFP-␤1b plus G␤2 (lower). Values
were fitted to y ⫽ mx ⫹ q with no constraints and are given in the figure. c, FRETmax values calculated from the fits from the single experiments
as shown in b for the interaction of CFP-␣12.1-fl ⫹ YFP-␤1b and CFP-␣12.1-fl ⫹ YFP-␤1b plus G␤2. Data were fitted according to y ⫽ mx ⫹ q. Only
experiments with r ⬎ 0.65 were considered. d, schematic diagram of the interaction between N-terminal CFP-tagged ␤1b subunit with N-terminal
YFP-tagged ␣12.1-fl in the presence of untagged G␤2 subunits. e, FRET measurements and calculation of FRETmax of a single transfection of
YFP-␣12.1-fl ⫹ CFP-␤1b (upper) and YFP-␣12.1-fl ⫹ CFP-␤1b plus G␤2 (lower). Values were fitted to y ⫽ mx ⫹ q with no constraints and are given
in the figure. f, FRETmax values calculated from the fits from the single experiments as shown in e for the interaction of YFP-␣12.1-fl ⫹ CFP-␤1b
and YFP-␣12.1-fl ⫹ CFP-␤1b plus G␤2. Data were fitted according to y ⫽ mx ⫹ q. Only experiments with r ⬎ 0.5 were considered (see Fig. 6d).
Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p ⬍ 0.05; **, p ⬍ 0.01, two-tailed Student’s t test.
DISCUSSION
G proteins and Ca2⫹ channel ␤ subunits have overlapping
binding sites on the presynaptic Ca2⫹ channel ␣1 subunit and
induce antagonistic biophysical effects on the channel (5, 14 –
17, 42). As suggested by Bean in 1989 (19) and supported by
detailed biophysical analysis (e.g. Refs. 20 –24) and observed
for the ␣12.2a N-type channel splice variant (25), the G protein
may induce a new conformational state of the closed channel.
Because of these findings the question concerning the mechanism of G protein modulation of presynaptic Ca2⫹ channels is
whether Ca2⫹ channel ␤ subunits and G protein subunits influence each others’ binding on the ␣1 subunit, which may
G Protein Modulation of P/Q-type Ca2⫹ Channels
49397
induce a certain conformational channel state. Here we present
evidence that G protein ␤ and Ca2⫹ channel ␤ subunits interact
differently on the two separate binding sites of the ␣1 subunit
and on the full-length channel. Ca2⫹ channel ␤ subunit can
compete G protein ␤ subunit binding on the loopI–II (probably
on the AID-binding site) but does not interact with the C
terminus. In addition, we present for the first time that the G
protein subunit when coexpressed with the Ca2⫹ channel
FRET pair ␣12.1/␤ alters the FRET signal between these two
interacting proteins, suggesting that the G protein changes the
orientation and/or association between the ␣12.1 and ␤ subunit
of the Ca2⫹ channel complex.
Mammalian Two-hybrid and FRET Measurements for Detection and Quantification of Protein-Protein Interactions—We
applied two independent systems to gain inside information
about the interactions between the proteins involved in P/Q-
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FIG. 8. Influence of the ␣12.1 C terminus on G protein-mediated FRET increase for ␣12.1/␤1b FRET pairs. a, upper, schematic diagram
of the interaction between N-terminal YFP-tagged ␤1b subunit with N-terminal CFP-tagged ␣12.1-fl in the presence of untagged G␤2 subunits and
untagged ␣12.1 derived C termini. Lower, schematic diagram of the interaction between N-terminal YFP-tagged ␤1b subunit with N-terminal
CFP-tagged ␣12.1 subunit, where the C terminus was deleted, in the presence of untagged G␤2 subunits. b, average FRET measurements for the
following FRET pairs: CFP-␣12.1-fl ⫹ YFP-␤1b; CFP-␣12.1-fl ⫹ YFP-␤1b ⫹ C terminus; CFP-␣12.1-fl ⫹ YFP-␤1b ⫹ G␤2; CFP-␣12.1-fl ⫹ YFP-␤1b⫹
C terminus ⫹ G␤2; CFP-␣12.1-fl-⌬C terminus ⫹ YFP-␤1b; CFP-␣12.1-fl-⌬C terminus ⫹ YFP-␤1b ⫹ G␤2. The following control was also analyzed but
is not shown in the diagram: CFP-␣12.1-fl-⌬C terminus ⫹ YFP ⫹ ␤1b. c, FRET measurements and calculation of FRETmax of a single transfection
of CFP-␣12.1-fl-⌬C terminus ⫹ YFP-␤1b (upper) and CFP-␣12.1-fl-⌬C terminus ⫹ YFP-␤1b plus G␤2 (lower). Values were fitted to y ⫽ mx ⫹ q with
no constraints and are given in the figure. d, FRETmax values calculated from the fits from the single experiments as shown in c for the interaction
of CFP-␣12.1-fl ⫹ YFP-␤1b; CFP-␣12.1-fl ⫹ YFP-␤1b ⫹ C terminus; CFP-␣12.1-fl ⫹ YFP-␤1b ⫹ G␤2; CFP-␣12.1-fl ⫹ YFP-␤1b⫹ C terminus ⫹ G␤2;
CFP-␣12.1-fl-⌬C terminus ⫹ YFP-␤1b and CFP-␣12.1-fl-⌬C terminus ⫹ YFP-␤1b ⫹ G␤2. Data were fitted according to y ⫽ mx ⫹ q. Only experiments
with r ⬎ 0.65 were considered. Values for shown experiments and controls are given in the Supplemental Material, Table b. **, p ⬍ 0.01, two-tailed
Student’s t test.
49398
G Protein Modulation of P/Q-type Ca2⫹ Channels
type channel modulation. First, we developed a modified MTH
system for detection and relative quantification of proteinprotein interactions. By using two fluorescent reporters, CFP
and YFP located on one expression plasmid, we monitored the
induced YFP-mediated fluorescence relative to constitutive
CFP fluorescence. This system was established in OK cells but
can also be applied to other mammalian cell lines. In order to
demonstrate that our system is applicable for detection of various protein-protein interactions, we analyzed the association
between G protein ␤ and ␥ subunits and the interaction between the G protein ␤ and Ca2⫹ channel ␤ subunits with the
intracellular domain of the Ca2⫹ channel ␣12.1 subunit. By
establishing FRET measurements according to the method of
Erickson et al. (32) between the two interacting proteins, the
data observed with the MTH system could be verified. Thus,
the MTH system allows the detection of protein-protein interaction, where the strength of the detected signals corresponds
to the measured FRET signals. Because the FRET method is
more sensitive and more direct, we used this technique to gain
insight into the mechanism of Ca2⫹ channel modulation. The
results are summarized in Table I.
FRET Measurements between the Ca2⫹ Channel ␤ Subunit,
G Protein Subunit, and the Isolated Binding Sites (LoopI–II
and C Terminus) of the P/Q-type Channel ␣1 Subunit Confirm
Biochemical Data of Ca2⫹ Channel G-Protein Interactions—We
first analyzed the interaction on the separated Ca2⫹ channel
␣12.1-binding sites, i.e. the loopI–II and the C terminus. The
interaction of P/Q-type ␣12.1-loopI–II with the Ca2⫹ channel ␤
subunit induced a stronger FRET signal than the interaction of
loopI–II with the G protein ␤ subunit. Whereas untagged Ca2⫹
channel ␤ subunits can compete or alter the interaction between G protein ␤ subunit and loopI–II, G␤ cannot compete the
interaction between Ca2⫹ channel ␤ subunit and loopI–II, at
least at concentrations used in the experiments. This finding
suggests that during modulation the G protein may not be able
to replace the Ca2⫹ channel ␤ subunit at the ␤ subunit-binding
site of the ␣1 subunit. This finding can be explained by the fact
that G protein ␤␥ subunits have two binding sites on the
␣12.1-loopI–II, whereas Ca2⫹ channel ␤ subunits only have one
(5, 7, 10, 43). The N-terminal-binding site (Fig. 10), which is the
overlapping binding site between Ca2⫹ channel ␤ and G protein
␤ subunit, may therefore have a higher affinity for ␤ subunits
than for G protein ␤␥ subunits (reduction in FRET signal for
loopI–II/G␤ interaction in the presence of ␤ subunits). Indeed,
De Waard et al. (7) described that the affinity of G␤␥ on this
particular binding site is 10 –20 times lower in comparison to
the Ca2⫹ channel ␤ subunit interaction. Thus, in the presence
of Ca2⫹ channel ␤ subunits, the preferable binding of G protein
␤ subunits may therefore be the C-terminal-binding site of the
loopI–II (Fig. 10). This is in agreement with the facts that the
C-terminal-binding site of loopI–II has a lower Kd value for
G␤␥ binding than the N-terminal-binding site of loopI–II (7)
and is sensitive to protein kinase C phosphorylation (10).
At the ␣12.1-C terminus only the G protein ␤2 subunit, but
not the Ca2⫹ channel ␤1b subunit, induced a strong FRET
signal. This correlates well with the observation of Walker et
al. (40) that Ca2⫹ channel ␤4 and ␤2a, but not ␤1b and ␤3,
subunits interact with the C terminus of the P/Q-type channel.
Thus, competition for binding between ␤1b and G␤2 is not likely
to occur at the C terminus but seems possible for the Ca2⫹
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FIG. 9. Interaction of Ca2ⴙ channel ␤1b with G protein ␤2 subunits on the full-length P/Q-type channel ␣12.1 subunit. a, schematic
diagram of the interaction between N-terminal CFP-tagged ␤1b subunit with N-terminal YFP-tagged G␤2 subunit on the full-length ␣12.1 subunit.
b, average FRET measurements for the following FRET pairs: CFP-␤1b ⫹ YFP-G␤2 and CFP-␤1b ⫹ YFP-G␤2 ⫹ ␣12.1-fl. The following control was
also analyzed but is not shown in the diagram: CFP-␤1b ⫹ YFP. c, FRET measurements and calculation of FRETmax of a single transfection of
CFP-␤1b ⫹ YFP-G␤2 ⫹ ␣12.1. Values were fitted to y ⫽ mx ⫹ q with no constraints and given in the figure. d, FRETmax values calculated from the
fits from the single experiments as shown in c for the interaction of CFP-␤1b ⫹ YFP-G␤2 ⫹ ␣12.1-fl. Data were fitted according to y ⫽ mx ⫹ q. Right,
only experiments with r ⬎ 0.65 were considered. Values for shown experiments and controls are given in the Supplemental Material, Table b. *,
p ⬍ 0.05; **, p ⬍ 0.01, two-tailed Student’s t test.
G Protein Modulation of P/Q-type Ca2⫹ Channels
49399
TABLE I
Summary of FRETmax values for the interaction of loopI–II, C terminus, full-length channel, full-length channel
2⫹
with C terminus deleted, and Ca channel ␤1b and ␤4 subunits as donor and/or acceptor proteins and
influence of coexpressed untagged proteins on average FRET and/or FRETmax values
Arrows indicate whether FRET was increased (1), decreased (2), or not changed (7) during the coexpression of the untagged subunits.
Unlabeled
Donor
FRETmax
␣12.1-loopI–II-CFP
␤1b-YFP
G␤2-YFP
3.63 ⫾ 0.37 (n ⫽ 6)
2.6 ⫾ 0.14 (n ⫽ 10)
␣12.1-C terminus-CFP
␤1b-YFP
G␤2-YFP
␤4-YFP
␤1b-YFP
G␤2-YFP
␤1b-YFP
⫺
3.48 ⫾ 0.5 (n ⫽ 7)
3.15 ⫾ 0.62 (n ⫽ 12)
6.48 ⫾ 0.84 (n ⫽ 8)a
2.93 ⫾ 0.34 (n ⫽ 9)
3.27 ⫾ 0.39 (n ⫽ 6)
␣12.1-fl-YFP
(⫹ ␤1b)
5.36 ⫾ 0.38 (n ⫽ 7)
4.17 ⫾ 0.18 (n ⫽ 7)
G␤2-YFP
1.84 ⫾ 0.22 (n ⫽ 4)
␣12.1-fl-CFP
(⫹ ␤1b)
␣12.1-fl-⌬C terminus-CFP
␤1b-CFP
G␤2-CFP
␤1b-CFP (⫹ ␣12.1-fl)
a
␤1b
G ␤2
2
2
7
2
1
7
1
Value from Fig. 7.
FIG. 10. Model of G protein modulation of P/Q-type Ca2ⴙ channels assembled by ␣12.1 and ␤1b subunits according to MTH and
FRET measurements. Left, the Ca2⫹ channel ␤1b subunit interacts
with the ␣1 subunit on the N-terminal region of the intracellular loop
I–II, the AID. Right, after activation of the G protein, the G protein ␤
subunit binds to its binding sites on the ␣1 subunit, i.e. C terminus (gray
box) and to the C-terminal region of the intracellular loopI–II (gray box)
of the ␣1 subunit. Binding of the G protein ␤ subunit to the channel
changes either the orientation and/or the affinity of ␣1 full-length subunit to the Ca2⫹ channel ␤ subunit, inducing a new conformational
state of the channel.
channel ␤4 and ␤2a subunits. Binding of G protein ␤␥ and Ca2⫹
channel ␤ to a short binding domain within the C terminus of
N, P/Q, and R-type channels has been described by several
groups (6, 9, 40). For example, Qin et al. (9) demonstrated that
Ca2⫹ channel ␤2a can compete G␤␥ binding on the C terminus
of ␣12.3 and that truncation of the C terminus eliminates the G
protein modulation of ␣12.3 assembled channels in Xenopus
oocytes. The interactions of G protein ␤ and Ca2⫹ channel ␤
subunit on the C terminus and the loopI–II suggest that the
primary binding site of Ca2⫹ channel ␤ subunits on the ␣1
subunit is the N-terminal region of loopI–II, whereas the primary binding site of the G protein ␤ subunit is the C terminus
and the C-terminal region of loopI–II. This finding agrees with
the role of the Ca2⫹ channel ␤ subunit in plasma membrane
targeting of the ␣1 subunit. Here, an endoplasmic reticulum
retention signal within the binding site of ␤ on ␣1-loopI–II is
shielded by the ␤ subunit, which allows transport out of the
endoplasmic reticulum (41).
FRET Measurements between the Ca2⫹ Channel ␤ Subunit,
G Protein Subunit, and the Full-length P/Q-type Channel ␣1
Subunit Suggest a Mechanism for P/Q-type Channel Modulation—We next analyzed the protein interactions on the full-
length ␣12.1 subunit. The interaction of the full-length ␣12.1
subunit with Ca2⫹ channel ␤ subunit induced a larger FRET
signal than the interaction of ␣12.1-fl with the G␤ subunit. This
effect was observed for using the ␣12.1 subunit as either acceptor or donor within the FRET pair. Because Ca2⫹ channel ␤
subunits are necessary for transport of the ␣1 subunit to the
plasma membrane, the experiments of the interaction between
␣1-fl and G protein ␤ subunits were performed in the presence
of an untagged Ca2⫹ channel ␤ subunit. It seems reasonable
that Ca2⫹ channel ␤ and G protein ␤ subunits bind at the same
time to the ␣12.1 subunit during modulation, because cotransfection of untagged ␣1 and tagged Ca2⫹ channel ␤ and G protein ␤ subunits induced a FRET signal. The existence of two
binding sites on the ␣1 subunit for G protein and ␤ subunits
may also suggest that two G protein ␤ or two Ca2⫹ channel ␤
subunits bind simultaneously to the ␣1 subunit. However, direct intracellular application of purified G protein ␤␥ subunits
and kinetic studies on N-type channels rather suggest that
modulation of one channel requires one G protein (22, 44). The
situation is more difficult for the binding of Ca2⫹ channel ␤
subunits to the ␣1 subunit. Birnbaumer and co-workers (18, 39)
suggested a two Ca2⫹ channel ␤-binding site model for transport and modulation of Ca2⫹ channels. The role of a second
modulating ␤ subunit binding has been suggested further by
several groups (45– 48). Recently, the intracellular application
of purified ␤ subunits to membrane vesicles of the skeletal
muscle supported the view of the modulation of pre-existing
Ca2⫹ channels independent of the role of ␤ subunits for targeting (49, 50). However, we did not observe a significant increase
in FRET values for ␤1b-CFP and ␤1b-YFP interaction on the
␣12.1 full-length channel (data not shown). Thus, the interaction of two Ca2⫹ channel ␤ subunits on one ␣1 subunit may not
be resolvable in our assays or only one ␤ subunit binds to the
channel. Further FRET analysis, in particular with single or
multiphoton microscopy, may shed light on this interesting
phenomenon.
Another interesting observation of our study is that ⬃25% of
all ␤ subunits interact with ␣1, whereas up to 70% of ␣1 subunits interact with ␤1b in the heterologous expression system.
The percentage of ␣1 associated with other ␤ subunits was even
higher (up to 90% (data not shown)), indicating that ␣1 subunits need ␤ subunits for transport and insertion into the
plasma membrane and support the findings of Bichet et al. (41)
for the role of ␤ subunits in ␣1 subunit trafficking.
The important finding on the full-length ␣1 subunit is that
the FRET signal between Ca2⫹ channel ␤ and ␣1 subunit is
increased in the presence of G protein ␤ subunits, which in-
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Acceptor
49400
G Protein Modulation of P/Q-type Ca2⫹ Channels
Acknowledgments—We are grateful to Drs. T. P. Snutch,
E. Perez-Reyes, and M. I. Simon for cDNAs, and Drs. L. Landmesser,
V. Lemmon, and S. Jones for reading the manuscript. We also thank
Karin Geckle for excellent technical assistance.
REFERENCES
1. Herlitze, S., Garcia, D. E., Mackie, K., Hille, B., Scheuer, T., and Catterall,
W. A. (1996) Nature 380, 258 –262
2. Ikeda, S. R. (1996) Nature 380, 255–258
3. Catterall, W. A. (2000) Annu. Rev. Cell Dev. Biol. 16, 521–555
4. Dolphin, A. C., Page, K. M., Berrow, N. S., Stephens, G. J., and Canti, C. (1999)
Ann. N. Y. Acad. Sci. 868, 160 –174
5. Jarvis, S. E., and Zamponi, G. W. (2001) Trends Pharmacol. Sci. 22, 519 –525
6. Zhang, J. F., Ellinor, P. T., Aldrich, R. W., and Tsien, R. W. (1996) Neuron 17,
991–1003
7. De Waard, M., Liu, H., Walker, D., Scott, V. E., Gurnett, C. A., and Campbell,
K. P. (1997) Nature 385, 446 – 450
8. Herlitze, S., Hockerman, G. H., Scheuer, T., and Catterall, W. A. (1997) Proc.
Natl. Acad. Sci. U. S. A. 94, 1512–1516
9. Qin, N., Platano, D., Olcese, R., Stefani, E., and Birnbaumer, L. (1997) Proc.
Natl. Acad. Sci. U. S. A. 94, 8866 – 8871
10. Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997)
Nature 385, 442– 446
11. Page, K. M., Stephens, G. J., Berrow, N. S., and Dolphin, A. C. (1997) J. Neurosci. 17, 1330 –1338
12. Furukawa, T., Miura, R., Mori, Y., Strobeck, M., Suzuki, K., Ogihara, Y.,
Asano, T., Morishita, R., Hashii, M., Higashida, H., Yoshii, M., and Nukada,
T. (1998) J. Biol. Chem. 273, 17595–17603
13. Bourinet, E., Soong, T. W., Stea, A., and Snutch, T. P. (1996) Proc. Natl. Acad.
Sci. U. S. A. 93, 1486 –1491
14. Dolphin, A. C. (1998) J. Physiol. (Lond.) 506, 3–11
15. Hille, B. (1994) Trends Neurosci. 17, 531–536
16. Ikeda, S. R., and Dunlap, K. (1999) Adv. Second Messenger Phosphoprotein
Res. 33, 131–151
17. Jones, S. W., and Elmslie, K. S. (1997) J. Membr. Biol. 155, 1–10
18. Birnbaumer, L., Qin, N., Olcese, R., Tareilus, E., Platano, D., Costantin, J.,
and Stefani, E. (1998) Bioenerg. Biomembr. 30, 357–375
19. Bean, B. P. (1989) Nature 340, 153–156
20. Elmslie, K. S., Zhou, W., and Jones, S. W. (1990) Neuron 5, 75– 80
21. Boland, L. M., and Bean, B. P. (1993) J. Neurosci. 13, 516 –533
22. Patil, P. G., de Leon, M., Reed, R. R., Dubel, S. J., Snutch, T. P., and Yue, D. T.
(1996) Biophys. J. 71, 2509 –2521
23. Lee, H. K., and Elmslie, K. S. (2000) J. Neurosci. 20, 3115–3128
24. Colecraft, H. M., Brody, D. L., and Yue, D. T. (2001) J. Neurosci. 21, 1137–1147
25. Herlitze, S., Zhong, H., Scheuer, T., and Catterall, W. A. (2001) Proc. Natl.
Acad. Sci. U. S. A. 98, 4699 – 4704
26. Tsien, R. Y. (1998) Annu. Rev. Biochem. 67, 509 –544
27. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M.,
and Tsien, R. Y. (1997) Nature 388, 882– 887
28. Ellenberg, J., Lippincott-Schwartz, J., and Presley, J. F. (1999) Trends Cell
Biol. 9, 52–56
29. Truong, K., and Ikura, M. (2001) Curr. Opin. Struct. Biol. 11, 573–578
30. Ruiz-Velasco, V., and Ikeda, S. R. (2001) J. Physiol. (Lond.) 537, 679 – 692
31. Mark, M. D., Liu, Y., Wong, S. T., Hinds, T. R., and Storm, D. R. (1995) J. Cell
Biol. 130, 701–710
32. Erickson, M. G., Alseikhan, B. A., Peterson, B. Z., and Yue, D. T. (2001) Neuron
31, 973–985
33. Mark, M. D., Wittemann, S., and Herlitze, S. (2000) J. Physiol. (Lond.) 528,
65–77
34. Wittemann, S., Mark, M. D., Rettig, J., and Herlitze, S. (2000) J. Biol. Chem.
275, 37807–37814
35. Fields, S., and Song, O. (1989) Nature 340, 245–246
36. Fields, S., and Sternglanz, R. (1994) Trends Genet. 10, 286 –292
37. Shioda, T., Andriole, S., Yahata, T., and Isselbacher, K. J. (2000) Proc. Natl.
Acad. Sci. U. S. A. 97, 5220 –5224
38. Pragnell, M., De Waard, M., Mori, Y., Tanabe, T., Snutch, T. P., and Campbell,
K. P. (1994) Nature 368, 67–70
39. Tareilus, E., Roux, M., Qin, N., Olcese, R., Zhou, J., Stefani, E., and Birnbaumer, L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1703–1708
40. Walker, D., Bichet, D., Campbell, K. P., and De Waard, M. (1998) J. Biol.
Chem. 273, 2361–2367
41. Bichet, D., Cornet, V., Geib, S., Carlier, E., Volsen, S., Hoshi, T., Mori, Y., and
De Waard, M. (2000) Neuron 25, 177–190
42. Zamponi, G. W., and Snutch, T. P. (1998) Curr. Opin. Neurobiol. 8, 351–356
43. Walker, D., and De Waard, M. (1998) Trends Neurosci. 21, 148 –154
44. Zamponi, G. W., and Snutch, T. P. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,
4035– 4039
45. Canti, C., Davies, A., Berrow, N. S., Butcher, A. J., Page, K. M., and Dolphin,
A. C. (2001) Biophys. J. 81, 1439 –1451
46. Gerster, U., Neuhuber, B., Groschner, K., Striessnig, J., and Flucher, B. E.
(1999) J. Physiol. (Lond.) 517, 353–368
47. Hohaus, A., Poteser, M., Romanin, C., Klugbauer, N., Hofmann, F., Morano, I.,
Haase, H., and Groschner, K. (2000) Biochem. J. 348, 657– 665
48. Yamaguchi, H., Hara, M., Strobeck, M., Fukasawa, K., Schwartz, A., and
Varadi, G. (1998) J. Biol. Chem. 273, 19348 –19356
49. Garcia, R., Carrillo, E., Rebolledo, S., Garcia, M. C., and Sanchez, J. A. (2002)
J. Physiol. (Lond.) 545, 407– 419
50. Jones, S. W. (2002) J. Physiol. (Lond.) 545, 334
51. Simen, A. A., and Miller, R. J. (2000) Mol. Pharmacol. 57, 1064 –1074
52. Stephens, G. J., Canti, C., Page, K. M., and Dolphin, A. C. (1998) J. Physiol.
(Lond.) 509, 163–169
53. Canti, C., Page, K. M., Stephens, G. J., and Dolphin, A. C. (1999) J. Neurosci.
19, 6855– 6864
54. Clapham, D. E., and Neer, E. J. (1997) Annu. Rev. Pharmacol. Toxicol.
167–203
55. Mende, U., Schmidt, C. J., Yi, F., Spring, D. J., and Neer, E. J. (1995) J. Biol.
Chem. 270, 15892–15898
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volves the C terminus of the ␣1 subunit. This suggests that G
protein binding to the channel induces a new conformational
state of the channel, which may correspond to the reluctant
state proposed by Bean in 1989 (19). Because Ca2⫹ channel
modulation not only involves the binding of the G protein to the
loopI–II and the C terminus but requires other channel domains, i.e. domain I (51, 52) and the N terminus, a reorientation of channel protein domains (in particular the N terminus
where the CFP is attached) seems likely (53). Thus, the next
step in elucidating the G protein modulation of presynaptic
Ca2⫹ channels will be the dynamic analysis of the interaction of
the involved proteins during transmitter application.
In summary, by using a modified MTH system and FRET
measurements, we present evidence for several new findings on
modulation of P/Q-type channels. Ca2⫹ channel ␤ subunits
interact differently than G protein ␤ subunits with ␣1-binding
sites, i.e. the loopI–II, the C terminus, as well as the full-length
␣1 subunit. Ca2⫹ channel ␤ can alter the binding of G␤ on the
loopI–II as indicated by the decrease in FRET, when Ca2⫹
channel ␤ subunits are coexpressed with the ␣1-loopI–II/G␤
FRET pair. On the full-length channel G protein ␤ subunits
alter the interaction between Ca2⫹ channel ␣1 and ␤ subunits,
which probably involves the binding of the G protein to the C
terminus of the Ca2⫹ channel ␣1 subunit. These results imply
that the G protein induces a new closed state of the channel,
which was proposed in the biophysical experiments for presynaptic Ca2⫹ channel modulation (19 –25).