Supplemental Material can be found at:
http://www.jbc.org/cgi/content/full/M414078200/DC1
THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 280, No. 25, Issue of June 24, pp. 23945–23959, 2005
Printed in U.S.A.
G Protein ␤2 Subunit-derived Peptides for Inhibition and
Induction of G Protein Pathways
EXAMINATION OF VOLTAGE-GATED Ca2⫹ AND G PROTEIN INWARDLY RECTIFYING K⫹ CHANNELS*□
S
Received for publication, December 14, 2004, and in revised form, February, 28, 2005
Published, JBC Papers in Press, April 11, 2005, DOI 10.1074/jbc.M414078200
Xiang Li‡, Alexander Hümmer§, Jing Han‡, Mian Xie‡, Katya Melnik-Martinez‡,
Rosa L. Moreno‡, Matthias Buck¶, Melanie D. Mark‡, and Stefan Herlitze‡储
From the ‡Department of Neurosciences and the ¶Department of Physiology and Biophysics, Case Western Reserve
University, School of Medicine, Cleveland, Ohio 44106 and §Flyion GmbH, Waldhäuserstrasse 64,
D-72076 Tübingen, Germany
Transmitter-mediated activation of G proteins causes inhibition of voltage-gated Ca2⫹ channels of the N-, P/Q-, and
R-type. The inhibition is mediated by G protein ␤␥ subunits (1,
2) and involves the direct binding of the G protein to the
channel. Ca2⫹ channels consist of a pore-forming ␣1 subunit
* This work was supported by National Institutes of Health Grant
R01 NS42623 (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 a Table and Figs. 1– 4.
储 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].
This paper is available on line at http://www.jbc.org
and several attached ancillary subunits (for review see Refs.
3–5). During binding to the channel complex, the G protein
presumably stabilizes a conformational state of the channel,
which causes the reluctant opening of the channel (6). This
biophysical model is supported by the fact that the G protein
alters the FRET 1 signal between the FRET pair, the Ca2⫹
channel ␣1, and the Ca2⫹ channel ␤ subunit (7, 8). This mechanism was proposed in earlier biophysical studies on G protein
modulation of the Ca2⫹ channels (9 –14). The binding sites of
the G protein on the channel have been carefully characterized.
G protein ␤␥ subunits bind to the intracellular loop I–II and to
the C terminus of the pore-forming ␣1 subunit (15–21). G protein modulation also involves the N terminus of the Ca2⫹
channel (22). Most surprisingly, little is known about the interaction of the G protein with the channel and how the G
protein domains are involved in modulation. Earlier studies
characterized single point mutations on the G protein ␤ subunit, suggesting that the entire G protein surface rather than a
single amino acid may determine specific effector functions (23,
24). Recently, Doering et al. (25) identified several structural
components of the G protein ␤ subunit as important for the
specificity of G protein modulation of N-type channels.
In this study, we addressed whether we could identify specific G protein domains that either inhibited or induced G
protein modulation of different channel (effector) proteins. By
applying electrophysiological studies and FRET measurements, we identified two structural components of the G protein important for ion channel modulation. Peptides derived
from the N terminus of G␤2, including the G protein ␤␥ interaction site, inhibit G protein modulation of GIRK and voltagedependent Ca2⫹ channels. The inhibitory effect is correlated
with a change in the interaction between G protein ␤ and ␥
subunits. In contrast, protein domains derived from the C
terminus of G␤2 induce G protein modulation of GIRK and
voltage-dependent Ca2⫹ channels. This effect is correlated with
an increase in FRET between GIRK channel subunits mediated
by either the G␤␥ complex or the modulatory peptide. Our data
suggest that peptides derived from G protein ␤ subunits can act
as independent domains for G protein ␤␥ effector protein such
as ion channels. These peptides will thus provide useful tools
1
The abbreviations used are: FRET, fluorescence resonance energy
transfer; GIRK, G protein inwardly rectifying potassium channel; FR,
FRET ratio; YFP, yellow fluorescent protein; CFP, cyan fluorescent
protein; HEK, human embryonic kidney; ACh, acetylcholine; AChR,
acetylcholine receptor; TTX, tetrodotoxin; ANOVA, analysis of variance; TEA, triethanolamine; GTP␥S, guanosine 5⬘-3-O-(thio)triphosphate; PMA, 4␣-phorbol 12-myristate 13-acetate; PKC, protein kinase
C; HA, hemagglutinin.
23945
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Voltage-gated Ca2ⴙ channels of the N-, P/Q-, and Rtype and G protein inwardly rectifying Kⴙ channels
(GIRK) are modulated via direct binding of G proteins.
The modulation is mediated by G protein ␤␥ subunits.
By using electrophysiological recordings and fluorescence resonance energy transfer, we characterized the
modulatory domains of the G protein ␤ subunit on the
recombinant P/Q-type channel and GIRK channel expressed in HEK293 cells and on native non-L-type Ca2ⴙ
currents of cultured hippocampal neurons. We found
that G␤2 subunit-derived deletion constructs and synthesized peptides can either induce or inhibit G protein
modulation of the examined ion channels. In particular,
the 25-amino acid peptide derived from the G␤2 N terminus inhibits G protein modulation, whereas a 35amino acid peptide derived from the G␤2 C terminus
induced modulation of voltage-gated Ca2ⴙ channels and
GIRK channels. Fluorescence resonance energy transfer (FRET) analysis of the live action of these peptides
revealed that the 25-amino acid peptide diminished the
FRET signal between G protein ␤2␥3 subunits, indicating a reorientation between G protein ␤2␥3 subunits in
the presence of the peptide. In contrast, the 35-amino
acid peptide increased the FRET signal between
GIRK1,2 channel subunits, similarly to the G␤␥-mediated FRET increase observed for this GIRK subunit
combination. Circular dichroism spectra of the synthesized peptides suggest that the 25-amino acid peptide is
structured. These results indicate that individual G protein ␤ subunit domains can act as independent, separate
modulatory domains to either induce or inhibit G protein modulation for several effector proteins.
23946
Peptides for Modulating Ion Channels and G Protein Pathways
for characterizing and manipulating G protein pathways in
living cells.
MATERIALS AND METHODS
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Construction of pYFP-nuc/HA-C1 for Co-expression Studies—All G
protein constructs were cloned into a new expression vector (pYFP-nuc/
HA-C1). In this vector, YFP is under the control of a second and
separate cytomegalovirus promoter and tagged with a nuclear localization signal, which allows the constitutive expression and transport of
YFP to the nucleus as well as the expression of the nonfused protein or
peptide of interest. Thus, cells with a nuclear targeted YFP contain the
cDNA encoding for the peptide of interest. For constructing the pYFPnuc/HA-C1 vector used for co-expression of the G␤2-derived deletion
constructs, two SV40 nuclear localization signals were added 3⬘ inframe into the YFP coding sequence in pEYFP-C1 (Clontech) (pYFPnuc). Then the cytomegalovirus promoter, polylinker region, and bovine
growth hormone polyadenylation signals were PCR-amplified from
pcDNA3.1 and subcloned into the AflII site of the pYFP-nuc-C1. An HA
tag polylinker was directly cloned into the Nhe-PmeI site of the amplified polylinker via blunt end cloning.
Cell Culture—Human embryonic kidney (HEK) 293 cells (tsA201
cells) were transfected with 2 ␮g of the indicated DNAs in each set of
experiments with Quantum PrepR CytofecteneTM Transfection Reagent
(Bio-Rad) and incubated for 24 –36 h. For non-live cell FRET measurements cells were embedded in Fluoromount (133 mM Tris/HCl, 30%
glycerol, 11% Mowiol, 2% 1,4-diazabicyclo[2.2.2]octaue).
Constructs—Human G protein ␤2 constructs (G␤2-full-length; 1–100,
101–200, 201–340, 1–50, 51–100, 201–270, 270 –340, 1–25, 26 –50, 201–
235, 236 –270, 271–305, and 306 –340) were PCR-amplified and subcloned in-frame into pYFP-nuc/HA-C1 for electrophysiological recordings and into pEYFP-C1 for localization within the cell. The cellular
localization of the YFP-tagged G␤ constructs is shown in the supplemental Figs. 1–3. Other constructs, i.e. G␥3 and GIRK1 and GIRK2,
were either PCR-amplified or if restriction sites were suitable cloned
into either pECFP-C1, pECFP-C2, pECFP-N1, pEYFP-C1, and
pEYFP-N1 and as non-tagged versions into pcDNA3.1. All PCR-amplified products were verified by sequencing, and protein expression was
verified by Western blot analysis after expression of the constructs in
COS-7 cells. Western blots of transfected COS-7 cells (with G␤ constructs) were performed with a polyclonal anti-green fluorescent protein antibody (Molecular Probes, Inc., Eugene, OR) according to standard procedures as described by Mark et al. (26). Muscarinic AChR M2
(human) was cloned into pcDNA3.1(⫹) and purchased from UMR cDNA
resource center (Rolla, MO). Reverse transcription and PCR amplification were used to clone ␥2 (stargazin) from rat brain RNA using the
following oligonucleotide primers: sense, 5⬘atggggctgtttgatcgaaggtgttcaaatg3⬘; antisense, 5⬘tcatacgggcgtggtccggcggttggctgt3⬘. The nucleotide sequence was verified to the rat ␥2 (GenBankTM accession number
NM_053351) by cDNA sequencing.
FRET Measurements—FRET measurements were performed by using a modified version of the 3-cube FRET method (27) as described in
Hümmer et al. (7). Briefly, the average FRET signal (FRET ratio) was
calculated using the following equation: FR ⫽ (FRETfluorescence ⫺ RD1 ⫻
CFPfluorescence)/(RA1 ⫻ (YFPfluorescence)), where the constants RD1 and
RA1 were derived from the CFP and YFP constructs expressed alone in
HEK293 cells. The average FRET signal was confirmed for each FRET
pair by acceptor photobleaching. Here FRET samples were exposed 30
min to fluorescent light through the YFP filter cube (excitation, HQ
500/20; band pass filter 535/30; beam splitter JP4 PC 535/30), and the
FRET efficiency (Eff) was calculated according to the increase in CFP
emission: Eff ⫽ (1 ⫺ CFPfluorescence before/CFPfluorescence after) ⫻ 100%.
In our experimental setting, we calculated that the YFP signal was
reduced by 70% after 30 min of light exposure. CFP, FRET, and YFP
pictures were analyzed with Volocity software (Improvision).
For live cell FRET measurements, cells were incubated in extracellular solution containing (in mM) 172 NaCl, 2.4 KCl, 10 HEPES, 10
glucose, 4 CaCl2, 4 MgCl2 (pH 7.3). CFP, FRET, and YFP pictures were
taken before and 30 min after peptide application. For calculation of the
FRET ratio over the whole image, images were saved as TIFF files and
processed with Igor software (WaveMetrics, Inc.). The FRET equation
(FR ⫽ (FRETfluorescence ⫺ RD1 ⫻ CFPfluorescence)/(RA1 ⫻ (YFPfluorescence))
was applied to every pixel of the image for all three images. FR within
these images was encoded by 256 gray values. These values were
converted into color values, where the intensity of the color represents
the FR value. For FR calculation, the gray scale value outside of the cell
of interest was determined (typically under ⬍33 gray scale values). This
value was subtracted from the whole image. The FR image of Figs. 6 and
7 without background subtraction are shown in the supplemental Fig. 4.
Fluorescence was detected with a conventional fluorescence microscope
(Leica DMLFSA) equipped with one filter wheel on the emission site
controlled by a Sutter Lambda10-2 and a Sutter DG5 light source on the
excitation site. For CFP and YFP detection, the following filters were
used: CFP, excitation, D436/10; emission, D470/30; YFP, excitation, HQ
500/20; band pass filter 535/30; beam splitter JP4 PC. All filters were
obtained from Chroma. The following objectives were used: a 63⫻ water
corrected 1.2NA objective for fixed FRET samples and a 63⫻ water
corrected long distance 0.9 NA objective for live cell recordings.
Peptide Delivery—The synthesized peptides (pep1–25, pep271–305,
and pep1–25scrambled) were dissolved in H2O and kept as 10 mM stock
solution at ⫺20 °C. From this stock solution further dilutions (100 ␮M)
were prepared in the intracellular solutions used for GIRK, P/Q-type,
and neuronal non-L-type channel recordings for the intracellular application of the peptides. The peptides have the following sequences:
pep1–25, MSELEQLRQEAEQLRNQIRDARKAC; pep1–25scrambled,
MRLQAEQRNCSLEIRLEQDKEQARA; and pep271–305, CGITSVAFSRSGRLLLAGYDDFNCNIWDAMKGDRA.
For peptide transfection, the Chariot transfection reagent (Active
Motif, Carlsbad, CA) was used. According to the instructions manual of
the manufacturer, 500 ng of peptide was mixed in the transfection
reagent, incubated for 30 min at room temperature, and then added
directly to the imaged cells in the recording chamber. Unless otherwise
indicated, electrophysiological recordings were performed 20 min after
chariot/peptide application, and FRET measurements were performed
30 min after chariot/peptide application.
Circular Dichroism Spectra—Peptides were dissolved in H2O. Peptide concentration were 29 ␮M for pep1–25 and 13 ␮M for pep271–305.
Far-UV circular dichroism spectra were measured on an Aviv instrument in a thermostatted 0.3-cm quartz cell (Helma). Measurements
were carried out at 25 and 5 °C. A single scan was acquired at 0.5 nm
intervals between 250 and 185 nm with an averaging time of 2 s. The
signal of buffer without peptide was subtracted from each measurement. Molar ellipticity was calculated as [⌰] ⫽ CD signal in millidegree
(peptide concentration M ⫻ cell length cm ⫻ number of residues). The
helical content was estimated from the ellipticity at 222 nm using the
procedure of Rohl and Baldwin (28).
Electrophysiology—For P/Q-type channel recordings in HEK293
cells, Ca2⫹ channel subunits (␣12.1, ␤1b, and ␣2␦) and the G protein
subunit (G␤2) or G protein ␤ fragments were co-expressed in tsA201
cells, and Ca2⫹ channel-mediated Ba2⫹ currents were measured and
analyzed as described previously (1, 29, 30). The following bath pipette
solutions were used: for external solution (mM), 100 Tris, 4 MgCl2, 10
BaCl2 with pH adjusted to 7.3 with methanesulfonic acid; for internal
solution (mM), 120 aspartic acid, 5 CaCl2, 2 MgCl2, 10 HEPES, 10 EGTA
and 2 Mg-ATP with pH adjusted to 7.3 with CsOH. Endogenous G
proteins were activated by adding 0.6 mM GTP␥S to the internal solution. Tail currents (supplemental Table) were fitted with a single exponential using Igor software.
PKC inhibitor peptide (Sigma) was dissolved in the intracellular
recording solution at a concentration of 100 ␮M. 4␣-Phorbol 12-myristate 13-acetate (PMA) (Sigma) was dissolved in Me2SO as a 1 mM stock
solution. 2 ␮l of this solution was added to 2 ml of the extracellular
bathing solution 5 min prior to the experiment (final concentration 1 ␮M).
For GIRK channel recordings in HEK293 cells, GIRK channel subunits (GIRK1 and GIRK2) were co-expressed with or without
mAChR-M2 receptors in tsA201 cells, and GIRK channel-mediated K⫹
currents were elicited by 50-ms voltage ramps from ⫺100 to ⫹50 mV.
The holding potential was 0 mV. ACh (Sigma) was dissolved in the
external solution to a final concentration of 10 ␮M. The following solutions were used: for external solution (mM), 20 NaCl, 120 KCl, 2 CaCl2,
1 MgCl2, 10 HEPES-NaOH, pH 7.3 (KOH); for internal solution (mM),
100 potassium aspartate, 40 KCl, 5 MgATP, 10 HEPES-KOH, 5 NaCl,
2 EGTA, 2 MgCl2, 0.01 GTP, pH 7.3 (KOH).
For non-L-type channel recordings in cultured hippocampal neurons,
Sprague-Dawley rat pups (Zivic Miller Inc.) were sacrificed at postnatal
day 1, and hippocampal neurons were dissociated and cultured on
coverslips according to a modified protocol from Bekkers et al. (31) and
described in Wittemann et al. (30). 10 –14-Day-old neurons were used
for Ca2⫹ channel recordings. The following solutions were used: for
internal solution (mM), 120 N-methyl-D-glucamine, 20 TEACl⫺, 10
HEPES, 1 CaCl2, 14 phosphocreatine (Tris), 4 Mg-ATP, 0.3 Na2GTP, 11
EGTA, pH 7.2, with methanesulfonic acid; for external solution (mM),
145 TEA, 10 HEPES, 10 CaCl2, 15 glucose, pH 7.4, with methanesulfonic acid. In addition, 1 ␮M TTX (Sigma) and 5 ␮M nimodipine (Sigma)
Peptides for Modulating Ion Channels and G Protein Pathways
23947
were added to the external solution to block voltage-dependent Na⫹
channels and L-type Ca2⫹ channels.
All whole cell patch clamp recordings (32) were performed with an
EPC9 amplifier (HEKA). Currents were digitized at 10 kHz and filtered
with the internal 10-kHz three-pole Bessel filter (filter 1) in series with
a 2.9-kHz 4-pole Bessel filter (filter 2) of the EPC9 amplifier. Series
resistances were partially compensated between 70 and 90%. Leak and
capacitative currents were subtracted by using hyperpolarizing pulses
from ⫺60 to ⫺70 mV with the p/4 method.
Statistical significance throughout the experiments was tested with
ANOVA using Igor Software. Standard errors are mean ⫾ S.E.
RESULTS
G protein modulation of N-, P/Q-, and R-type channels is
mediated by G protein ␤␥ subunits (1, 2) and most likely
involves the direct binding of the G protein to the channel (16,
18, 19). Co-expression of G␤2 together with the P/Q- or N-type
channel ␣1, ␤1b, and ␣2␦ subunits results in channel modulation, i.e. a shift in the voltage dependence of activation, a more
shallow activation curve, and prepulse facilitation (1, 29, 33).
In this study we addressed whether deletion fragments of the G
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FIG. 1. Induction of P/Q-type channel G protein modulation by G protein ␤2 deletion constructs. A, examples of Ba2⫹ current traces
in the presence and absence of G protein ␤ peptides with ATP in the intracellular recording solution. Ba2⫹ currents were elicited from a holding
potential of ⫺60 mV by a 5-ms test pulse to ⫹20 mV. After 1 s a 10-ms prepulse to ⫹150 mV was applied, and a second 5-ms test pulse to ⫹20
mV was elicited after stepping back for 2 ms to ⫺60 mV. The facilitation ratios throughout the experiments were determined by dividing the peak
current of test pulse 2 by the peak current of test pulse 1. The current traces indicate that in the presence of G␤2 full-length (middle trace) and
G␤-(271–305) (lower trace) facilitation is increased over facilitation in the absence of activated G proteins (upper trace). B, facilitation ratios were
determined as described in A for whole cell patch clamp recordings of P/Q-type channels expressed in HEK293 cells transfected without or with
G␤2 or the indicated G␤2 peptide fragments. Numbers on the left indicate the length of the G␤2 deletion constructs in amino acids, i.e. 70 or 35
amino acids. The number in parentheses indicates the number of experiments. **, p ⬍ 0.01, ANOVA. C, voltage dependence of activation for
P/Q-type channels expressed in HEK293 cells co-expressed with or without G protein ␤ peptides. Voltage pulses were elicited from a holding
potential of ⫺60 mV to various step potentials in 5-mV steps. Tail currents were related to the largest tail current and plotted in %. The voltage
dependence of activation was determined by a Boltzmann fit according to I/Imax ⫽ 1/(1 ⫹ exp((Vh ⫺ V)/K))), where I/Imax is the normalized tail
current; Vh is the half-activation voltage, and K determines the steepness of the curve. The current traces and plots indicate that in the presence
of G␤-(201–340) and for the control where GTP␥S was used in the intracellular solution, the voltage-dependent activation curve of the expressed
P/Q-type channels is shifted to more positive potentials in comparison to currents recorded with an intracellular solution containing only ATP. The
inset shows the tail currents of the traces above at ⫹20 and ⫹65 mV at a larger time scale to illustrate that tail currents were fast throughout the
experiments (see also the supplemental table). The activation curves represent single examples of cells for the indicated condition. D, voltage
dependence of activation (half-activation voltage (Vh)) for P/Q-type channels transfected without or with G␤2 or the indicated G␤2 peptide
fragments. Numbers on the left indicate the length of the G␤2 deletion constructs in amino acids, i.e. 70- or 35-amino acids. The number in
parentheses indicates the number of experiments. *, p ⬍ 0.05; **, p ⬍ 0.01, ANOVA.
23948
Peptides for Modulating Ion Channels and G Protein Pathways
FIG. 2. Reduction of P/Q-type channel inhibition by G protein
␤2 deletion constructs. A, Ba2⫹ current traces in the presence or
absence of G protein ␤ deletion constructs with GTP␥S in the intracellular recording solution. Ba2⫹ currents and facilitation ratio were elicited and determined as in Fig. 1A. The current traces indicate that the
P/Q-type channel facilitation ratio is decreased in the presence of G␤(1–25) but not in the presence of G␤-(51–100). B, facilitation ratios were
determined as described in Fig. 1A for P/Q-type channels expressed in
HEK293 cells in the presence of GTP␥S in the absence or presence of
the indicated G␤2 deletion constructs. The number in parentheses indicates the number of experiments. **, p ⬍ 0.01, ANOVA.
P/Q-type channel modulation was not altered in the presence of
the G␤-(101–200) and G␤-(201–340) deletion constructs.
We next analyzed smaller deletion constructs of the first 100
amino acids of G␤2. Whereas the 1–50 construct reduced facilitation significantly, the 51–100 peptide did not. Furthermore,
the two 25 amino acids long constructs, G␤-(1–25) and G␤-(26 –
50) (Fig. 2B) were sufficient for a reduction in modulation.
Thus, our data suggest that the N-terminal region of the G
protein ␤ subunit can diminish P/Q-type channel modulation in
HEK293 cells.
Application of Synthesized G Protein ␤2-derived Peptides,
Pep1–25 and Pep271–305, Inhibit or Induce G Protein Modulation of P/Q-type, Neuronal Non-L-type, and GIRK Channels—
In our overexpression study, we found that the N-terminal
region of the G protein ␤2 subunit inhibits G protein modulation of P/Q-type channels, whereas the C-terminal part induces
modulation. We next investigated if direct application of synthesized peptides reproduced the effects observed with the
overexpression of the G␤ constructs. We therefore synthesized
one 25-amino acid-long peptide derived from the N terminus of
G␤2 containing amino acids 1–25 (pep1–25) and one 35-amino
acid long peptide derived from the C terminus containing
amino acids 271–305. The reason for choosing these regions
was that we observed strong effects on inhibition and induction
of P/Q-type channel modulation for the corresponding cDNA
constructs (see Figs. 1 and 2). As a control peptide, we synthesized a peptide that contained the amino acids of pep1–25 in a
random order (pep1–25scrambled). In addition to testing these
peptides on the P/Q-type channel modulation in HEK293 cells,
we also wanted to investigate if these peptides are specific for
P/Q-type channels or if other G protein ␤␥-modulated channels,
e.g. neuronal non-L-type channels and GIRKs, are also
affected.
We first tested the effects of the synthesized peptides on
Ca2⫹ channel modulation, and we applied the peptides to the
intracellular side of the P/Q-type channels expressed in
HEK293 cells. Intracellular application of pep1–25 reduced the
GTP␥S-mediated P/Q-type channel facilitation significantly,
whereas neither the pep271–305 nor the pep1–25scrambled
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protein ␤ subunit were able to either induce or block G protein
modulation of the P/Q-type channels when co-expressed together in a heterologous expression system. We divided the
human G protein ␤2 subunit consisting of 340 amino acids into
three domains encoding amino acids 1–100, 101–200, and 201–
340. We then further subdivided these protein domains into
smaller fragments to identify small protein regions that can be
synthesized and directly applied to ion channels (e.g. GIRK
channels and neuronal Ca2⫹ channels) and to investigate the
potential of these peptides in manipulating G protein-modulated effector proteins. The distribution of the YFP-tagged G␤
constructs in HEK293 cells is shown in the supplemental
Figs. 1–3.
C-terminally Derived G Protein ␤2 Deletion Constructs Induce P/Q-type Channel Modulation in HEK293 Cells—We first
investigated if G protein ␤2-derived constructs induce modulation of P/Q-type channels comparable with levels observed with
the G protein ␤2 subunit alone (Fig. 1, A, middle trace, and B).
G protein modulation was measured as prepulse facilitation, a
characteristic of G protein-mediated voltage-dependent inhibition of the Cav2 channel family (9). To investigate prepulse
facilitation, we compared the peak current amplitude of currents elicited by the two test pulses throughout the experiments. The test pulses were separated by a depolarizing pulse
to relief G protein modulation from the Ca2⫹ channel.
As indicated in Fig. 1, A and B, prepulse facilitation of
P/Q-type channels was observed in the presence of the G␤(201–340) but not in the presence of G␤-(1–100) and G␤-(101–
200). Thus, the C terminus of the G protein ␤2 subunit induces
P/Q-type channel modulation.
We further divided the G␤-(201–340) region into smaller
constructs: the first is 70 amino acids long, and the second is 35
amino acids long. As shown in Fig. 1B, the 70-amino acid-long
G␤-(201–270) and G␤-271–340 peptides both increased P/Qtype channel facilitation. Investigation of the 35-amino acidlong constructs on P/Q-type channel modulation revealed that
only G␤-(236 –270), G␤-(271–305), and G␤-(305–340) induced
significant modulation over background facilitation, whereas
the G␤-(201–235) had no effect.
We next investigated whether the modulatory G␤2-derived
constructs induce a shift in the voltage dependence of activation, as would be predicted for the voltage-dependent G protein
modulation of P/Q-type channels. In order to do so we analyzed
the tail currents after the voltage pulse for the applied voltage
pulse protocols. Indeed, as shown in Fig. 1, C and D, an ⬃8-mV
shift in the voltage dependence of activation to more depolarized potentials was observed for P/Q-type channels expressed
with G␤-(201–340), G␤-(201–270), G␤-(271–340), G␤-(271–
305), and G␤-(305–340) in comparison to channels expressed
without the G␤ fragments. In contrast, G␤-(1–100), G␤-(101–
200), and G␤-(201–235), which did not induce facilitation, also
did not shift the voltage dependence of activation to more
positive potentials. G␤-(236 –270) shifted P/Q-type channel activation slightly to more positive potentials. The data suggest
that co-expression of the protein fragments derived from the
C-terminal end of the G protein ␤2 subunits induce P/Q-type
channel modulation comparable with the modulatory effects
described for the full-length G␤2 subunit.
N-terminally Derived G Protein ␤2 Deletion Constructs Reduce P/Q-type Channel Modulation in HEK293 Cells—We next
investigated whether the modulation of P/Q-type channels is
reduced in the presence of G protein ␤ fragments when endogenous G proteins are activated by GTP␥S. As shown in Fig. 2B, we
observed that the first 100 amino acids (G␤-1–100) of the G
protein ␤ subunit decreased significantly G protein modulation
when endogenous G proteins were activated by GTP␥S, whereas
Peptides for Modulating Ion Channels and G Protein Pathways
23949
had any effect (Fig. 3, A and B). To rule out the possibility that
pep1–25 acts through activation of PKC, which would also
result in a reduction in P/Q-type channel modulation (14, 34),
we performed the pep1–25 experiments in the presence of the
PKC inhibitor peptide. In these experiments P/Q-type facilitation was still reduced when pep1–25 and PKC inhibitor peptide
were co-applied to the channel, suggesting that pep1–25 does
not reduce channel modulation via activation of PKC. To demonstrate that the PKC inhibitor peptide can antagonize the
action of PKC on the P/Q-type channel, we tested the PKC
inhibitor peptide in the presence of the phorbol ester PMA.
Extracellular application of 1 ␮M PMA to P/Q-type channels 5
min prior to the experiments reduced GTP␥S-mediated modulation to control levels, whereas in the presence of 100 ␮M PKC
inhibitor peptide in the intracellular solution and 1 ␮M PMA in
the extracellular solution the GTP␥S-mediated facilitation was
not reduced (Fig. 3B). We next tested the effects of pep271–305.
As shown in Fig. 3, A and B, pep271–305 increased P/Q-type
facilitation comparable with the facilitation ratio measured in
the presence of the full-length G␤2 subunits, whereas pep1–25
or pep1–25scrambled had no effect. The time course of the
peptide-mediated channel modulation occurred within 1 min
after seal formation and was significantly faster than the inhibitory effect of pep1–25 (Fig. 3C). We next investigated if the
modulatory effect of pep271–305 and G␤2 is occluded by the
pep1–25 peptide. Application of pep1–25 neither significantly
reduced the pep271–305 nor the G␤2 full-length (fl) induced
prepulse facilitation of the P/Q-type channels, suggesting that
pep271–305 acted independently from pep1–25. The results
indicate that the pep1–25 inhibits and the pep271–305 induces
P/Q-type channel modulation.
The pep271–305-mediated P/Q-type channel modulation re-
sembles channel modulation induced by G␤2 subunits. To investigate if the effects on channel modulation are analogous to
the action of G␤2fl, we characterized more extensively the
properties of the pep271–305 peptide on Ca2⫹ channel modulation according to studies performed previously (see Refs. 35
and 36). We first compared the shift in the voltage dependence
of activation between GTP␥S-, G␤2fl-, and pep271–305-modulated channels, and we found that in all cases the voltage
dependence of activation is shifted to more positive potentials
(Fig. 4, A and B). Application of high positive prepulses to ⫹150
mV shifted the voltage dependence of activation curve for
GTP␥S-, G␤2fl-, and pep271–305-modulated channels by 7–9
mV to more negative potentials, and the curves became steeper
(Fig. 4, C and D). This result indicates that the amount of
inhibition relief is comparable between G␤2fl and pep271–305.
We next compared the voltage-dependent relief from channel
inhibition by increasing the voltage of the prepulse. In this
experiment, we increased the test pulse duration to 20 ms to
reveal possible changes on the steady state currents by
pep271–305 in comparison to GTP␥S and G␤2fl (Fig. 4E). 50%
of G protein relief is caused by prepulse voltages around 60 mV
for GTP␥S-, G␤2fl-, and pep271–305-modulated P/Q-type channels (Fig. 4F). No differences in the facilitation ratios of
GTP␥S-, G␤2fl-, and pep271–305-modulated channels were observed when ratios where taken after 5 or 20 ms during the test
pulse after the prepulse (Fig. 4G). According to Arnot et al. (36),
the G␤2 subunits induce a kinetic slowing of P/Q-type channel
currents by a factor of around 1.4 when the activation time of
the test pulse after the prepulse was compared with the activation time of the test pulse before the prepulse. Comparing
5-ms test pulses to ⫹20 mV before and after application of a
prepulse to ⫹150 mV between channels modulated by G␤2 or
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
FIG. 3. G protein ␤2-derived peptides (pep1–25 and pep271–305) reduce or induce P/Q-type channel
modulation in HEK293 cells. A,
pep1–25 reduces and pep271–305 induces
P/Q-type channel modulation in HEK293
cells. Ba2⫹ current traces are shown in
the presence or absence of G protein ␤
peptides without (upper trace) or with
GTP␥S (lower trace) in the intracellular
recording solution. Ba2⫹ currents and facilitation ratios were elicited and determined as in Fig. 1A. The current traces
indicate that in the presence of pep271–
305 P/Q-type channel facilitation is induced, whereas pep1–25 reduces the
GTP␥S-mediated P/Q-type channel facilitation. Peptides were applied through the
patch pipette in a concentration of 100
␮M. B, facilitation ratios were determined
as described in Fig. 1A for P/Q-type channels expressed in HEK293 cells in the
absence or presence of GTP␥S and of the
indicated G␤2-derived peptides (pep1–25
and pep271–305). Peptides were applied
through the patch pipette in a concentration of 100 ␮M. The number in parentheses
indicates the number of experiments. **,
p ⬍ 0.01, ANOVA. C, example traces of
the time course of increase and decrease
of P/Q-type channel facilitation in the
presence of pep1–25 (GTP␥S in pipette
solution) and pep271–305. Facilitation
was determined as described in Fig. 1A.
Each point represents the peak current
facilitation at the given time point. D,
time constants of the increase and decrease of P/Q-type channel facilitation in
the presence of pep1–25 (GTP␥S in pipette solution) and pep271–305. The
number in parentheses indicates the number of experiments. **, p ⬍ 0.01, ANOVA.
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Peptides for Modulating Ion Channels and G Protein Pathways
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
FIG. 4. G protein ␤2-derived peptide pep271–305 and full-length G␤2 induce analogous effects on P/Q-type channel modulation. A,
voltage dependence of activation for P/Q-type channels expressed in HEK293 cells in the presence or absence (control) of GTP␥S, G␤2fl, or
pep271–305. Voltage pulses were elicited from a holding potential of ⫺60 mV to various step potentials in 5-mV steps. Tail currents were related
to the largest tail current and plotted in %. The voltage dependence of activation was determined by a Boltzmann fit according to I/Imax ⫽ 1/(1 ⫹
exp((Vh ⫺ V)/K))), where I/Imax is the normalized tail current; Vh is the half-activation voltage, and K determines the steepness of the curve. The
plots indicate that in the presence of G␤2fl or pep271–305 and where GTP␥S was used in the intracellular solution the voltage-dependent activation
curve of the expressed P/Q-type channels is shifted to more positive potentials in comparison to currents recorded with an intracellular solution
containing only ATP (see also supplemental data for tail current kinetics). The activation curves represent single examples of cells for the indicated
condition. B, voltage dependence of activation (half-activation voltage (Vh)) for P/Q-type channels expressed in HEK293 cells in the presence or
absence (control) of GTP␥S, G␤2fl, or pep271–305. The number in parentheses indicates the number of experiments. **, p ⬍ 0.01, ANOVA. C,
voltage dependence of prepulse facilitation for P/Q-type channels expressed in HEK293 cells in the presence of G␤2fl or pep271–305. A 5-ms voltage
pulse (test pulse 1) was elicited from a holding potential of ⫺60 mV. After 1 s, a 10-ms prepulse to ⫹150 mV was applied. After stepping back for
2 ms to ⫺60 mV, a second 5-ms voltage pulse was elicited. The potential of the test pulse was increased in 5-mV steps from ⫺10 to ⫹65 mV. Tail
currents were related to the largest tail current and plotted in %. As described in A, the voltage dependence of activation was determined by a
Boltzmann fit for the activation curve before and after the prepulse. The plots indicate that the prepulse shifts the voltage dependence of activation
to more negative potentials for P/Q-type channels co-expressed with G␤2fl or for channels where the pep271–305 was applied through the patch
Peptides for Modulating Ion Channels and G Protein Pathways
difficult, and the results have to be interpreted carefully. However, the effects of the peptides on neuronal Ca2⫹ channels
resemble the effects observed on P/Q-type channels expressed
in HEK293 cells.
We next analyzed the action of these peptides on the G
protein modulation of GIRK channels. We co-expressed GIRK1
and GIRK2 subunits together with the muscarinic AChR-M2,
which couples to pertussis toxin-sensitive G proteins and activates GIRK channels (38). We first showed that application of
10 ␮M ACh increased inward basal GIRK currents by 54%
measured at ⫺80 mV (Fig. 6A, top trace). Direct application of
the pep1–25 peptide through the patch pipette decreased the
basal GIRK current by 15% (Fig. 6A, middle trace). In the presence of pep1–25, application of ACh only increased the GIRK
current by 4%, indicating that the pep1–25 peptide blocks G
protein-coupled receptor-mediated activation of GIRK channels. This effect was peptide-specific, because application of
pep1–25scrambled reduced basal GIRK current by 2%, but
extracellular application of ACh still increased the GIRK current by 31%. In contrast to the inhibitory action of pep1–25 on
GIRK channel activity, the C-terminally derived peptide
pep271–305 increased the basal GIRK current by 26%. Application of ACh 10 min after peptide application further increased the GIRK current (Fig. 6A, bottom trace) by 60%. The
time constants for the peptide delivery through the patch pipette was significantly faster for pep271–305 in comparison to
the pep1–25, whereas peptide delivery via chariot transfection
reagent had a time constant of ⬃200 s for both peptides
(pep1–25 and pep271–305) (Fig. 6D). These results indicate
that the pep271–305 increases basal GIRK currents.
In summary, the G␤2-derived N-terminal 25-amino acid-long
peptide reduces GIRK and Ca2⫹ channel modulation, whereas
the synthesized G␤2-derived C-terminal 35-amino acid-long
peptide induces GIRK activation and Ca2⫹ channel modulation. The effects seem to be peptide-specific because the inhibition of Ca2⫹ channel modulation is only achieved by pep1–25
but not by pep271–305 or pep1–25scrambled, whereas the induction of Ca2⫹ channel modulation is only mediated by
pep271–305 but not by the pep1–25 or pep1–25scrambled.
Peptide Pep1–25 Decreases the FRET Signal between G Protein ␤2 and ␥3 Subunits in Living HEK293 Cells—In order to
investigate a possible mechanism for the reduction in GIRK
pipette (see also supplemental data for tail current kinetics). The activation curves represent single examples of cells for the indicated condition.
D, shift in the voltage dependence of activation between activation curves before and after the prepulse for non-modulated P/Q-type channels or
channels modulated by GTP␥S, G␤2fl, or pep271–305. For calculating ⌬Vh (mV), the half-activation voltage of the activation curve after the
prepulse was subtracted from the half-activation voltage of the activation curve before the prepulse (⌬Vh ⫽ Vh2 ⫺ Vh1). The number in parentheses
indicates the number of experiments. **, p ⬍ 0.01, ANOVA. E, voltage dependence of relief from G protein inhibition for P/Q-type channels
expressed in HEK293 cells in the presence of G␤2fl or pep271–305. From a holding potential of ⫺60 mV, a prepulse was elicited for 10 ms to various
step potentials (step size 5 mV). After 10 ms, a 20-ms test pulse to ⫹20 mV was elicited. Test current traces are shown for prepulses to ⫺10, ⫹20,
⫹120, and ⫹150 mV for P/Q-type channels modulated by G␤2fl and pep271–305. F, voltage dependence of relief from G protein inhibition for
P/Q-type channels expressed in HEK293 cells in the presence of GTP␥S, G␤2fl, or pep271–305. The amplitude of test currents was measured after
15 ms and normalized to the current amplitude produced by the most positive prepulse (⫹150 mV). The curves were fitted with a sigmoidal curve,
and the voltages to elicit half-maximal current increase were determined (see supplemental Table). No difference between the voltage-dependent
relief from G protein inhibition was observed. G, comparison between facilitation ratios measured after 5 or 20 ms during the test pulse for P/Q-type
channels expressed in HEK293 cells in the presence or absence (control) of GTP␥S, G␤2fl, or pep271–305. From a holding potential of ⫺60 mV, 10
ms after a 10-ms prepulse to ⫺10 or ⫹150 mV, a test pulse was applied to 20 mV for 20 ms. Facilitation ratio was determined at different time
points of the test pulse (5 and 20 ms) by dividing the test pulse current after a prepulse to ⫺10 mV by the test pulse current after a prepulse to
⫹150 mV (see E). The number in parentheses indicates the number of experiments. H, comparison between the re-inhibition and recovery from G
protein inhibition of P/Q-type channels modulated by G␤2fl or pep271–305. To investigate the re-inhibition of P/Q-type channels, Ba2⫹ currents
were elicited from a holding potential of ⫺60 mV by a test pulse to ⫹20 mV. After 1 s a 10-ms prepulse to ⫹150 mV was applied, and a second 5-ms
test pulse to ⫹20 mV was elicited after stepping back to ⫺60 mV. The time between prepulse and second test pulse was increased between 1 and
195 ms by 1 ⫹ 1.5^x, where x is the number of sweeps. The facilitation ratio was plotted versus the time interval between prepulse and test pulse.
A single exponential fit of this curve was used to determine the time constant of re-inhibition. To investigate the recovery from inhibition, the same
voltage protocol as described above was used with two alterations. The time of the prepulse but not the time interval between prepulse and test
pulse was increased between 1 and 20 ms by 1 ⫹ 1.5^x, where x is the number of sweeps. The time interval between prepulse and test pulse was
2 ms. The facilitation ratio was plotted versus the time interval between prepulse and test pulse. A single exponential fit of this curve was used
to determine the time constant of recovery of P/Q-type channel inhibition. The re-inhibition and recovery curves represent single examples of cells
for the indicated condition. I, time constants for the re-inhibition and recovery from inhibition for P/Q-type channels modulated by GTP␥S, G␤2fl,
or pep271–305. Time constants were determined as described in H. The number in parentheses indicates the number of experiments. *, p ⬍ 0.05,
ANOVA.
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
pep271–305, the activation kinetics were slowed by a factor of
1.2 (supplemental Table). We next compared the inhibition
kinetics between the modulated channels. The recovery of P/Qtype channel modulation was analyzed by increasing the prepulse duration. For GTP␥S-, G␤2fl-, and pep271–305-modulated channels, the recovery time constants were 7– 8 ms (Fig.
4, G and H) These values are in agreement with results published previously for G␤2fl-mediated inhibition of P/Q-type
channels (36). The time course of G protein re-inhibition was
analyzed by increasing the time between prepulse and the
following test pulse. Re-inhibition was faster for channels modulated by pep271–305 in comparison to G␤2fl and GTP␥S. The
re-inhibition of the P/Q-type channel is predicted to depend on
the amount of applied or available G proteins (37). It is therefore likely that the peptide concentration delivered through the
patch pipette on the channel, at least in this set of experiments,
is higher than the G␤2fl subunit concentration in the cell. Thus
our data revealed no significant differences on the voltage
dependence of activation, the voltage-dependent relief of the G
protein modulation, the effect on steady state current, and the
recovery kinetics of the modulation between G␤2fl and pep271–
305, suggesting that pep271–305 mimics the action of the
G␤2fl subunit.
We next investigated if these peptides also act on neuronal
voltage-gated Ca2⫹ channels. 10 –14-Day-old cultured hippocampal neurons were analyzed for their expression of nonL-type Ca2⫹ channels. Non-L-type Ca2⫹ currents were isolated
by blocking Na⫹ and K⫹ channels with TTX and TEA and
voltage-gated L-type channels with nimodipine (Fig. 5A). Peptides were delivered by the chariot transfection reagent 20 min
before the recordings. As observed for the heterologously expressed recombinant P/Q-type channels and shown in Fig. 5,
pep1–25 decreased the facilitation ratio of neuronal non-L-type
Ca2⫹ currents in the presence of GTP␥S. Neither the pep271–
305 nor the chariot transfection reagent alone reduced significantly GTP␥S-mediated non-L-type channel modulation. In
contrast, application of the pep271–305 in the absence of
GTP␥S increased Ca2⫹ channel facilitation, whereas neither
the chariot transfection reagent alone nor the pep1–25 had an
effect on channel modulation. Because cultured hippocampal
neurons develop extensive dendritic and axonal processes,
proper voltage control of the somatic neuronal Ca2⫹ channels is
23951
23952
Peptides for Modulating Ion Channels and G Protein Pathways
and Ca2⫹ channel modulation in the presence of the peptide
pep1–25, we investigated the interaction between G protein ␤
and ␥ subunits. Considering the x-ray structure of the G protein, the N terminus (first 48 amino acids) of the G protein ␤
subunit is expected to interact with the G protein ␥ subunit
(Fig. 9) (39, 40). Thus, it is possible that the pep1–25 peptide
may act by perturbing the interaction between G protein ␤␥
subunits. To demonstrate the interaction and/or proximity between the G protein ␤2 and G protein ␥3 subunits, we performed fluorescent resonance energy transfer measurements
based on the method of Erickson et al. (27 and see Ref. 7). The
G protein ␤ subunit was used as the acceptor (tagged to YFP),
whereas the G protein ␥ subunit (tagged to CFP) was used as a
fluorescent donor in the FRET assay. Both fluorophores were
tagged to the N terminus of the G protein subunit. According to
the crystal structure, both fluorophores should be in close proximity because the N termini of the G protein ␤ and ␥ lie next to
each other. We first verified that both constructs localized to
the cell membrane when expressed in HEK293 cells (Fig. 7A).
We next analyzed if these two proteins were in close proximity
and thus able to induce FRET. The most accepted method to
demonstrate FRET is the dequenching of the CFP emission by
acceptor (YFP) photobleaching. As shown in Fig. 7B, 30 min of
photobleaching increased the CFP emission of G␤2-YFP- and
G␥3-CFP-transfected cells by 15%. As a positive control for the
acceptor bleaching experiments, we used a chameleon Ca2⫹
sensor. Within this protein CFP is fused to YFP via calmodulin
and the calmodulin-binding peptide of the myosin light chain
kinase (41). After 30 min of acceptor bleaching, the CFP fluorescence increased by 21%. In contrast the non-interacting
protein pairs (G␤2-YFP ⫹ CFP, G␥3-CFP ⫹ YFP, CFP alone,
and CFP ⫹ YFP) expressed in HEK293 cells revealed only a
slight increase in CFP emission. As an additional negative
control, we analyzed the FRET signal between a membranelocalized protein and the G protein subunits (i.e. ␥2(stargazin)CFP ⫹ G␤2-YFP and G␥3-CFP ⫹ ␥2(stargazin)-YFP). The constructs did not induce FRET. These results indicate that G
protein ␤␥ subunits induced a FRET signal.
We next analyzed if the FRET signal between G protein ␤␥
subunits in HEK293 cells is altered when the peptides
(pep1–25 and pep271–305) were delivered into the cells via the
chariot transfection reagent. We calculated first the average
FRET signal between G␤␥ subunits for transfected HEK293
cells and then applied the peptide dissolved in the chariot
transfection reagent to the cells (Fig. 7C). After 30 min of
incubation time we again calculated the FRET signal of the
same cells analyzed before. We found that 30 min after application of the pep1–25, the average FRET signal for G␤2␥3 was
reduced significantly in the presence of the pep1–25. This is
also demonstrated in Fig. 7D, where a strong FRET signal
between G␤2␥3 was detected at the outer rim of the cell, probably representing the cell membrane. This signal decreased
significantly after 30 min (Fig. 7D). In contrast neither the
pep271–305 nor the chariot transfection reagent alone altered
the average FRET signal between G protein ␤␥ subunits. These
data indicate that the peptide pep1–25 decreases the FRET
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FIG. 5. G protein ␤2-derived peptides (pep1–25 and pep271–305) reduce or induce neuronal non-L-type channel modulation. A,
example current trace of a non-L-type current elicited by a 500-ms-long voltage ramp protocol from ⫺60 to ⫹80 mV. The current was measured
in the presence of TTX, nimodipine, and TEA to block Na⫹, K⫹, and L-type Ca2⫹ channels. B, pep1–25 reduces and pep271–305 induces non-L-type
channel modulation in cultured hippocampal neurons. Ca2⫹ current traces in the presence or absence of G protein ␤ peptides with (upper trace)
or without GTP␥S (lower trace) in the intracellular recording solution. Ca2⫹ currents were elicited from a holding potential of ⫺60 mV by a 10-ms
test pulse to ⫹5 mV. After 2 s a 10-ms prepulse to ⫹100 mV was applied, and a second 10-ms test pulse to ⫹5 mV was elicited after stepping back
for 10 ms to ⫺60 mV. Facilitation ratios were determined as described in Fig. 1A. The current traces indicate that in the presence of pep271–305
P/Q-type channel facilitation is induced, whereas pep1–25 reduces the GTP␥S-mediated P/Q-type channel facilitation. 20 min before the
recordings, peptides were delivered via the chariot transfection reagent in a concentration of 10 ␮M. C, facilitation ratios for non-L-type channels
of cultured hippocampal neurons in the absence or presence of GTP␥S and of the indicated G␤2-derived peptides (pep1–25 and pep271–305) 20 min
before the recordings peptides were delivered via the chariot transfection reagent in a concentration of 10 ␮M. The extracellular solution contained
1 ␮M TTX and 5 ␮M nimodipine to isolate non-L-type Ca2⫹ currents. The number in parentheses indicates the number of experiments. **, p ⬍ 0.01,
ANOVA.
Peptides for Modulating Ion Channels and G Protein Pathways
23953
signal between G protein ␤2 and ␥3 subunits probably by changing the orientation or distance between the attached CFP
and YFP.
Peptide Pep271–305 Increases the FRET Signal between
GIRK1/2 Subunits in Living HEK293 Cells—We next investigated the effects of pep271–305 on GIRK channel subunit interaction. Recently, it has been shown that activation of the
muscarinic ACh receptor M2 or co-expression of G protein ␤␥
altered the FRET signal between CFP and YFP-tagged GIRK
subunits (42). We therefore asked whether we could change the
FRET signal between YFP-tagged GIRK1 and CFP-tagged
GIRK2 subunits when we co-expressed the G protein ␤␥ subunits and whether the G protein-mediated FRET change could
be elicited by pep271–305. Again we first verified the FRET
signals for the given protein interactions with acceptor bleaching experiments (Fig. 8B), and we compared the results to the
measured average FRET signal for the given interaction (Fig.
8C). These experiments were performed on transfected fixed
HEK293 cells. We found that co-expressed GIRK2-CFP and
GIRK1-YFP induced a robust average FRET signal, and that
after YFP bleaching the CFP emission increased for this FRET
pair by 9%. Both constructs localized to the same areas in
HEK293 cells as indicated in Fig. 8A. Co-expression of G protein ␤␥ increased the FRET signal between GIRK1/2 significantly, which correlated well with a significant increase in the
CFP emission to 15%. In contrast GIRK1-YFP co-expressed
with cytosolic CFP or membrane-targeted stargazin-CFP and
GIRK2-CFP co-expressed with cytosolic YFP or membranetargeted stargazin-YFP did not induce a FRET signal.
We next investigated in live HEK293 cells if the G protein
␤␥-mediated FRET increase between GIRK1/2 subunits could
be mediated by the pep271–305 peptide. We therefore calculated the FR for GIRK1/2 before and after application of the
pep271–305 peptide dissolved in the chariot transfection reagent, and we found that the pep271–305 increased FR significantly. In contrast the application of the pep1–25 or the chariot
transfection reagent alone did not induce a change in FR. We
further verified our results by analyzing the change in FR over
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FIG. 6. G protein ␤2-derived peptides (pep1–25 and pep271–305) reduce or induce GIRK channel modulation in HEK293 cells. A, K⫹
current traces of GIRK channels expressed in HEK293 cells in the absence and presence of the G protein ␤ peptides pep1–25 and pep271–305 before
and after GIRK channel activation via muscarinic ACh receptors. GIRK channel subunits 1 and 2 were co-expressed with muscarinic AChR-M2
in HEK293 cells. K⫹ currents through GIRK channels were elicited by a voltage ramp from ⫺100 to ⫹50 mV with a duration of 50 ms (control;
thick line). 10 ␮M ACh was applied after 30 s to activate mAChR-M2. In the absence of G␤2-derived peptides, activation of mAChR-M2 increased
the GIRK current amplitude (⫹ACh; thin line; upper trace). Peptides were applied through the patch pipette in a concentration of 100 ␮M, and
GIRK currents were recorded immediately after whole cell configuration was achieved (control; thick line). Peptides were allowed to diffuse into
the cell for 5–10 min until the GIRK current amplitude was stable (⫹peptide; dotted line). After stabilization of the GIRK current amplitude in the
presence of the peptide, ACh was applied (⫹ACh⫹peptide; thin line). In the presence of the pep1–25 peptide the control current was reduced, and
ACh application only caused a small increase in GIRK currents (middle trace). In contrast, in the presence of the pep271–305 peptide GIRK current
amplitude was increased, and ACh-mediated GIRK currents were further increased (lower trace). B, current change (increase or decrease) in GIRK
current amplitude in the presence and absence of G protein ␤2-derived peptides (pep1–25 and pep271–305) before and after activation of
mAChR-M2. Positive current indicates an increase, and negative current indicates a decrease in GIRK current amplitude measured at ⫺80 mV.
⫹pep indicates the GIRK current change in the presence of peptide. ⫹ACh indicates the GIRK current change after application of 10 ␮M ACh in
the absence or presence of pep1–25, pep271–305, or pep1–25scrambled. The number in parentheses indicates the number of experiments. C,
example traces of the time course of decrease and increase of GIRK currents in the presence of pep1–25 during ACh application (upper trace) and
pep271–305 before ACh application (lower trace). Currents were elicited as described in A. Each point represents the current measured at ⫺80 mV
within the ramp protocol. D, time constants of the increase and decrease of GIRK currents in the presence of pep1–25 during ACh application and
pep271–305 before ACh application applied through the patch pipette or via the chariot transfection reagent. The number in parentheses indicates
the number of experiments. **, p ⬍ 0.01, ANOVA.
23954
Peptides for Modulating Ion Channels and G Protein Pathways
time. HEK293 cells were transfected with GIRK1/2 subunits,
and cells were monitored before and after application of the
pep271–305 peptide (Fig. 8, D and E). As indicated in Fig. 8D
an increase in the FR was measured after application of the
pep271–305 but not pep1–25. This is also demonstrated in Fig.
8E. Here a larger FR was detected in particular at the outer
edge of the cell (probably membrane region) after application of
the peptide. These results indicate that the pep271–305 peptide changes the FRET signal between GIRK1/2 subunits probably by slightly reorienting the GIRK1/2 subunits.
Circular Dichroism Spectra Predicts ␣-Helical Structures for
Pep1–25—In order to investigate whether the synthesized peptides used in our experiments are structured, we analyzed the
CD spectra of the peptides. Far-UV CD spectra of pep1–25
showed features that are characteristic for polypeptides that
are significantly helical with a maximum near 190 and minima
at 208 and 222 nm (Fig. 9B). However, the data demonstrate
also that this peptide is not fully helical, because the minimum
at 205 is slightly shifted and the ratio of ellipticity at 222 and
208 nm is still not close or is greater than 1. Estimates for the
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FIG. 7. G protein ␤2 subunit-derived peptide, pep1–25, reduces the FRET signal between YFP-tagged G protein ␤2 and CFP-tagged
G protein ␥3 subunit. A, distribution of YFP-tagged G protein ␤2 and CFP-tagged G protein ␥3 subunits in HEK293 cells. Deconvolved images
of the co-distribution of YFP-G␤2 (left) and CFP-G␥3 (right) in HEK293 cells 24 h after transfection. Note fluorescence for both constructs is brighter
at the edges, suggesting membrane localization of G␤2 and G␥3. B, acceptor photobleaching for FRET pairs co-transfected in HEK293 cells:
CFP-G␥3 ⫹ YFP-G␤2; CFP ⫹ YFP-G␤2; CFP-G␥3 ⫹ YFP; CFP; CFP ⫹ YFP; the chameleon Ca2⫹ sensor protein; CFP-G␥3 ⫹ YFP-(stargazin)␥2 and
CFP-(stargazin)␥2 ⫹ YFP-G␤2. CFP fluorescence for each FRET pair was compared before and after 30 min of YFP photobleaching. The efficiency
reflects the increase in CFP fluorescence. The number in parentheses indicates the number of experiments. *, p ⬍ 0.05; **, p ⬍ 0.01, ANOVA. C,
FRET ratio for the FRET pair CFP-G␥3 ⫹ YFP-G␤2 before and after application of G protein ␤2-derived peptides pep1–25 and pep271–305. CFP,
FRET, and YFP pictures were taken before application of the peptide, and the average FR was calculated (see “Materials and Methods”). Peptides
dissolved in the chariot transfection reagent were applied to the live cells in a 10 ␮M concentration. Cells exposed to the peptides were incubated
for 30 min at room. After 30 min, CFP, FRET, and YFP pictures were taken again, and the average FR was calculated. The control FRET pairs
CFP ⫹ YFP-G␤2, CFP-G␥3 ⫹ YFP, CFP-G␥3 ⫹ YFP-(stargazin)␥2, and CFP-(stargazin)␥2 ⫹ YFP-G␤2 were measured in fixed cells. (RD1 and RA1
constants used: G␤␥ ⫹ pep1–25 (0.554;0.03); G␤␥ ⫹ pep271–305 (0.551;0.028); G␤␥ ⫹ chariot (0.558;0.036); G␤ ⫹ CFP and G␥ ⫹ YFP (0.568;0.054);
CFP-G␥3 ⫹ YFP-(stargazin)␥2 (0.597; 0.12); CFP-(stargazin)␥2 ⫹ YFP-G␤2 (0.59; 0.118).) **, p ⬍ 0.01, ANOVA. D, example of a change in the
YFP-G␤2/CFP-G␥3 FR over time in the presence of the G protein ␤2-derived peptide pep1–25. Upper, CFP, FRET, and YFP pictures taken from
HEK293 cells transfected with YFP-G␤2/CFP-G␥3 before peptide delivery. From these pictures an image was calculated (lower), where the FR per
pixel is encoded in color value. The arrow indicates that a high FR is detected on the outer edge of the cell and that the FR is larger before
application of the pep1–25 peptide. In contrast, the * indicates a cell with YFP-G␤2 expression but no or very little CFP-G␥3 expression and
therefore no change in the FR. The bar on the right relates the calculated FR to color scale values.
Peptides for Modulating Ion Channels and G Protein Pathways
helical content are 22% at 25 °C and 30% at 5 °C. The results
are in reasonable agreement with the 29% estimated from a
sequence-based prediction for the pep1–25 peptide (43). Both a
temperature increase and addition of 200 mM NaCl reduce
helical content slightly, as may be expected from a weakening
of intrapeptide hydrogen bonds and salt bridges, respectively.
In contrast pep271–305 showed no appreciable ellipticity at
222 nm (Fig. 9B). Both estimates and predictions (also for
pep1–25scrambled) gave helix content of less than 3%. Addition of 50% trifluoroethanol, a co-solvent that stabilizes helical
structure, had only a marginal effect on pep271–305, confirming that they have little or no intrinsic propensity to adopt
helical structure (44). The results are in agreement with the
structure of the corresponding protein region in the G protein
␤␥ complex (Fig. 9A).
DISCUSSION
tide, FRET between G protein ␤ and ␥ subunits was diminished, indicating that the orientation and/or distance between
these two subunits had been altered in the presence of the
peptide. Because the pep1–25 peptide comprises part of the
binding site between G protein ␤␥ and shows a high degree of
␣-helical structure, it is most likely that this peptide acts like a
wedge to separate the two proteins. In the presence of the
peptide, we showed that modulation of GIRK and Ca2⫹ channels was reduced. This may have important implications for
the function of the G␤␥ subunit interaction site for the correct
orientation and placement of the G protein ␤␥ complex in the
membrane to allow protein target modulation.
However, other possibilities have to be considered, which
may account for the loss of channel modulation in the presence
of pep1–25. For example the peptide may interfere with GTP␥S
binding and/or nucleotide-dependent stimulation of the endogenous G proteins, the peptide may terminate or accelerate the
G protein signal, or pep1–25 may interfere with the G protein
binding to the channel itself. We tried to address and rule out
at least some of these issues in our studies. For example, we
increased the GTP␥S concentration in the pipette 2-fold to 1.2
mM, but we could still observe the blocking effect on channel
modulation of pep1–25 (data not shown), suggesting that at
least at these GTP␥S concentrations the pep1–25 most likely
does not interfere with the activation of endogenous G protein.
We also tried to exclude the possibility that pep1–25 activates
PKC, which has been described as a mechanism to counteract
N- and P/Q-type channel modulation (14, 34). We therefore
performed the experiments in the presence of the PKC inhibitor peptide but could still block GTP␥S-mediated Ca2⫹ channel
modulation with pep1–25, suggesting that pep1–25 does not
act via PKC activation or competes with the PKC inhibitor
peptide on PKC. It has to be mentioned at this point that other
possibilities terminating G protein signaling such as up-regulation of RGS proteins or stimulation or stabilization of the
reassociation of the heterotrimeric G protein (G␣␤␥) may still
be able to occur, and we cannot exclude that these processes are
involved in the reduction of channel modulation. We also tested
if the pep1–25 may interfere with G protein ␤␥ binding to the
channel. If this would be the case, then pep1–25 would interfere with the modulatory action of G␤2 or pep271–305 if these
two proteins/peptides modulate P/Q-type channels via direct
binding to the channel. We tested this possibility by applying
pep1–25 together with pep271–305 and in the presence of exogenously expressed G␤2 subunits. In both cases modulation
was still observed arguing against the interference of pep1–25
for binding of G␤ subunits or domains to the channel but rather
point to an important interaction between G protein ␤ and ␥
subunits for G protein modulation. The results concerning the
action of G␤2 or pep271–305 independent of G␥ and the change
in G␤␥ interaction/orientation with loss in G protein signaling
are puzzling. One interpretation might be that in isolation the
modulatory domains within G␤2 can act independently on the
channel, but in the G protein complex G␤␥ has to be oriented
precisely on the effector protein.
The importance of the N-terminal G␤␥ interaction site is also
demonstrated by the fact that Asn-35 and/or Asn-36 in the G␤1
subunit are necessary for sensing the PKC-mediated phosphorylated state of the N-type channel leading to stronger Ca2⫹
channel inhibition (25). This suggests that this part of the G␤
subunit comes in close proximity to the binding site on the Ca2⫹
channel intracellular loop I–II.
Until now, it has remained unclear how G␥ subunits influence effector function. One possibility is that G protein ␥ subunits have direct effects on channel modulation. Recent studies
by Logothetis and co-workers (48) showed that the C-terminal
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We demonstrate in this study that the G␤2 N-terminally
derived G protein ␤␥ interacting domain reduces G protein
modulation of GIRK and Ca2⫹ channels and shows that the
first 25 amino acids of this domain alters the interaction between the G protein ␤ and ␥ subunits. In addition we demonstrate that the G␤2 C-terminally derived peptide pep271–305
induces G protein modulation of GIRK and Ca2⫹ channels, and
we further show that this peptide increases the FRET signal
between interacting GIRK channel subunits. Thus, both peptides act on multiple G protein-modulated target proteins. Possible mechanisms for these effects are discussed below.
Circular Dichroism Suggest That Pep1–25 Is Partly ␣-Helical, whereas Pep271–305 Is Unstructured in Solution—By contrast to the recent advent of designer peptides, which have
been optimized for very high helical propensity, peptide fragments from proteins are generally known to contain only poorly
stabilized secondary structures. Thus it is surprising that
pep1–25 has a relatively strong helical propensity, which may
aid folding or recognition, both in a kinetic and thermodynamic
sense. A helix that is populated to 50% relative to unstructured
state has a thermodynamic stability of ⌬G ⬃0 kcal/mol as ⌬G ⫽
⫺RT ln (fraction helical). At first this value may appear inconsequential, but it is significant compared with peptides, such as
pep271–305, that have no tendency to form helical structure,
even in the helix-stabilizing co-solvent trifluoroethanol. In
these cases additional interactions of considerably magnitude
may be needed to stabilize the secondary structures. By comparison, pep1–25 is kinetically and thermodynamically primed
to form a helical structure, which may be of considerable importance for the interaction of this region with the remainder of
G␤, G␥, or the channels.
N-terminally Derived G Protein ␤ Peptides Reduce Ion Channel Modulation and Change the Interaction between G Protein
␤ and ␥ Subunits—G proteins consist of three subunits (␣, ␤,
and ␥), which work together to confer extracellular transmitter
signals to intracellular effectors (45). The crystal structure has
recently been resolved and confirms the biochemical data that
G protein ␤ and ␥ subunits tightly interact (39, 40). Both N
termini lie next to each other (Fig. 9A). Therefore, one would
predict that fusion of suitable fluorophore combinations allowing FRET (e.g. CFP and YFP) to the N terminus of both G
protein ␤ and ␥ subunits would demonstrate that the fluorophores come into close contact resulting in energy transfer. In
fact, FRET between N-terminally YFP-tagged G␤1 and N-terminally CFP-tagged G␥2 was recently demonstrated by RuizVelasco and Ikeda (46) and was also observed for the G protein
subunits used in our study (G␤2␥3). In addition, biomolecular
fluorescence complementation was achieved by fusing the separated N and C termini of YFP to the N termini of G␤ and G␥
(47). By applying the G␤2 N-terminally derived pep1–25 pep-
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Peptides for Modulating Ion Channels and G Protein Pathways
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FIG. 8. G protein ␤2 subunit-derived peptide pep271–305 increases the FRET signal between YFP-tagged GIRK1 and CFP-tagged
GIRK2 subunit. A, distribution of YFP-tagged GIRK1 and CFP-tagged GIRK2 subunits in HEK293 cells. Deconvolved images of the codistribution of YFP-GIRK1 (left) and CFP-GIRK2 (right) in HEK293 cells 24 h after transfection. Note fluorescence for both constructs is brighter
at the edges of the cells, suggesting membrane localization of GIRK1/2. B, acceptor photobleaching for FRET pairs co-transfected in HEK293 cells:
CFP-GIRK2 ⫹ YFP-GIRK1; CFP-GIRK2 ⫹ YFP-GIRK1 ⫹ G␤2␥3; CFP ⫹ YFP-GIRK1; CFP-GIRK2 ⫹ YFP; CFP-(stargazin)␥2 ⫹ YFP-GIRK1; and
CFP-GIRK2 ⫹ YFP-(stargazin)␥2. CFP fluorescence for each FRET pair was compared before and after 30 min of YFP photobleaching. The
efficiency reflects the relative increase in CFP fluorescence. The number in parentheses indicates the number of experiments. **, p ⬍ 0.01, ANOVA.
C, FRET ratio for the FRET pair CFP-GIRK2 ⫹ YFP-GIRK1 in the presence and absence of co-expressed G protein ␤2␥3 subunits. HEK293 cells
were co-transfected with the indicated constructs and fixed after 24 h of incubation time. CFP, FRET, and YFP pictures were taken, and the
average FR was calculated (see “Materials and Methods”). The following FRET pairs were analyzed: CFP-GIRK2 ⫹ YFP-GIRK1; CFP-GIRK2 ⫹
YFP-GIRK1 ⫹ G␤2␥3; CFP ⫹ YFP-GIRK1; CFP-GIRK2 ⫹ YFP; CFP-(stargazin)␥2 ⫹ YFP-GIRK1; and CFP-GIRK2 ⫹ YFP-(stargazin)␥2. The
number in parentheses indicates the number of experiments. **, p ⬍ 0.01, ANOVA. (RD1 and RA1 constants used: GIRK1/2 (0.636;0.103); GIRK1/2
⫹G␤␥ (0.626;0.103); GIRK1 ⫹ CFP (0.592;0.096); GIRK2 ⫹ YFP (0.568;0.092); CFP-(stargazin)␥2 ⫹ YFP-GIRK1 (0.597;0.12); and CFP-GIRK2 ⫹
YFP-(stargazin)␥2 (0.598;0.12)). D, FRET ratio (FR) for the FRET pair CFP-GIRK2 ⫹ YFP-GIRK1 before and after application of G protein
␤2-derived peptides pep1–25 and pep271–305. CFP, FRET, and YFP pictures were taken before application of the peptide, and the average FRET
signal was calculated (see “Materials and Methods”). Peptides dissolved in the chariot transfection reagent were applied to the live cells in a 10
␮M concentration. Cells exposed to the peptides were incubated for 30 min at room. After 30 min, CFP, FRET, and YFP picture were taken again,
and the average FRET signal was calculated. (RD1 and RA1 constants used: (0.508;0.045).) *, p ⬍ 0.05, ANOVA. E, example of a change in the
YFP-GIRK1/CFP-GIRK2 FR over time in the presence of the G protein ␤2-derived peptide pep271–305 (upper). CFP, FRET, and YFP pictures taken
Peptides for Modulating Ion Channels and G Protein Pathways
23957
half-of G␥2 is required for GIRK channel activation. In this
study G␤␥ complexes assembled with the yeast G␥ subunit
blocked GIRK channel activation (48). In addition, Myung and
Garrison (54) revealed that G␤1␥2 dimers but not G␤1␥1 or
G␤1␥11 dimers activate adenylate cyclase type II, suggesting
that the G␥ subunit determines the activity of the G␤␥ effector
complex. In addition, a peptide derived from G protein ␥5 has
been shown to inhibit specifically muscarinic receptor signaling, including modulation of N-type Ca2⫹ currents in sympathetic neurons (49).
Given our observation that pep1–25 decreases the FRET
signal between G␤␥ and reduces G protein modulation, the G␤␥
interaction site is most likely important for precise orientation
of the G␤␥ complex on the target protein. It is interesting to
note that the N-terminal part of the intracellular loop III–IV of
the P/Q-type Ca2⫹ channel ␣1 subunit reveals structural similarities to the G protein ␥2 subunit (50).
C-terminally Derived G Protein ␤ Peptides Induce Ion Channel Modulation and Change the Interaction between the Ion
Channel Subunits—Several groups have tried to identify the
amino acids within the G␤ subunit responsible for specificity
and/or target protein function. One mutagenesis study analyzed G protein ␤-alanine mutations on various effector functions (23, 51). These mutations could either increase, decrease,
or abolish G␤␥-dependent interactions and were distributed
over the entire G protein with clusters of amino acid residues in
positions 51–100. Here, in particular the point mutations L55A
and I80A largely increased the prepulse facilitation ratio of
N-type Ca2⫹ channels (23). Most interestingly, the L55A G␤
mutant attenuated specifically the modulation of P/Q- but not
N-type channels (51). The position of this mutation is adjacent
to the interaction site between G protein ␤␥, and mutations at
this position may therefore alter the orientation of the G␤
protein relative to the G␥ subunits. Most of the identified
amino acids are located on the large surface area of G␤, suggesting that this site of the G␤ subunit anchors the G protein to
the channel. The recent study by Doering et al. (25) analyzed
the G␤ subunit specificity for N-type channel modulation by
creating chimeras between the G␤1 and G␤5 subunits. The
authors found two 20-amino acid-long regions (positions 140 –
168 and 186 –204) and an Asn-Tyr-Val motif that contributed to
the specific action of the G protein for N-type channel modulation. Most interestingly, exchange of the N terminus (amino
acid 1– 47) and the C terminus (amino acid 280-C terminus)
between the two subunits did not change the subunit specificity
in Ca2⫹ channel modulation. These are the two regions that
were identified in our deletion approach. In particular, we
found that the C-terminal region of the G protein ␤2 subunit
was able to induce Ca2⫹ channel modulation as well as GIRK
channel activation. The three-dimensional structure reveals
that the C terminus contains the WD domains WD6 and WD7.
Most interestingly, this C-terminal site is distinct from the
from HEK293 cells transfected with YFP-GIRK1/CFP-GIRK2 before peptide delivery. From these pictures an image was calculated (lower) where
the FR per pixel is encoded in the color values (see “Materials and Methods”). The arrow indicates that a high FR is detected on the outer edge
of the cell and that the FR is further increased after application of the pep271–305 peptide. The bar on the right relates the calculated FRET ratio
to color scale values.
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
FIG. 9. Circular dichroism spectra and location of modulatory peptides within the G protein ␤␥ protein complex. A, structure of the
G protein ␤␥ subunit complex (upper and lower surface and side view of the G protein shown as 180° and 90° rotations) and location of the
modulatory peptides within the G protein ␤ subunit: G protein ␤ subunit (light gray) with amino acids 1–25 (red) and 271–305 (orange); G protein
␥ subunit (black). Structure is from Refs. 39 and 40. B, circular dichroism spectra of pep1–25 and pep271–305 at 25 °C.
23958
Peptides for Modulating Ion Channels and G Protein Pathways
complex nervous system disorders (57). For example, the most
effective drug in the treatment of schizophrenia (clozapine)
interacts with at least four classes of different G protein-coupled receptors (i.e. serotonin, ACh, adrenergic, and other biogenic amine receptors). We predict that the G protein ␤ subunit-derived peptides will modulate these intracellular
signaling pathways.
Acknowledgments—We are grateful to Drs. T. P. Snutch,
E. Perez-Reyes, M. I. Simon, and R. Y. Tsien for cDNAs. We thank
Drs. L. Landmesser and S. W. 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. Bean, B. P. (1989) Nature 340, 153–156
7. Hümmer, A., Delzeith, O., Gomez, S. R., Moreno, R. L., Mark, M. D., and
Herlitze, S. (2003) J. Biol. Chem. 278, 49386 – 49400
8. Sandoz, G., Lopez-Gonzalez, I., Grunwald, D., Bichet, D., Altafaj, X., Weiss, N.,
Ronjat, M., Dupuis, A., and de Waard, M. (2004) Proc. Natl. Acad. Sci.
U. S. A. 101, 6267– 6272
9. Elmslie, K. S., Zhou, W., and Jones, S. W. (1990) Neuron 5, 75– 80
10. Boland, L. M., and Bean, B. P. (1993) J. Neurosci. 13, 516 –533
11. 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
12. Lee, H. K., and Elmslie, K. S. (2000) J. Neurosci. 20, 3115–3128
13. Colecraft, H. M., Brody, D. L., and Yue, D. T. (2001) J. Neurosci. 21, 1137–1147
14. Herlitze, S., Zhong, H., Scheuer, T., and Catterall, W. A. (2001) Proc. Natl.
Acad. Sci. U. S. A. 98, 4699 – 4704
15. Zhang, J. F., Ellinor, P. T., Aldrich, R. W., and Tsien, R. W. (1996) Neuron 17,
991–1003
16. De Waard, M., Liu, H., Walker, D., Scott, V. E., Gurnett, C. A., and Campbell,
K. P. (1997) Nature 385, 446 – 450
17. Herlitze, S., Hockerman, G. H., Scheuer, T., and Catterall, W. A. (1997) Proc.
Natl. Acad. Sci. U. S. A. 94, 1512–1516
18. Qin, N., Platano, D., Olcese, R., Stefani, E., and Birnbaumer, L. (1997) Proc.
Natl. Acad. Sci. U. S. A. 94, 8866 – 8871
19. Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997)
Nature 385, 442– 446
20. Page, K. M., Stephens, G. J., Berrow, N. S., and Dolphin, A. C. (1997) J. Neurosci. 17, 1330 –1338
21. 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
22. Canti, C., Page, K. M., Stephens, G. J., and Dolphin, A. C. (1999) J. Neurosci.
19, 6855– 6864
23. Ford, C. E., Skiba, N. P., Bae, H., Daaka, Y., Reuveny, E., Shekter, L. R., Rosal,
R., Weng, G., Yang, C. S., Iyengar, R., Miller, R. J., Jan, L. Y., Lefkowitz,
R. J., and Hamm, H. E. (1998) Science 280, 1271–1274
24. Mirshahi, T., Mittal, V., Zhang, H., Linder, M. E., and Logothetis, D. E. (2002)
J. Biol. Chem. 277, 36345–36350
25. Doering, C. J., Kisilevsky, A. E., Feng, Z. P., Arnot, M. I., Peloquin, J., Hamid,
J., Barr, W., Nirdosh, A., Simms, B., Winkfein, R. J., and Zamponi, G. W.
(2004) J. Biol. Chem. 279, 29709 –29717
26. Mark, M. D., Liu, Y., Wong, S. T., Hinds, T. R., and Storm, D. R. (1995) J. Cell
Biol. 130, 701–710
27. Erickson, M. G., Alseikhan, B. A., Peterson, B. Z., and Yue, D. T. (2001) Neuron
31, 973–985
28. Rohl, C. A., and Baldwin, R. L. (1997) Biochemistry 36, 8435– 8442
29. Mark, M. D., Wittemann, S., and Herlitze, S. (2000) J. Physiol. (Lond.) 528,
65–77
30. Wittemann, S., Mark, M. D., Rettig, J., and Herlitze, S. (2000) J. Biol. Chem.
275, 37807–37814
31. Bekkers, J. M., and Stevens, C. F. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,
7834 –7838
32. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981)
Pfluegers Arch. 391, 85–100
33. Garcia, D. E., Li, B., Garcia-Ferreiro, R. E., Hernandez-Ochoa, E. O., Yan, K.,
Gautam, N., Catterall, W. A., Mackie, K., and Hille, B. (1998) J. Neurosci.
18, 9163–9170
34. Zamponi, G. W. (2001) Cell Biochem. Biophys. 34, 79 –94
35. Currie, K. P., and Fox, A. P. (1997) J. Neurosci. 17, 4570 – 4579
36. Arnot, M. I., Stotz, S. C., Jarvis, S. E., and Zamponi, G. W. (2000) J. Physiol.
(Lond.) 527, 203–212
37. Zamponi, G. W., and Snutch, T. P. (1998) Proc. Natl. Acad. Sci. U. S. A. 95,
4035– 4039
38. Herlitze, S., Ruppersberg, J. P., and Mark, M. D. (1999) J. Physiol. (Lond.) 517,
341–352
39. Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H., and Sigler,
P. B. (1996) Nature 379, 311–319
40. Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E., and Sigler, P. B. (1996)
Nature 379, 369 –374
41. Miyawaki, A., Llopis, J., Heim, R., McCaffery, J. M., Adams, J. A., Ikura, M.,
and Tsien, R. Y. (1997) Nature 388, 882– 887
Downloaded from www.jbc.org at MEDIZINISCHE EINRICHTUNGE on September 7, 2009
contact sites between G␤ and G␣ subunits in heterotrimeric G
proteins (39, 52), and it overlaps with the location of the binding site of phosducin, which inhibits G protein action (53). In
addition, mutations in C-terminal peptide regions of G␤, which
undergo a conformational change during phosducin binding
and come into contact with the farnesyl group of G␥, led to
decreases in activity on phospholipase C-␤ and adenylyl cyclase
type II (54). Thus, the surface area including the WD repeat 6
and 7 (C-terminal end) may determine the effector function for
several proteins, i.e. phosducin, phospholipase C-␤, adenylyl
cyclase type II, GIRK, and voltage-gated Ca2⫹ channels. It
seems likely that during activation of the G protein, a conformational change within the G protein (55) induces a conformational change of the target protein, such as ion channels. A
G␤␥-mediated change of a FRET signal has been demonstrated
for P/Q-type channel ␣1 and ␤ subunits (7, 8) and for the GIRK
channel subunits (42). We used the GIRK1/2 interaction to
demonstrate that the pep271–305 can induce a change in the
FRET signal between the channel subunits. The physiological
action of the peptide, which mimics the action of the G protein
itself, and the relatively fast time course of its action on channel modulation in comparison to the pep1–25 suggest a direct
interaction between the peptide and the channel proteins (i.e.
GIRK and Ca2⫹ channel). However, we do not know at this
point if the peptide directly interacts with the channel to induce modulation, because we were not able to co-immunoprecipitate the HA-tagged 271–305 deletion construct with the
P/Q-type Ca2⫹ channel co-expressed in HEK293 cells (data not
shown). Another critical point for induction of modulation by G
protein-derived peptides is that for the overexpressed peptides
it might be considered that differences in the targeting of the
construct, aggregation within the cell, or different expression
levels may account for the differential effects seen for modulation of the channels. Therefore, we looked at the localization
and possible aggregation of the constructs when tagged to YFP.
The constructs were either localized to the membrane or within
the cytoplasm, and no aggregation of the constructs was observed within the analyzed time period, i.e. 24 h of expression.
This excludes that G protein aggregation within the endoplasmic reticulum or Golgi might be the cause of the modulatory
effects seen on the channels (supplemental Figs. 1–3). In addition, application of brefeldin A, which leads to 90% Golgi disassembly within 20 min (56), did not change the pep1–25 or
pep271–305 action on GIRK currents, suggesting that these
peptides do not block or activate G protein transport from Golgi
structures to the plasma membrane (data not shown). Because
the C-terminal region of the G␤ subunit also comes into contact
with the G␥ subunit (see Fig. 9) and the G␣ subunit, we cannot
rule out that the C-terminally derived peptides change the
orientation between G protein ␤ and ␥ subunits relative to the
target protein or release G protein ␤␥ subunits from the G␣␤␥
complex, which could explain the modulatory effect of the
pep271–305. However, in our experiments (Fig. 7) we did not
detect a change in the FRET signal between G␤␥ in the presence of pep271–305.
In summary, we have identified the structural domains of
the G protein ␤2 subunit, which can either suppress (i.e. the
G␤2 N-terminal G␥ interacting domain) or induce ion channel
modulation (i.e. the G␤2 C-terminal domain). By using the
synthesized peptides of these domains, we revealed that the
peptides most likely change the orientation between the G
protein ␤␥ subunits (pep1–25) or the GIRK channel subunits
(pep271–305). Besides their importance for understanding ion
channel modulation, the peptides will provide useful tools for
manipulating the amount of G protein activation within a cell
and could therefore be used as magic shotguns for treatment of
Peptides for Modulating Ion Channels and G Protein Pathways
42. Riven, I., Kalmanzon, E., Segev, L., and Reuveny, E. (2003) Neuron 38,
225–235
43. Munoz, V., and Serrano, L. (1994) Nat. Struct. Biol. 1, 399 – 409
44. Buck, M. (1998) Q. Rev. Biophys. 31, 297–355
45. Neer, E. J., and Smith, T. F. (1996) Cell 84, 175–178
46. Ruiz-Velasco, V., and Ikeda, S. R. (2001) J. Physiol. (Lond.) 537, 679 – 692
47. Hynes, T. R., Tang, L., Mervine, S. M., Sabo, J. L., Yost, E. A., Devreotes, P. N.,
and Berlot, C. H. (2004) J. Biol. Chem. 279, 30279 –30286
48. Peng, L., Mirshahi, T., Zhang, H., Hirsch, J. P., and Logothetis, D. E. (2003)
J. Biol. Chem. 278, 50203–50211
49. Akgoz, M., Azpiazu, I., Kalyanaraman, V., and Gautam, N. (2002) J. Biol.
Chem. 277, 19573–19578
50. Fathallah, M., Sandoz, G., Mabrouk, K., Geib, S., Urbani, J., Villaz, M., Ronjat,
M., Sabatier, J. M., and de Waard, M. (2002) Eur. J. Neurosci. 16, 219 –228
23959
51. Agler, H. L., Evans, J., Colecraft, H. M., and Yue, D. T. (2003) J. Gen. Physiol.
121, 495–510
52. Wall, M. A., Coleman, D. E., Lee, E. C., Iniguez-Lluhi, J. A., Posner, B. A.,
Gilman, A. G., and Sprang, S. R. (1995) Cell 83, 1047–1058
53. Gaudet, R., Bohm, A., and Sigler, P. B. (1996) Cell 87, 577–588
54. Myung, C. S., and Garrison, J. C. (2000) Proc. Natl. Acad. Sci. U. S. A. 97,
9311–9316
55. Bunemann, M., Frank, M., and Lohse, M. J. (2003) Proc. Natl. Acad. Sci.
U. S. A. 100, 16077–16082
56. Sciaky, N., Presley, J., Smith, C., Zaal, K. J., Cole, N., Moreira, J. E., Terasaki,
M., Siggia, E., and Lippincott-Schwartz, J. (1997) J. Cell Biol. 139,
1137–1155
57. Roth, B. L., Sheffler, D. J., and Kroeze, W. K. (2004) Nat. Rev. Drug Discov. 3,
353–359
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