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J. Biol. Chem., Vol. 278, Issue 49, 49386-49400, December 5, 2003

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Competitive and Synergistic Interactions of G Protein ₂ and Ca²⁺ Channel _1b Subunits with Ca_v2.1 Channels, Revealed by Mammalian Two-hybrid and Fluorescence Resonance Energy Transfer Measurements^*

Alexander Hümmer, Oliver Delzeith, Shannon R. Gomez, Rosa L. Moreno, Melanie D. Mark, and Stefan Herlitze

From the Department of Neurosciences, Case Western Reserve University, School of Medicine, Cleveland, Ohio 44106-4975

Received for publication, June 23, 2003 , and in revised form, September 22, 2003.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Presynaptic Ca²⁺ channels are inhibited by metabotropic receptors. A possible mechanism for this inhibition is that G protein subunits modulate the binding of the Ca²⁺ channel subunit on the Ca²⁺ channel complex and induce a conformational state from which channel opening is more reluctant. To test this hypothesis, we analyzed the binding of Ca²⁺ channel and G protein subunits on the two separate binding sites, i.e. the loopI–II and the C terminus, and on the full-length P/Q-type ₁2.1 subunit by using a modified mammalian two-hybrid system and fluorescence resonance energy transfer (FRET) measurements. Analysis of the interactions on the isolated bindings sites revealed that the Ca²⁺ channel _1b subunit induces a strong fluorescent signal when interacting with the loopI–II but not with the C terminus. In contrast, the G protein subunit induces FRET signals on both the C terminus and loopI–II. Analysis of the interactions on the full-length channel indicates that Ca²⁺ channel _1b and G protein subunits bind to the ₁ subunit at the same time. Coexpression of the G protein increases the FRET signal between ₁/_1b FRET pairs but not for ₁/_1b FRET pairs where the C terminus was deleted from the ₁ subunit. The results suggest that the G protein alters the orientation and/or association between the Ca²⁺ channel and ₁2.1 subunits, which involves the C terminus of the ₁ subunit and may corresponds to a new conformational state of the channel.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Voltage-gated Ca²⁺ channels of the N-, P/Q-, and R-type are inhibited by G protein-coupled receptors. The voltage-dependent inhibition is mediated by G protein subunits (1, 2) but is also induced by coexpression of the G protein subunit alone (1). Ca²⁺ channels consist of at least three subunits: the pore-forming ₁ and several auxiliary subunits such as the intracellulary located subunit and the transmembrane subunit ₂. The ₁ subunit consists of four channel domains, which are connected via intracellular peptide loops (for review see Refs. 3–5). G protein subunits modulate the channel via interaction with the intracellular peptide domain (loop I–II) and the C terminus of the ₁ subunit (6–12). Interestingly, the G subunit-binding sites overlap with the Ca²⁺ channel subunit-binding sites on the ₁ channel subunit (5, 7, 9, 10). In addition to the overlapping binding sites, G protein and Ca²⁺ channel subunits induce antagonistic effects on defined biophysical properties of the channel. For example, Ca²⁺ channel subunits (with the exception of ₂ subunits) shift the voltage dependence of activation to more hyperpolarized potentials, whereas G subunits have the opposite effects, i.e. a depolarizing shift (e.g. Refs. 13–18). The overlapping binding sites on the channel as well as their antagonistic effects on the voltage dependence of channel activation suggest that Ca²⁺ channel and G protein subunits may compete for binding sites on the ₁ subunits during G protein modulation. Early biophysical analysis of the Ca²⁺ channel G protein modulation suggested that Ca²⁺ channels are stabilized in a certain conformational state during G protein modulation from which channel opening is more difficult to achieve (19). According to this model, G protein subunits may induce and stabilize this reluctant state of the channel (20–24). In addition, one splice variant of the N-type channel mimics a G protein-modulated channel in the absence of activated G proteins, supporting the idea that the G protein binding to the channel induces and stabilizes an intrinsic state of the Ca²⁺ channel (25).

Green fluorescent protein (GFP)¹ has become an important fluorescent tag to study the localization, targeting, and interaction of proteins (26). Visualization of GFP does not require any cofactor or enzymatic reaction and is therefore suitable as a reporter gene for an immediate read out in a two-hybrid interaction assay, for example. Because of the existence of several spectrally distinguishable variants of GFP, two reporter genes can be used to record and compare the expression and interaction of two independent proteins. Dual-color imaging and fluorescence resonance energy transfers (FRET) were performed in various studies with promising results by using CFP and YFP as fluorescent pairs (27–30).

By using a modified mammalian two-hybrid system (MTH) and FRET, we asked how Ca²⁺ channel and G protein subunits interact at the binding sites on the ₁ subunit and whether the G protein induces a new conformational state of the channel. Our results suggest that Ca²⁺ channel _1b and G protein subunits differentially interact with the two isolated binding sites of the ₁ subunit (i.e. loopI–II and C terminus). On the functional full-length channel the Ca²⁺ channel _1b subunits may interact more strongly than G protein subunits, because the FRET signals were larger for ₁/_1b FRET pairs. Interestingly, when G protein subunits were coexpressed with Ca²⁺ channel ₁ and subunits, there was an increase in the fluorescence signal between the Ca²⁺ channel subunits. This increase in FRET was abolished when the C terminus was deleted from the ₁ subunit or overexpressed in the untagged form. The results suggest that the G protein induces an altered conformational state of the P/Q-type channel, which probably involves the binding of the G protein to the C terminus of the ₁ subunit.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Mammalian Two-hybrid System
Construction of DNA-BD and AD Fusion Proteins and pHASH-3—The following cloning and reporter vectors of the mammalian Matchmaker two-hybrid assay kit (Clontech) were used: pM as cloning vector to construct the GAL4 DNA-BD fusion constructs; pVP16 as cloning vector for AD fusion constructs; pM3-VP16, pM-53, and pVP16-T as positive control vectors; pVP16 and pM without insert as negative control vectors. G₂, G₃, and the Ca²⁺ channel constructs _1b, ₁2.1-loop I–II, and ₁1.2-loop I–II were amplified by a single PCR, and constructs were subcloned in-frame into pM and pVP16. All amplified products were verified by sequencing. For construction of pHASH-3, five consensus GAL4-binding sites (UASG17-mer (x5)) and an adenovirus E1b minimal promoter were amplified by a single PCR and were subcloned into pEYFP-C1 (Clontech). This vector was called pHASH-1. Then two nuclear localization signals were added 3' in-frame into the YFP gene. This vector was called pHASH-2. For creating pHASH-3, two nuclear localization signals were also added 3' to the CFP gene in pECFP (pECFP-NLS). The inducible YFP gene was cut out from pHASH2 using BspTI and subsequently subcloned into the BspTI site of pECFP-NLS. The 5' BspTI site was introduced into pHASH-2 with the inducible promoter via PCR.

Cell Culture and Immunohistochemistry—Opossum kidney (OK), Chinese hamster ovary, and human embryonic kidney (HEK) 293 cells were transfected with the indicated DNAs in each set of experiments with Effectene^TM Transfection Reagent (Qiagen GmbH, Germany). 0.25 µg of each DNA in combination with 0.5 µg of the reporter plasmids (pHASH-3) were used for transfection. Equal amounts of total DNA within one set of experiments were used by adding unrelated DNA (plasmid pBF-1) to the transfection mixture when necessary. For example in Fig. 1 a maximum of 5 different DNAs were transfected in a 2:1:1:1:1 (with pHASH-3 added at twice the molar ratio than the other DNAs). When only 3 or 4 different DNAs were transfected, the missing DNA was replaced by pBF-1 DNA at the same ratio.

View larger version (34K): [in this window] [in a new window]   FIG. 1. A YFP/CFP-based mammalian two-hybrid system to detect protein-protein interactions. a, schematic representation of the YFP/CFP-based mammalian two-hybrid system. Upper, transfection of the vectors pM (expressing the interacting protein X fused to the GAL4 DNA-BD), pVP16 (expressing the interacting protein Y fused to the VP16-AD), and the reporter plasmid pHASH-3 (encoding for constitutively expressed CFP and induced YFP) are cotransfected into a mammalian cell line. Middle, if protein X interacts with protein Y, transcription of YFP is induced via the GAL4 DNA-BD and VP16-AD. The YFP gene carries two nuclear localization signals at the 3' end and is cloned 3' to four GAL4-binding sites and an adenovirus E1b minimal promoter. CFP is constitutively expressed and is under cytomegalovirus promoter control. The CFP gene also carries two nuclear localization signals (NLS) at the 3' end. Both YFP and CFP genes are located on one vector (pHASH-3). Lower, interaction of protein X with protein Y leads to the induction of YFP expression and targeting of YFP to the nucleus. Because CFP is constitutively expressed and also localized to the nucleus, CFP fluorescence can be compared with the YFP fluorescence. The intensity values for CFP and YFP fluorescence were determined by imaging techniques in combination with analysis programs. Intensity values were defined as the sum of the gray scale values for all pixels in a defined object area. The intensity values from the nucleus were subtracted from the background intensity values, and the ratio between the corrected intensity values between YFP and CFP was determined. b–d, time dependence of YFP/CFP ratios. OK cells were transfected with pHASH-3 and positive control constructs (interacting proteins or fusion proteins that induce YFP expression) in b with pM3-VP16 (•), pM53/pVP16-T (), and pM-G2, pVP16-G3 (shaded triangle), or negative controls (no induction of YFP) in c with pHASH-3 alone (), pM and VP16 separated (shaded diamond) and pM53/pVP16-CP () both transfected with pHASH-3. The numbers of cells with YFP fluorescence was divided by the number of cells with CFP fluorescence at the indicated time point. d shows the averaged relative cell number of detectable YFP fluorescence relative to CFP fluorescence for positive controls in b (•) and negative controls in c (shaded triangle). Optimal transfection/signal efficiencies were reached between 18 and 24 h incubation time (gray bar). Standard errors are mean ± S.E. e and f, ratio of YFP/CFP intensity ratio for 18 h (e) and 36 h (f) incubation time. OK cells were transfected with pM53/pVP16-T (white bar) (18 h, 1 ± 0.11 (n = 20); 36 h, 1 ± 0.1 (n = 30)) and pM-G2, pVP16-G3 (gray bar) (18 h, 0.52 ± 0.08 (n = 21); 36 h, 0.29 ± 0.05 (n = 54); *, p < 0.05) together with pHASH-3; for negative controls with pM and VP16 separated (18 h, 0.04 ± 0.01 (n = 17); 36 h, 0.04 ± 0.005 (n = 48)) and pM53/pVP16-CP (18 h, 0.03 ± 0.01 (n = 27); 36 h, 0.01 ± 0.004 (n = 41)) both transfected with pHASH-3. Intensity values at 18 and 36 h are normalized to the positive control (pM-53/pVP16-T). The results indicate that the YFP/CFP intensity ratio after 18 h is higher for the pM-G2 and pVP16-G3 interaction than after 36 h. Number of cells analyzed are indicated in parentheses. *, p < 0.05, Student's t test for G23-induced intensities after 18 and 36 h. g, Western blot and induction of YFP/CFP fluorescence of fusion proteins expressed in OK cells from studies in b–f. Proteins were detected with a monoclonal antibody against p53 and VP16, respectively. All fusion proteins used produced proteins of the correct size: pM53 (16.17 kDa), G3 (51.81 kDa), VP16-T (82.76–90.89 kDa), G2 (46.94 kDa). Positive control combinations pM-53/pVP16-T and G3/G2 induced YFP fluorescence, whereas negative control pM/pVP16 did not. h, detection and relative quantification of the interaction of Ca2+ channel 1-loopI–II with Ca2+ channel and G protein subunits. OK cells were transfected with the indicated vector combinations including pHASH-3, and fluorescence intensity values were analyzed after 24 h of transfection. Left, YFP/CFP intensity ratios were calculated for the following cotransfections: pM53 and pVP16-T (0.99 ± 0.06 (n = 47)); pM-12.1-loopI–II (aa 369–418), pVP16-1b (0.45 ± 0.025 (n = 53)); pM-12.1-loopI–II (aa 369–418), pVP16-G2 (0.24 ± 0.04 (n = 35)); pM-12.1-loopI–II (aa 369–418), pVP16-G2 and pcDNA3-3 (aa 1–73) (0.22 ± 0.062 (n = 31)); pM-11.2.-loopI–II (aa 408–520), pVP16-G2 (0.013 ± 0.009 (n = 66)) and pM/pVP16 (0.018 ± 0.005 (n = 55)). Right, Western blot of fusion proteins expressed in OK cells from studies in h. Proteins were detected as described in g. All fusion proteins used produced proteins of the correct size: 12.1-loopI–II (aa 369–418) (21.56 kDa), 11.2.-loopI–II (29.52 kDa), 1b (74.94 kDa), and G2 (46.94 kDa). Western blots are from the same cell preparation. Number of cells analyzed are indicated in parentheses. Note: for G3 subunit expression in the MTH system a C-terminal truncation of G3 was used. The C terminus of G contains a CAAX motif that directs prenylation of the molecule and is responsible for anchoring the G complex to the plasma membrane (54). It has been shown that C-terminal truncations of G do not interfere with its ability to bind G subunits (55). *, p < 0.05, Student's t test.

Immunocytochemistry and Quantification of YFP and CFP Signals—Cells were embedded in Fluoromount (133 mM Tris/HCl, 30% glycerol; 11% Mowiol, 2% diazabicyclo[2.2.2]octane). Fluorescence was detected with a conventional fluorescence microscope (Axioskop; Carl Zeiss, Oberkochen, Germany). For CFP and YFP detection, the following filter sets were used: CFP, excitation, short-pass D436/10; beamsplitter 460DCLP and emission, bandpass filter 480/30; YFP, excitation, short-pass HQ 500/20; beamsplitter Q515LP and emission, bandpass filter 535/30. All filters were obtained from AHF Analysentechnik AG, Germany.

Intensity ratios between nuclear YFP and CFP fluorescence were calculated by dividing the mean intensity values for YFP by the mean intensity values for CFP. Mean intensity values of YFP and CFP fluorescence were calculated by subtracting the intensity values measured from the extracellular background from the intensity values measured from the fluorescence in the nucleus of the individual cells. Intensity values are defined as the sum of the gray scale values for all pixels contained in a defined object area. For every fluorophore in each set of experiments the optimal exposure time for the YFP and the CFP fluorescence signals was determined for the strongest signal of the positive control (i.e. pM53/pVP16-T/pHASH-1–3). Fluorescence intensities were compared with the fluorescence signal at the defined exposition time. In addition, CFP fluorescence background after YFP excitation was measured by expressing pHASH-3 alone, and values were subtracted from all experiments performed for the same transfection. Images were captured with a CCD camera (RTE/CCD-1300-Y/HS Princeton Instruments; Tucson, AZ), and pictures were analyzed with MetaMorph 4.01 (Visitron Systems GmbH, Puchheim, Germany). All experiments described were performed at least in triplicate, and data were presented as means ± S.E. Colors used for YFP, CFP, and DAPI in Fig. 1 are computer-generated colors (Adobe Photoshop 5.5).

Fluorescence Resonance Energy Transfer
Constructs—Constructs ₁2.1-loopI–II (residues 369–418), ₁2.1-loopI–II-Y/S, ₁1.2-loopI–II (residues 406–520), ₁2.1-C terminus (residues 1766–2212), ₁2.1 full-length, ₁2.1 full-length-C terminus (residues 1–1857), _1b, ₄, G₂, G₃ were either PCR-amplified or if restriction sites were suitable cloned into either pECFP-C1, pEYFP-C1 (Clontech), pECFP-C2, and pEYFP-C2 (derived from pEGFP-C2) or as non-tagged versions into pcDNA1, -3, or pcDNA3.1. All PCR-amplified products were verified by sequencing.

FRET Measurements—For the calculation of FRET values and FRET-derived values, a two-step approach was used, which is based on the formalism and procedures of Erickson et al. (32). In the first step, the constants RD1 and RA1 were determined by a multilinear regression (MLR) of the type FRET_fl = x CFP_fl + x YFP_fl + [MLR] (where fl indicates fluorescence). A simple manipulation of (FRET = (FRET_fl - RD1 x CFP_fl)/(RA1 x (YFP_fl - RD2 x CFP_fl))) yields the relations RD1 = + FRET x RA1 x RD2 and RA1 = /FRET. Because the term FRET x RA1 x RD2 turns out to be exceedingly small in comparison to RD1 (see Table in the Supplemental Material), the constant RD1 was estimated by to a good degree of approximation. This regression method has the advantage of producing the results completely independent of any additive adjustments of the basic input data usually necessary because of background variation. Furthermore, the correlation coefficient r of [MLR] can be calculated. In case r is close to 1or -1, it indicates the appropriateness of a linear relation between the variables, as predicted by the theory given in Erickson et al. (32).

Considering the data of cells expressing donor (X-CFP) only, the constant RD1 was set to be in [MLR]. By using the data of the cells expressing acceptor protein (X-YFP) only, RA1 was determined as of the [MLR], because in this case the FRET ratio (FRET) is equal to 1 by definition (FRET = (FRET_fl (from FRET pair) + FRET_fl (from YFP))/FRET_fl (from YFP)) (32).

In the second step the constants FR_max and FRET_max were calculated by an ordinary linear fit of the data of cells expressing both donor (X-CFP) and acceptor protein (X-YFP). As suggested by equation FRET = FR_max x Ab + 1 (32), the data were linearly fitted according to the free type FRET = m x Ab + c (y = mx + q). The predicted value FRET of the FRET ratio was given according to FRET = (FRET_fl - RD1 x CFP_fl)/(RA1 x (YFP_fl - RD2 x CFP_fl)) (see above) (32) using the original data and the R constants calculated in the first step. The percentage Ab of bound acceptors was calculated according to Ab = (CFP_est + YFP_est + K_d(Eff) - ((CFP_est + YFP_est + K_d(EFF))² - 4 x CFP_est x YFP_est)^1/2]/(2 x YFP_est) [A34] (with CFP_est = CFP_fl/M_D; YFP_est = YFP_fl/M_A; M_A and M_D set as in Erickson et al. (32), K_D(EFF) set 0), where Eff indicates efficiency and est indicates estimate. The quality of the linear fit was measured by the correlation coefficient r shown in the figures.

Throughout the experiments statistical significance (p) was determined with a two-tailed Student's t test with p < 0.05 (^*) and p < 0.01 (^**). Standard errors are the mean ± S.E.

Electrophysiology—CFP- and YFP-tagged Ca²⁺ channel subunits (₁2.1, _1b) and the G protein subunit (G₂) were coexpressed in tsA201 cells, and Ca²⁺ channel-mediated Ba²⁺ currents were measured and analyzed as described previously (1, 33, 34).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
A Modified Mammalian Two-hybrid System to Detect Protein-Protein Interactions—The yeast two-hybrid system is based on the finding by Fields and Song (35) that eukaryotic trans-acting transcription factors like GAL4 can be divided into two physically separated but still functional independent domains. Both domains are normally part of a nuclear protein, which binds to a specific activation sequence of the target genes and regulates their transcription. Therefore, the DNA binding domain (DNA-BD) binds to certain upstream activating sequences (UAS) in close proximity to the promoter of the gene. One or several activation domains (AD) increase the transcription rate by directing the RNA polymerase II complex for downstream action. The AD and DNA-BD have to be in close physical proximity for efficient gene transcription. Separating AD from DNA-BD results in loss of gene transcription, whereas tethering AD to the DNA-BD by fusion of interacting proteins restores the function of the transcription factor. The possibility to separate the two domains and fuse them to putative interacting proteins allows one to monitor the interaction via expression of a certain reporter gene (36) (Clontech and Stratagene). Recently, GFP has been used for monitoring protein interactions in an MTH system, which is based on the same principles as the yeast system (37).

One problem concerning the detection of especially weak protein interactions in the MTH system is the identification of positively transfected cells and the detection of a signal relative to background. In addition, the induced fluorescence intensity among several cells within a single assay may vary because of unequal numbers of reporter plasmids within the cell, different expression times after transfection, or because of the cell type. To overcome these problems, we introduced a second constitutively expressed reporter, the cyan fluorescence protein (CFP), into a vector where YFP is under the control of the GAL4-inducible promoter. The CFP is under CMV promoter control and is also transported to the nucleus. CFP-mediated fluorescence therefore indicates a positively transfected cell. The induction of YFP fluorescence can now be compared with the fluorescence signals of CFP, which are both expressed in the same restricted area, i.e. the nucleus (Fig. 1a). Thus, induced YFP signals can be detected and monitored relative to the CFP signals within single cells.

As shown in Fig. 1, b–d, we first determined the optimal expression time for detection of protein-protein interactions in the MTH system. This was necessary because the YFP reporter plasmid gene reveals low expression over time in the absence of interacting proteins. We therefore analyzed the signal ratio between YFP-induced fluorescence and the constitutive CFP fluorescence for positive and negative controls at various expression times after transfection. As shown in Fig. 1, b–d, the signal ratio between YFP and CFP fluorescence depends on the incubation time after transfection. YFP fluorescence was first detected 12 h after transfection in 20–50% of constitutively CFP-expressing cells for the positive control constructs, i.e. pM3-VP16 (fused GAL-4-DNA-BD and VP16-AD), interacting p53 protein/SV40 large T-antigen (pM53/pVP16-T), and G₂/G₃ interaction (Fig. 1, b–e). The relative cell number in which YFP fluorescence was detected in CFP-positive cells increased to 75–100% 24 h after transfection (Fig. 1b). In contrast, for the negative controls (non-interacting pairs pM/pVP16, pM53/pVP16, and pHASH-3 alone), YFP fluorescence was only detected after 18 h in 15% of the CFP-positive cells, and the cell number with YFP fluorescence further increased to a saturating level after 36–48 h of expression (Fig. 1c). Forty-eight h after transfection 60–70% of the CFP-positive cells revealed YFP fluorescence due to the leakage of the GAL4/E1b promoter (Fig. 1c). As shown in Fig. 1d, the optimal signal to noise ratio for the detection of YFP fluorescence and quantification of protein interactions occurred after 18–24 h of expression for OK cells. To demonstrate further that the signal to noise ratio decreased for incubation times longer than 24 h, we compared the YFP/CFP fluorescence ratios from cells incubated for 18 and 36 h after transfection. As shown in Fig. 1, e and f, the YFP/CFP fluorescence intensity ratios decline significantly for G₂/G₃ interaction from 0.52 ± 0.08 (n = 21) to 0.29 ± 0.05 (n = 54) (p < 0.05, Student's t test). Thus, optimal YFP/CFP fluorescence ratios are obtained between 18 and 24 h following transfection, which is the time where background fluorescence is minimized.

Interaction of the Ca²⁺ Channel Subunit and the G Protein Subunit with the Intracellular Loop I–II of the Ca²⁺ Channel ₁ Subunits—The binding sites of the Ca²⁺ channel and G protein subunits are localized at the intracellular domain connecting domain I and II and the C terminus of the Ca²⁺ channel ₁ subunit (6–12). We first analyzed the interaction of both proteins (Ca²⁺ channel and G protein subunit) on the ₁-loopI–II of the P/Q-type channel with the MTH system (Fig. 1h). P/Q-type channel loopI–II and G₂ (0.24 ± 0.04 (n = 35)) induced a YFP fluorescent signal, which was significantly weaker than the signal induced with _1b (0.45 ± 0.03 (n = 53)). In contrast, no YFP fluorescent signal was detected for coexpression of L-type channel ₁1.2-loopI–II and G protein ₂ subunits. This result was expected, because G protein subunits do not interact with the L-type channel ₁ subunits.

To verify the results observed with the MTH system, we compared and analyzed the direct interaction of the ₁-loop-I–II with the Ca²⁺ channel and G protein subunits using the three cube FRET method between CFP-tagged donor proteins (loopI–II) and YFP-tagged acceptor proteins (_1b/G₂) (Fig. 2). We calculated independently two FRET-based values according to a modified version of Erickson et al. (32). First, we determined the average FRET value (Fig. 2b); second, we determined the maximal FRET (FRET_max) value for the interacting protein pairs (Fig. 2, c + d) to qualitatively compare the protein interaction with other interactions examined. Differences in the FRET_max values correspond to a difference in the affinity of the interaction or the distance and/or orientation between the donor relative to the acceptor protein.

View larger version (26K): [in this window] [in a new window]   FIG. 2. Interaction of Ca2+ channel 1b and G protein 2 subunits with the intracellular loopI–II of P/Q-type 1 subunit. a, schematic diagram of the interaction of N-terminal YFP-tagged 1b or G2 subunit with N-terminal CFP-tagged intracellular loopI–II of the 1 subunit. b, average FRET measurements for the following FRET pairs: CFP-12.1-loopI–II + YFP-1b; CFP12.1-loopI–II + YFP-G2; 11.2-loopI–II + YFP-G2. The following controls were also analyzed but are not shown in the diagram: CFP-12.1-loopI–II + YFP; CFP-11.2-loopI–II + YFP; CFP + YFP-1b; CFP + YFP-G2; CFP +YFP; "chameleon" Ca2+ indicators localized to the cytoplasm and localized to the nucleus. c, FRET measurements and calculation of FRETmax of a single transfection of CFP-12.1-loopI–II + YFP-1b (upper) CFP-12.1-loopI—II + YFP-G2 (lower). Values were fitted to y = mx + q with no constraints and given in the figure. d, FRETmax values calculated from the fits from the single experiments as shown in c for the interaction of CFP-12.1-loopI–II + YFP-1b and CFP-12.1-loopI–II + YFP-G2. Data were fitted according to y = mx + q with no constraints. Only experiments with r >0.65 were considered. e, competition of acceptors (YFP-1b) for donor (12.1-loopI–II) binding. Average FRET measurements for the following FRET pairs: CFP-12.1-loopI–II + YFP-1b; CFP-12.1-loopI–II + YFP-1b + 1b; CFP-12.1-loopI–II + YFP-1b + G2. f, competition of acceptors (YFP-G2) for donor (12.1-loopI–II) binding. Average FRET measurements for the following FRET pairs: CFP-12.1-loopI–II + YFP-G2; CFP-12.1-loopI–II + YFP-G2 + 1b; CFP-12.1-loopI–II + YFP-G2 + G2. For all FRET experiments shown in Figs. 2, 3, 4, 5, 6, 7, 8 and described throughout the text, the following control experiments were performed. The acceptor protein (protein or protein domain fused to YFP) was expressed with untagged CFP, and the donor protein (protein or protein domain fused to CFP) was expressed with untagged YFP, and the average FRET values were determined. The average FRET values were around 1 and are given in the Supplemental Material, Table b. *, p < 0.05; **, p < 0.01, two-tailed Student's t test.

The average FRET value depends on the percentage of acceptor protein interacting with donor protein within one cell and is maximal (FRET_max) when all acceptors within one cell interact with one donor. Because this is not the case for nonfused proteins, the actual FRET value is related to the amount of acceptor to donor association, which was calculated by Erickson et al. (32) and is given by the Ab value (32). Thus, the Ab value corresponds to the fraction of YFP-tagged proteins that are preassociated with CFP-tagged proteins. For non-fused donor and acceptor proteins, Ab values between 0 and 1 are expected by definition. The distribution may vary according to the incubation time, the protein pair analyzed, the cell type, the amount of binding sites, and other factors. The distribution should be fitted with a straight line (32). Fig. 2c shows the distribution of a single experiment for the interaction between ₁2.1-loopI–II-CFP cotransfected with _1b-YFP (upper panel) or G₂-YFP (lower panel). The results indicate that the data can be fitted with a straight line. The interaction between P/Q-type channel ₁2.1-loopI–II with _1b induced a stronger FRET_max value (3.63 ± 0.37 (n = 6)) than the interaction between P/Q-type channel ₁2.1-loopI–II and G₂ (2.6 ± 0.14 (n = 10)) (Fig. 2d).

As suggested by the antagonistic function and the overlapping binding sites on the ₁ subunit between the Ca²⁺ channel subunit and G protein subunit, we asked whether Ca²⁺ channel subunits compete with G protein subunits for binding on the ₁2.1-loopI–II. To analyze whether competition occurs between G₂ or _1b on the ₁2.1-loopI–II, we coexpressed first equal DNA amounts of CFP-tagged ₁2.1-loop-I-II and YFP-tagged _1b or G₂ with untagged _1b and/or G₂ subunits (Fig. 2, e and f). For competition of acceptor to donor binding, a reduction in the interacting pairs should result in a reduction in the average FRET value. In our experiments a significant reduction in the fluorescence intensity ratio for the average FRET values was detected when the acceptor molecule was coexpressed with its untagged version, i.e. tagged ₁2.1-loop-I–II/_1b coexpressed with untagged _1b (Fig. 2e) and tagged ₁2.1-loop-I-II/G₂ coexpressed with untagged G₂ (Fig. 2f). Thus, the corresponding untagged protein reduces the number of available acceptor proteins for FRET. In addition, a significant decrease was observed for the FRET signal of ₁2.1-loopI–II/G₂ coexpressed with untagged _1b (Fig. 2f), whereas even a 5-fold excess of untagged G₂ could not significantly reduce the FRET signal in the ₁2.1-loopI–II/_1b interaction (Fig. 2e). A further increase in the G₂ concentration resulted in loss of viable cells necessary for FRET measurements probably due to toxic cell effects of G₂. The results suggest that the Ca²⁺ channel _1b subunit interacts differently than the G protein ₂ subunit on the ₁2.1-loopI–II. This is consistent with the fact that subunits bind to the AID on the loopI–II, whereas G protein subunits interact with the AID and with higher affinity on a second, more C-terminally located binding site of loopI–II.

As described in our earlier work (1, 8, 33), G protein subunits when coexpressed alone with P/Q-type channels in HEK293 cells induce G protein modulation. To rule out the possibility that G protein subunits alter the interaction with G interacting proteins, we cotransfected G protein ₃ subunits in equal concentrations and 5 times higher concentrations. At equal concentration the G protein ₃ subunit did not alter the fluorescence intensity ratios in the MTH (Fig. 1h; ₁2.1-loopI–II + G₂, 0.24 ± 0.04 (n = 35), and ₁2.1-loopI–II + G₂ + G₃, 0.22 ± 0.06 (n = 31)) and FRET measurements for G₂ interaction with ₁2.1-loopI–II (₁2.1-loopI–II-CFP + G₂-YFP, 3.31 ± 0.24 (n = 39) and (₁2.1-loopI–II-CFP + G₂-YFP + G₃, 3.76 ± 0.25 (n = 33)), and in FRET measurements for interaction with the full-length (fl) ₁ subunit (₁2.1-fl-CFP + G₂-YFP, FRET 1.35 ± 0.09 (n = 94) ₁2.1-fl-CFP + G₂-YFP + G₃, 1.33 ± 0.03 (n = 102)). Higher concentrations of G protein ₃ subunits reduced the FRET and also the Ab signals (₁2.1-fl-CFP + G₂-YFP, FRET 1.35 ± 0.09; Ab 0.149 ± 0.01 (n = 94) ₁2.1-fl-CFP + G₂-YFP +5 times G₃, 1.16 ± 0.02; Ab 0.02 ± 0.003 (n = 50)). The reduction in FRET and Ab signals further support the idea that G protein subunits are expressed and act as a G protein subunit sink as already observed when G protein subunits were coexpressed with P/Q-type channels. Expression of the G₃ subunit abolished GTPS-mediated modulation of P/Q-type channels (1). Because we did not see a significant change in the interaction between G₂ and ₁2.1-loopI–II or ₁2.1-full-length subunit in the presence of equal molar concentrations of G₃, we did not cotransfect G₃ in the other experiments.

To verify further that the FRET³ method is capable of detecting affinity changes between interacting proteins, we introduced a point mutation (Tyr to Ser exchange) into the AID-binding site of the loopI–II. The mutation within the loopI–II has been described in biochemical assays to reduce binding between loopI–II and the Ca²⁺ channel subunits as well as G protein subunits (7, 38). As expected, the loopI–II Tyr-Ser mutation reduced the FRET signal significantly for the interaction with _1b and G protein subunit in comparison to wild type loopI–II (₁2.1-loopI–II + _1b, 3.18 ± 0.13 (n = 97); ₁2.1-loopI–II-Y/S + _1b, 1.95 ± 0.13 (n = 85); ₁2.1-loopI—II + G₂, 2.25 ± 0.06 (n = 98); ₁2.1-loopI–II-Y/S + G₂, 1.55 ± 0.05 (n = 94)), indicating that FRET³ can detect affinity changes within protein-protein interactions (Fig. 3).

View larger version (15K): [in this window] [in a new window]   FIG. 3. Interaction of Ca2+ channel 1b and G protein 2 subunits with wild type and mutated intracellular loopI–II of the P/Q-type 1 subunit. a, schematic diagram of the AID within the loopI–II and the location of the point mutation (Tyr/Ser) which reduces the binding affinity of Ca2+ channel and G protein subunits. b, average FRET measurements for the following FRET pairs: CFP-12.1-loopI–II + YFP-1b; CFP-12.1-loopI–II-Y/S + YFP-1b; CFP-12.1-loopI–II + YFP-G2; CFP-12.1-loopI–II-Y/S + YFP-G2. The following control was also analyzed but is not shown in the diagram: CFP-12.1-loopI–II-Y/S + YFP. Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p < 0.05; **, p < 0.01, two-tailed Student's t test.

Interaction of the Ca²⁺ Channel Subunit and the G Protein Subunit with the C Terminus of the ₁2.1 Subunit—The second binding site of the Ca²⁺ channel subunit and the G protein subunit on the P/Q-type channel is located within the C terminus of the ₁ subunit (6, 9, 39, 40). However, this binding site seems to be subunit type-specific. It has been described for the P/Q-type channel C terminus (rabbit) that _2a and ₄ subunits but not _1b and ₃ subunits interact with the C-terminal binding domain (9, 40). Therefore, we wanted to analyze how Ca²⁺ channel and G protein subunits interact with the C terminus of the rat ₁2.1 subunit (Fig. 4). Coexpression of the C terminus and the G protein subunit induced a consistent, average FRET signal (FRET 2.63 ± 0.05 (n = 1041)) with a FRET_max value of 3.48 ± 0.5 (n = 7). In contrast, coexpression of the C terminus and _1b subunit did induce a small average FRET signal (FRET 1.14 ± 0.02 (n = 767)), which could not be fitted with a straight line, indicating that the _1b/C-terminal signal is unspecific. As a positive control of Ca²⁺ channel subunit interaction with the C terminus, we analyzed the interaction between the Ca²⁺ channel ₄ subunit and the C terminus. Coexpression of YFP-₄ with the CFP-tagged C terminus induced an average FRET signal of 1.97 ± 0.02 (n = 146) and a FRET_max signal of 3.15 ± 0.62 (n = 12), which was not significantly different from the FRET_max signal observed for the interaction between the C terminus and the G protein. Thus, the FRET measurements correlate with the described biochemical data for the interaction of G₂, ₄, and _1b subunits on the ₁2.1 P/Q-type channel and support the view that _1b subunits bind to the loopI–II (AID), whereas G₂ bind to both the C terminus and the loopI–II of the P/Q-type channel.

View larger version (26K): [in this window] [in a new window]   FIG. 4. Interaction of Ca2+ channel 1b and G protein 2 subunits with the intracellular C terminus of the P/Q-type 1 subunit. a, schematic diagram of the interaction of N-terminal-YFP tagged 1b and G2 subunit with N-terminal CFP-tagged intracellular C terminus of the 12.1 subunit. b, average FRET measurements for the following FRET pairs: CFP-12.1-C terminus + YFP-1b; CFP-12.1-C terminus + YFP-G2; CFP-12.1-C terminus + YFP-4. The following controls were also analyzed but are not shown in the diagram: CFP-12.1-C terminus + YFP and CFP + YFP-4. c, FRET measurements and calculation of FRETmax of a single transfection of CFP-12.1-C terminus + YFP-G2 and CFP-12.1-C terminus + YFP-4. Values were fitted to y = mx + q with no constraints and given in the figure. d, FRETmax values calculated from the fits from the single experiments as shown in c for the interaction of CFP-12.1-C terminus + YFP-G2 and CFP-12.1-C terminus + YFP-4. Data were fitted according to y = mx + q. Only experiments with r > 0.65 were considered. Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p < 0.05; **, p < 0.01, two-tailed Student's t test.

View larger version (39K): [in this window] [in a new window]   FIG. 5. Interaction of Ca2+ channel 1b and G protein 2 subunits with the full-length P/Q-type channel 12.1 subunit. a, P/Q-type channel-mediated Ba2+ currents measured in tsA-201 cells. CFP-tagged 12.1 subunits were coexpressed with YFP-tagged 1b and YFP-tagged G2 subunits in tsA-201 cells, and currents were measured after 48–72 h after transfection with patch clamp measurements. Upper trace, a 500-ms voltage ramp from a holding potential of -70 to + 100 mV elicit a whole cell Ba2+ current. Lower trace, prepulse facilitation of P/Q-type channels was determined by comparing two 5-ms test pulses to the indicated test potential separated by a 1-s interval at the holding potential of -70 mV. 2 ms before the second test pulse a 10-ms conditioning prepulse to +100 mV was applied which resulted in an increase in the peak and tail current. b, distribution and FRET signal of N-terminal CFP-tagged full-length 12.1 subunits coexpressed with YFP-tagged 1b or YFP-tagged G2 subunits and untagged 1b subunits. Top, detected CFP fluorescence; middle, detected FRET fluorescence; bottom, detected YFP fluorescence for the cotransfected FRET protein pairs. c, schematic diagrams for the interactions of N-terminal YFP-tagged 1b and G2 subunits with N-terminal CFP-tagged full-length 12.1 subunit (upper) and N-terminal CFP-tagged 1b and G2 subunit with N-terminal YFP-tagged full-length 12.1 subunit (lower). d, average FRET measurements for the following FRET pairs: CFP-12.1-fl + YFP-1b; CFP-12.1-fl + YFP-G2 + 1b; YFP-12.1-fl + CFP-1b; YFP-12.1-fl + CFP-G2 + 1b. The following controls were also analyzed but are not shown in the diagram: CFP-12.1-fl + YFP + 1b and YFP-12.1-fl + CFP + 1b. Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p < 0.05; **, p < 0.01, two-tailed Student's t test.

View larger version (33K): [in this window] [in a new window]   FIG. 6. Interaction of Ca2+ channel 1b and G protein 2 subunits with the full-length P/Q-type channel 12.1 subunit. a, schematic diagram for the interactions of N-terminal YFP-tagged 1b and G2 subunits with N-terminal CFP-tagged full-length 12.1 subunit (upper). FRET measurements and calculation of FRETmax of a single transfection of CFP-12.1-fl + YFP-1b (middle) and CFP-12.1-fl + YFP-G2 + 1b (lower). Values were fitted to y = mx + q with no constraints and are given in the figure. b, schematic diagram for the interactions of N-terminal CFP-tagged 1b and G2 subunit with N-terminal YFP-tagged full-length 12.1 subunit (upper). FRET measurements and calculation of FRETmax of a single transfection of YFP-12.1-fl + CFP-1b (middle) and YFP-12.1-fl + CFP-G2 + 1b (lower). Values were fitted to y = mx + q with no constraints and are given in the figure. c, average Ab values calculated according to Erickson et al. (32) for the following FRET pairs: CFP-12.1-fl + YFP-1b; CFP-12.1-fl + YFP-G2 + 1b; YFP-12.1-fl + CFP-1b; YFP-12.1-fl + CFP-G2 + 1b. d, FRETmax values calculated from the fits from the single experiments as shown in a and b for the interaction of CFP-12.1-fl + YFP-1b; CFP-12.1-fl + YFP-G2 + 1b; YFP-12.1-fl + CFP-1b; YFP-12.1-fl + CFP-G2 + 1b. Data were fitted according to y = mx + q. Only experiments with r > 0.65 were considered except for YFP-12.1-fl + CFP-1b. Due to the fact that FRET values were clustered at Ab values of 1 for this particular FRET pair (see also Erickson et al. (32)), the correlation for this FRET pair was not as high as for the other FRET pairs. We therefore considered experiments with r > 0.5. Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p < 0.05; **, p < 0.01, two-tailed Student's t test.

Interaction of Ca²⁺ Channel ₁ and _1b Subunits in the Presence of G Protein ₂ Subunits—Activation of a G proteincoupled receptor leads to the dissociation of G protein and subunits, the active components of the G protein. It is assumed that G after its dissociation from G interacts with the Ca²⁺ channel to induce modulation. The binding of G to the channel complex could either result in the release of the Ca²⁺ channel subunit from the channel (which is not supported by our interaction studies (Fig. 2)) or could induce a conformational change in the channel complex. To mimic the G protein modulation of the channel complex in the heterologous expression system, we coexpressed untagged G₂ together with the FRET Ca²⁺ channel pair ₁/_1b. If the G protein competes the binding of Ca²⁺ channel _1b subunit to ₁, we should observe a decreased FRET value, whereas rearranging the ₁/_1b subunits could either result in an increase or decrease in the FRET signal depending on the reorientation of the fluorophores. We observed a significant increase in the average FRET value from 1.45 ± 0.03 (n = 200) to 1.62 ± 0.04 (n = 151) for the interaction between ₁2.1-fl-CFP and the _1b-YFP when coexpressed with untagged G₂. This indicates a change of the interaction between Ca²⁺ channel ₁2.1-fl and _1b subunits in the presence of G protein ₂ subunits. The result was confirmed by determination of the FRET_max values. In the presence of G₂ subunits, the FRET_max value was significantly increased for ₁2.1-fl/_1b interaction from FRET_max 4.81 ± 0.85 (n = 3) to FRET_max 9.26 ± 1.07 (n = 3) (Fig. 7, a–c). We next analyzed the effect of G₂ on the ₁/_1b FRET pair, when ₁ was the acceptor in FRET. Again we observed a significant increase in the average FRET value from 2.29 ± 0.07 (n = 240) to 2.54 ± 0.09 (n = 255) and FRET_max value from 5.79 ± 0.3 (n = 7) to 7.48 ± 0.46 (n = 7) (Fig. 7, d–f), indicating that G protein subunits change the orientation between Ca²⁺ channel ₁ and _1b subunits rather than competing for binding with the Ca²⁺ channel _1b subunits on the channel complex.

View larger version (31K): [in this window] [in a new window]   FIG. 7. Coexpression of G protein 2 subunits with the Ca2+ channel 12.1/1b FRET pairs. a, schematic diagram of the interaction between N-terminal YFP-tagged 1b subunit with N-terminal CFP-tagged 12.1-fl in the presence of untagged G2 subunits. b, FRET measurements and calculation of FRETmax of a single transfection of CFP-12.1-fl + YFP-1b (upper) and of CFP-12.1-fl + YFP-1b plus G2 (lower). Values were fitted to y = mx + q with no constraints and are given in the figure. c, FRETmax values calculated from the fits from the single experiments as shown in b for the interaction of CFP-12.1-fl + YFP-1b and CFP-12.1-fl + YFP-1b plus G2. Data were fitted according to y = mx + q. Only experiments with r > 0.65 were considered. d, schematic diagram of the interaction between N-terminal CFP-tagged 1b subunit with N-terminal YFP-tagged 12.1-fl in the presence of untagged G2 subunits. e, FRET measurements and calculation of FRETmax of a single transfection of YFP-12.1-fl + CFP-1b (upper) and YFP-12.1-fl + CFP-1b plus G2 (lower). Values were fitted to y = mx + q with no constraints and are given in the figure. f, FRETmax values calculated from the fits from the single experiments as shown in e for the interaction of YFP-12.1-fl + CFP-1b and YFP-12.1-fl + CFP-1b plus G2. Data were fitted according to y = mx + q. Only experiments with r > 0.5 were considered (see Fig. 6d). Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p < 0.05; **, p < 0.01, two-tailed Student's t test.

View larger version (36K): [in this window] [in a new window]   FIG. 8. Influence of the 12.1 C terminus on G protein-mediated FRET increase for 12.1/1b FRET pairs. a, upper, schematic diagram of the interaction between N-terminal YFP-tagged 1b subunit with N-terminal CFP-tagged 12.1-fl in the presence of untagged G2 subunits and untagged 12.1 derived C termini. Lower, schematic diagram of the interaction between N-terminal YFP-tagged 1b subunit with N-terminal CFP-tagged 12.1 subunit, where the C terminus was deleted, in the presence of untagged G2 subunits. b, average FRET measurements for the following FRET pairs: CFP-12.1-fl + YFP-1b; CFP-12.1-fl + YFP-1b + C terminus; CFP-12.1-fl + YFP-1b + G2; CFP-12.1-fl + YFP-1b+ C terminus + G2; CFP-12.1-fl-C terminus + YFP-1b; CFP-12.1-fl-C terminus + YFP-1b + G2. The following control was also analyzed but is not shown in the diagram: CFP-12.1-fl-C terminus + YFP + 1b. c, FRET measurements and calculation of FRETmax of a single transfection of CFP-12.1-fl-C terminus + YFP-1b (upper) and CFP-12.1-fl-C terminus + YFP-1b plus G2 (lower). Values were fitted to y = mx + q with no constraints and are given in the figure. d, FRETmax values calculated from the fits from the single experiments as shown in c for the interaction of CFP-12.1-fl + YFP-1b; CFP-12.1-fl + YFP-1b + C terminus; CFP-12.1-fl + YFP-1b + G2; CFP-12.1-fl + YFP-1b+ C terminus + G2; CFP-12.1-fl-C terminus + YFP-1b and CFP-12.1-fl-C terminus + YFP-1b + G2. Data were fitted according to y = mx + q. Only experiments with r > 0.65 were considered. Values for shown experiments and controls are given in the Supplemental Material, Table b. **, p < 0.01, two-tailed Student's t test.

Interaction of Ca²⁺ Channel _1b Subunits and G Protein ₂ Subunits on the Full-length P/Q-type Channel—

View larger version (19K): [in this window] [in a new window]   FIG. 9. Interaction of Ca2+ channel 1b with G protein 2 subunits on the full-length P/Q-type channel 12.1 subunit. a, schematic diagram of the interaction between N-terminal CFP-tagged 1b subunit with N-terminal YFP-tagged G2 subunit on the full-length 12.1 subunit. b, average FRET measurements for the following FRET pairs: CFP-1b + YFP-G2 and CFP-1b + YFP-G2 + 12.1-fl. The following control was also analyzed but is not shown in the diagram: CFP-1b + YFP. c, FRET measurements and calculation of FRETmax of a single transfection of CFP-1b + YFP-G2 + 12.1. Values were fitted to y = mx + q with no constraints and given in the figure. d, FRETmax values calculated from the fits from the single experiments as shown in c for the interaction of CFP-1b + YFP-G2 + 12.1-fl. Data were fitted according to y = mx + q. Right, only experiments with r > 0.65 were considered. Values for shown experiments and controls are given in the Supplemental Material, Table b. *, p < 0.05; **, p < 0.01, two-tailed Student's t test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Mammalian Two-hybrid and FRET Measurements for Detection and Quantification of Protein-Protein Interactions—We applied two independent systems to gain inside information about the interactions between the proteins involved in P/Q-type channel modulation. First, we developed a modified MTH system for detection and relative quantification of proteinprotein interactions. By using two fluorescent reporters, CFP and YFP located on one expression plasmid, we monitored the induced YFP-mediated fluorescence relative to constitutive CFP fluorescence. This system was established in OK cells but can also be applied to other mammalian cell lines. In order to demonstrate that our system is applicable for detection of various protein-protein interactions, we analyzed the association between G protein and subunits and the interaction between the G protein and Ca²⁺ channel subunits with the intracellular domain of the Ca²⁺ channel ₁2.1 subunit. By establishing FRET measurements according to the method of Erickson et al. (32) between the two interacting proteins, the data observed with the MTH system could be verified. Thus, the MTH system allows the detection of protein-protein interaction, where the strength of the detected signals corresponds to the measured FRET signals. Because the FRET method is more sensitive and more direct, we used this technique to gain insight into the mechanism of Ca²⁺ channel modulation. The results are summarized in Table I.

View this table: [in this window] [in a new window]   TABLE I Summary of FRETmax values for the interaction of loopI—II, C terminus, full-length channel, full-length channel with C terminus deleted, and Ca2+ channel 1b and 4 subunits as donor and/or acceptor proteins and influence of coexpressed untagged proteins on average FRET and/or FRETmax values Arrows indicate whether FRET was increased (), decreased (), or not changed () during the coexpression of the untagged subunits.

FRET Measurements between the Ca²⁺ Channel Subunit, G Protein Subunit, and the Isolated Binding Sites (LoopI–II and C Terminus) of the P/Q-type Channel ₁ Subunit Confirm Biochemical Data of Ca²⁺ Channel G-Protein Interactions—

View larger version (26K): [in this window] [in a new window]   FIG. 10. Model of G protein modulation of P/Q-type Ca2+ channels assembled by 12.1 and 1b subunits according to MTH and FRET measurements. Left, the Ca2+ channel 1b subunit interacts with the 1 subunit on the N-terminal region of the intracellular loop I–II, the AID. Right, after activation of the G protein, the G protein subunit binds to its binding sites on the 1 subunit, i.e. C terminus (gray box) and to the C-terminal region of the intracellular loopI–II (gray box) of the 1 subunit. Binding of the G protein subunit to the channel changes either the orientation and/or the affinity of 1 full-length subunit to the Ca2+ channel subunit, inducing a new conformational state of the channel.

FRET Measurements between the Ca²⁺ Channel Subunit, G Protein Subunit, and the Full-length P/Q-type Channel ₁ Subunit Suggest a Mechanism for P/Q-type Channel Modulation—We next analyzed the protein interactions on the fulllength ₁2.1 subunit. The interaction of the full-length ₁2.1 subunit with Ca²⁺ channel subunit induced a larger FRET signal than the interaction of ₁2.1-fl with the G subunit. This effect was observed for using the ₁2.1 subunit as either acceptor or donor within the FRET pair. Because Ca²⁺ channel subunits are necessary for transport of the ₁ subunit to the plasma membrane, the experiments of the interaction between ₁-fl and G protein subunits were performed in the presence of an untagged Ca²⁺ channel subunit. It seems reasonable that Ca²⁺ channel and G protein subunits bind at the same time to the ₁2.1 subunit during modulation, because cotransfection of untagged ₁ and tagged Ca²⁺ channel and G protein subunits induced a FRET signal. The existence of two binding sites on the ₁ subunit for G protein and subunits may also suggest that two G protein or two Ca²⁺ channel subunits bind simultaneously to the ₁ subunit. However, direct intracellular application of purified G protein subunits and kinetic studies on N-type channels rather suggest that modulation of one channel requires one G protein (22, 44). The situation is more difficult for the binding of Ca²⁺ channel subunits to the ₁ subunit. Birnbaumer and co-workers (18, 39) suggested a two Ca²⁺ channel -binding site model for transport and modulation of Ca²⁺ channels. The role of a second modulating subunit binding has been suggested further by several groups (45–48). Recently, the intracellular application of purified subunits to membrane vesicles of the skeletal muscle supported the view of the modulation of pre-existing Ca²⁺ channels independent of the role of subunits for targeting (49, 50). However, we did not observe a significant increase in FRET values for _1b-CFP and _1b-YFP interaction on the ₁2.1 full-length channel (data not shown). Thus, the interaction of two Ca²⁺ channel subunits on one ₁ subunit may not be resolvable in our assays or only one subunit binds to the channel. Further FRET analysis, in particular with single or multiphoton microscopy, may shed light on this interesting phenomenon.

Another interesting observation of our study is that 25% of all subunits interact with ₁, whereas up to 70% of ₁ subunits interact with _1b in the heterologous expression system. The percentage of ₁ associated with other subunits was even higher (up to 90% (data not shown)), indicating that ₁ subunits need subunits for transport and insertion into the plasma membrane and support the findings of Bichet et al. (41) for the role of subunits in ₁ subunit trafficking.

The important finding on the full-length ₁ subunit is that the FRET signal between Ca²⁺ channel and ₁ subunit is increased in the presence of G protein subunits, which involves the C terminus of the ₁ subunit. This suggests that G protein binding to the channel induces a new conformational state of the channel, which may correspond to the reluctant state proposed by Bean in 1989 (19). Because Ca²⁺ channel modulation not only involves the binding of the G protein to the loopI–II and the C terminus but requires other channel domains, i.e. domain I (51, 52) and the N terminus, a reorientation of channel protein domains (in particular the N terminus where the CFP is attached) seems likely (53). Thus, the next step in elucidating the G protein modulation of presynaptic Ca²⁺ channels will be the dynamic analysis of the interaction of the involved proteins during transmitter application.

In summary, by using a modified MTH system and FRET measurements, we present evidence for several new findings on modulation of P/Q-type channels. Ca²⁺ channel subunits interact differently than G protein subunits with ₁-binding sites, i.e. the loopI–II, the C terminus, as well as the full-length ₁ subunit. Ca²⁺ channel can alter the binding of G on the loopI–II as indicated by the decrease in FRET, when Ca²⁺ channel subunits are coexpressed with the ₁-loopI–II/G FRET pair. On the full-length channel G protein subunits alter the interaction between Ca²⁺ channel ₁ and subunits, which probably involves the binding of the G protein to the C terminus of the Ca²⁺ channel ₁ subunit. These results imply that the G protein induces a new closed state of the channel, which was proposed in the biophysical experiments for presynaptic Ca²⁺ channel modulation (19–25).

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant R01 NS42623-01A1 (to S. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains 2 tables.

To whom correspondence should be addressed: Dept. of Neurosciences, Case Western Reserve University, School of Medicine, Rm. E604, 10900 Euclid Ave., Cleveland, OH 44106-4975. Tel.: 216-368-1804; Fax: 216-368-4650; E-mail: sxh106{at}pop.cwru.edu.

¹ The abbreviations used are: GFP, green fluorescent protein; aa, amino acid(s); AD, activation domain; AID, ₁ interaction domain; BD, binding domain; CFP, cyan fluorescent protein; FRET, fluorescence resonance energy transfer; HEK, human embryonic kidney; MLR, multilinear regression; MTH, mammalian two-hybrid system; NLS, nuclear localization signal; YFP, yellow fluorescent protein; OK, opossum kidney; GTPS, guanosine 5'-3-O-(thio)triphosphate.

	ACKNOWLEDGMENTS

 
We are grateful to Drs. T. P. Snutch, E. Perez-Reyes, and M. I. Simon for cDNAs, and Drs. L. Landmesser, V. Lemmon, and S. Jones for reading the manuscript. We also thank Karin Geckle for excellent technical assistance.

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