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J. Biol. Chem., Vol. 280, Issue 25, 23945-23959, June 24, 2005

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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^*

Xiang Li, Alexander Hümmer, Jing Han, Mian Xie, Katya Melnik-Martinez, Rosa L. Moreno, Matthias Buck¶, Melanie D. Mark, and Stefan Herlitze||

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

Received for publication, December 14, 2004 , and in revised form, February 28, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Voltage-gated Ca²⁺ channels of the N-, P/Q-, and R-type and G protein inwardly rectifying K⁺ 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 Ca²⁺ currents of cultured hippocampal neurons. We found that G₂ 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 G₂ N terminus inhibits G protein modulation, whereas a 35-amino acid peptide derived from the G₂ C terminus induced modulation of voltage-gated Ca²⁺ 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 ₂₃ subunits, indicating a reorientation between G protein ₂₃ 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 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 G₂, including the G protein interaction site, inhibit G protein modulation of GIRK and voltage-dependent 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 G₂ 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 for characterizing and manipulating G protein pathways in living cells.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 pYFP-nuc/HA-C1 vector used for co-expression of the G₂-derived deletion constructs, two SV40 nuclear localization signals were added 3' in-frame into the YFP coding sequence in pEYFP-C1 (Clontech) (pYFP-nuc). 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 µg of the indicated DNAs in each set of experiments with Quantum Prep^R Cytofectene^TM 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 (G₂-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. G₃ 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 (GenBank^TM accession number NM_053351 [GenBank] ) 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 = (FRET_fluorescence - RD1 x CFP_fluorescence)/(RA1 x (YFP_fluorescence)), 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 - CFP_fluorescence before/CFP_fluorescence after) x 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 = (FRET_fluorescence - RD1 x CFP_fluorescence)/(RA1 x (YFP_fluorescence)) 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 63x water corrected 1.2NA objective for fixed FRET samples and a 63x 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 µM) 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 µM for pep1-25 and 13 µM 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 x cell length cm x 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 (G₂) 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 GTPS 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 µM. 4-Phorbol 12-myristate 13-acetate (PMA) (Sigma) was dissolved in Me₂SO as a 1 mM stock solution. 2 µl of this solution was added to 2 ml of the extracellular bathing solution 5 min prior to the experiment (final concentration 1 µM).

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 µM. 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 µM TTX (Sigma) and 5 µM nimodipine (Sigma) were added to the external solution to block voltage-dependent Na⁺ channels and L-type Ca²⁺ channels.

View larger version (31K): [in this window] [in a new window]   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 G2 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 G2 or the indicated G2 peptide fragments. Numbers on the left indicate the length of the G2 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 GTPS 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 G2 or the indicated G2 peptide fragments. Numbers on the left indicate the length of the G2 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.

Statistical significance throughout the experiments was tested with ANOVA using Igor Software. Standard errors are mean ± S.E.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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 G₂ 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 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 Ca_v2 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/Q-type channel facilitation. Investigation of the 35-amino acid-long 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.

View larger version (20K): [in this window] [in a new window]   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 GTPS 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 GTPS in the absence or presence of the indicated G2 deletion constructs. The number in parentheses indicates the number of experiments. **, p < 0.01, ANOVA.

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 GTPS. 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 GTPS, whereas 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 G₂. 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 G₂ 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 GTPS-mediated P/Q-type channel facilitation significantly, whereas neither the pep271-305 nor the pep1-25scrambled 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 µM PMA to P/Q-type channels 5 min prior to the experiments reduced GTPS-mediated modulation to control levels, whereas in the presence of 100 µM PKC inhibitor peptide in the intracellular solution and 1 µM PMA in the extracellular solution the GTPS-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 G₂ 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 G₂ is occluded by the pep1-25 peptide. Application of pep1-25 neither significantly reduced the pep271-305 nor the G₂ 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.

View larger version (29K): [in this window] [in a new window]   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 GTPS (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 GTPS-mediated P/Q-type channel facilitation. Peptides were applied through the patch pipette in a concentration of 100 µM. 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 GTPS and of the indicated G2-derived peptides (pep1-25 and pep271-305). Peptides were applied through the patch pipette in a concentration of 100 µM. 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 (GTPS 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 (GTPS in pipette solution) and pep271-305. The number in parentheses indicates the number of experiments. **, p < 0.01, ANOVA.

View larger version (37K): [in this window] [in a new window]   FIG. 4. G protein 2-derived peptide pep271-305 and full-length G2 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 GTPS, G2fl, 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 G2fl or pep271-305 and where GTPS 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 GTPS, G2fl, 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 G2fl 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 G2fl or for channels where the pep271-305 was applied through the patch 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 GTPS, G2fl, 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 G2fl 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 G2fl 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 GTPS, G2fl, 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 GTPS, G2fl, 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 G2fl 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 GTPS, G2fl, or pep271-305. Time constants were determined as described in H. The number in parentheses indicates the number of experiments. *, p < 0.05, ANOVA.

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 µM 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 G₂-derived N-terminal 25-amino acid-long peptide reduces GIRK and Ca²⁺ channel modulation, whereas the synthesized G₂-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 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 G₂-YFP- and G₃-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 (G₂-YFP + CFP, G₃-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 membrane-localized protein and the G protein subunits (i.e. ₂(stargazin)-CFP + G₂-YFP and G₃-CFP + ₂(stargazin)-YFP). The constructs did not induce FRET. These results indicate that G protein subunits induced a FRET signal.

View larger version (26K): [in this window] [in a new window]   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 GTPS (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 GTPS-mediated P/Q-type channel facilitation. 20 min before the recordings, peptides were delivered via the chariot transfection reagent in a concentration of 10 µM. C, facilitation ratios for non-L-type channels of cultured hippocampal neurons in the absence or presence of GTPS and of the indicated G2-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 µM. The extracellular solution contained 1 µM TTX and 5 µM nimodipine to isolate non-L-type Ca2+ currents. The number in parentheses indicates the number of experiments. **, p < 0.01, ANOVA.

View larger version (30K): [in this window] [in a new window]   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 µM ACh was applied after 30 s to activate mAChR-M2. In the absence of G2-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 µM, 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 µM 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.

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 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.

View larger version (48K): [in this window] [in a new window]   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-G2 (left) and CFP-G3 (right) in HEK293 cells 24 h after transfection. Note fluorescence for both constructs is brighter at the edges, suggesting membrane localization of G2 and G3. B, acceptor photobleaching for FRET pairs co-transfected in HEK293 cells: CFP-G3 + YFP-G2; CFP + YFP-G2; CFP-G3 + YFP; CFP; CFP + YFP; the chameleon Ca2+ sensor protein; CFP-G3 + YFP-(stargazin)2 and CFP-(stargazin)2 + YFP-G2. 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-G3 + YFP-G2 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 µM 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-G2, CFP-G3 + YFP, CFP-G3 + YFP-(stargazin)2, and CFP-(stargazin)2 + YFP-G2 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-G3 + YFP-(stargazin)2 (0.597; 0.12); CFP-(stargazin)2 + YFP-G2 (0.59; 0.118).) **, p < 0.01, ANOVA. D, example of a change in the YFP-G2/CFP-G3 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-G2/CFP-G3 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-G2 expression but no or very little CFP-G3 expression and therefore no change in the FR. The bar on the right relates the calculated FR to color scale values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
We demonstrate in this study that the G₂ 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 G₂ 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 G₁ and N-terminally CFP-tagged G₂ was recently demonstrated by Ruiz-Velasco and Ikeda (46) and was also observed for the G protein subunits used in our study (G₂₃). 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 G₂ N-terminally derived pep1-25 peptide, 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 GTPS 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 GTPS 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 GTPS 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 GTPS-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 G₂ 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 G₂ 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 G₂ 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 G₂ 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 G₁ 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 half-of G₂ 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 G₁₂ dimers but not G₁₁ or G₁₁₁ 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).

View larger version (22K): [in this window] [in a new window]   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 + G23; 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 23 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 + G23; 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 µM 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 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.

View larger version (83K): [in this window] [in a new window]   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.

In summary, we have identified the structural domains of the G protein ₂ subunit, which can either suppress (i.e. the G₂ N-terminal G interacting domain) or induce ion channel modulation (i.e. the G₂ 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 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.

	FOOTNOTES

 
^* 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.

The on-line version of this article (available at http://www.jbc.org) contains a Table and Figs. 1, 2, 3, 4.

^|| 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.: 216-368-1804; Fax: 216-368-4650; E-mail: sxh106{at}cwru.edu.

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; GTPS, guanosine 5'-3-O-(thio)triphosphate; PMA, 4-phorbol 12-myristate 13-acetate; PKC, protein kinase C; HA, hemagglutinin.

	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

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 

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