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Originally published In Press as doi:10.1074/jbc.M702063200 on August 7, 2007

J. Biol. Chem., Vol. 282, Issue 40, 29563-29573, October 5, 2007
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Snapin, a New Regulator of Receptor Signaling, Augments {alpha}1A-Adrenoceptor-operated Calcium Influx through TRPC6*Formula

Fumiko Suzuki1, Shigeru Morishima1, Takashi Tanaka, and Ikunobu Muramatsu2

From the Division of Pharmacology, Department of Biochemistry and Bioinformative Sciences, School of Medicine, University of Fukui, 23-3 Matsuoka-Shimoaizuki, Eiheiji, Fukui 910-1193, Japan

Received for publication, March 9, 2007 , and in revised form, August 6, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of Gq-protein-coupled receptors, including the {alpha}1A-adrenoceptor ({alpha}1A-AR), causes a sustained Ca2+ influx via receptor-operated Ca2+ (ROC) channels, following the transient release of intracellular Ca2+. Transient receptor potential canonical (TRPC) channel is one of the candidate proteins constituting the ROC channels, but the precise mechanism linking receptor activation to increased influx of Ca2+ via TRPCs is not yet fully understood. We identified Snapin as a protein interacting with the C terminus of the {alpha}1A-AR. In receptor-expressing PC12 cells, co-transfection of Snapin augmented {alpha}1A-AR-stimulated sustained increases in intracellular Ca2+ ([Ca2+]i) via ROC channels. By altering the Snapin binding C-terminal domain of the {alpha}1A-AR or by reducing cellular Snapin with short interfering RNA, the sustained increase in [Ca2+]i in Snapin-{alpha}1A-AR co-expressing PC12 cells was attenuated. Snapin co-immunoprecipitated with TRPC6 and {alpha}1A-AR, and these interactions were augmented upon {alpha}1A-AR activation, increasing the recruitment of TRPC6 to the cell surface. Our data suggest a new receptor-operated signaling mechanism where Snapin links the {alpha}1A-AR to TRPC6, augmenting Ca2+ influx via ROC channels.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracellular Ca2+ signaling is one of the most important cell responses induced by the activation of Gq-protein-coupled receptors (GqPCRs).3 Receptor activation results in the generation of inositol 1,4,5-triphosphate (IP3), which in turn via the IP3 receptor releases Ca2+ from endoplasmic reticulum stores (1). This rapid and transient increase in intracellular Ca2+ is followed by the sustained entry of extracellular Ca2+ that results in a prolonged elevation of intracellular Ca2+ (2). The sustained entry of Ca2+ is now considered to be mediated through Ca2+-permeable plasma membrane ion channels, which are activated either by the IP3-induced depletion of the internal Ca2+ store or by other receptor-operated processes that are as yet not well understood. The sustained entry of Ca2+ is thought to be caused by two different cation channels that have been designated as store-operated Ca2+ (SOC) and receptor-operated Ca2+ (ROC) channels, respectively (3, 4). Recent studies suggest that some, if not all, of these Ca2+ channels involved in the sustained entry of Ca2+ are members of the family of transient receptor potential canonical (TRPC) channels. Furthermore, certain proteins interacting with receptors or channels such as Homer (5), Junctate (6), and STIM1 (7-9) have been found to be important for Ca2+ signaling. Some ROC channels are now known to interact with phospholipase C-{gamma} (PLC-{gamma}) and to be regulated by PLC-{gamma}, forming a ROC signal complex (10, 11). However, there may be additional proteins involved in the ROC complex linking GqPCRs to TRPCs.

Using the {alpha}1A-AR as a prototype of GqPCRs because of its greater effect on Ca2+ signaling compared with the other {alpha}1-AR subtypes (12-14), we hypothesized that in addition to the previously known mechanisms, other receptor-accessory proteins might be involved in coupling the {alpha}1A-AR to enhanced cellular Ca2+ entry via the ROC channels. To test this hypothesis, we first sought to identify {alpha}1A-AR-interacting proteins using a yeast two-hybrid approach, with the receptor as bait in the setting of a brain-derived cDNA library. This screen identified Snapin as a protein that interacts with the {alpha}1A-AR; Snapin is better known as a synaptic vesicle-associated protein (15, 16). We next sought to determine the following: (i) if in a cell or tissue context, Snapin could indeed interact directly with the {alpha}1A-AR; (ii) if Snapin-{alpha}1A-AR interactions might account for the ability of the receptor to regulate the sustained phase of Ca2+ entry via ROC channels; (iii) if TRPCs might be involved in such an augmented Ca2+ influx; (iv) if in a cell or tissue context, TRPCs could indeed interact directly with Snapin; and (v) if the activation of the {alpha}1A-AR could influence the interaction of Snapin with TRPCs. Our results suggest a new ROC signaling mechanism wherein Snapin acts as an accessory GqPCR protein that plays a key role in {alpha}1A-AR-regulated Ca2+ influx via TRPC6.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Yeast Two-hybrid Screening and Colony-lift Filter Assay—Human {alpha}lA-AR (17) was subcloned into pGBKT7 and used as bait. A human brain cDNA library (Clontech, Mountain View, CA) was screened by a yeast two-hybrid method using the MATCHMAKER two-hybrid system 3 (Clontech). Full-length of Snapin, cloned by a yeast two-hybrid screening, was subcloned into pGADT7 (Clontech) for colony-lift assay and in vitro translation.

Colony-lift filter assays were also performed using MATCHMAKER two-hybrid system 3, based on the beta-galactosidase activities. More information is described in the supplemental Methods.

Antibodies—Custom-ordered affinity-purified rabbit anti-Snapin antibody was prepared by MBL, Nagoya, Japan, raised against the synthetic peptide corresponding to residues 117-136 of mouse Snapin. Rabbit anti-{alpha}lA-AR antibody (raised against the epitope corresponding to residues 331-466 of the human receptor) and anti-heat shock protein 90 (HSP90) antibody were from Santa Cruz Biotechnology (Santa Cruz, CA); rabbit anti-TRPC3, -TRPC5, and -TRPC6 antibodies were from Alomone Labs (Jerusalem, Israel); and anti-beta-actin antibody was from Sigma. For immunocytochemistry, mouse anti-FLAG monoclonal antibody and FITC-conjugated anti-rabbit IgG antibody were purchased from Santa Cruz Biotechnology, and TRITC-conjugated anti-mouse IgG antibody was from Sigma.

Co-immunoprecipitation Studies—In vitro synthesized proteins obtained from a reticulocyte lysate synthesis protocol and solubilized homogenates of rat brain cortex and PC12 cells were used for co-immunoprecipitation studies. HA-tagged Snapin and c-Myc-tagged {alpha}lA-AR were translated in vitro with [35S]methionine using the TNT T7 coupled reticulocyte lysate system (Promega, Madison, WI). Synthesized proteins were mixed and co-immunoprecipitated either by anti-c-Myc or anti-HA antibody using a MATCHMAKER co-immunoprecipitation kit (Clontech). For co-immunoprecipitation analyses of cell- or tissue-derived proteins, a solubilized membrane pellet fraction was obtained from homogenates of rat cortex or PC12 cells by ultracentrifugation. To examine the interaction of TRPC6 and Snapin promoted by {alpha}lA-AR activation, the total cell homogenate was used directly. The membrane fraction or the homogenate was solubilized in a buffer containing 1% Triton X-100 (Sigma) and was incubated with AminoLink Plus gel with immobilized anti-Snapin or anti-{alpha}1A-AR antibody (Seize primary immunoprecipitation kit, Pierce). Equal amounts (10 µg) of immunoprecipitated protein from the untreated and agonist/antagonist-treated cells were subjected to Western blot analysis, and the blot densities for TRPC6 and receptor were normalized to the densities observed for these components in the immunoprecipitates from agonist/antagonist-untreated cells. Biotinylation and separation of the cell surface protein were done using a cell surface protein biotinylation and purification kit (Pierce).

PC12 Cells and Transfection—Rat pheochromocytoma-derived PC12 cells were transfected with {alpha}lA-AR tagged with FLAG at its N terminus and/or Snapin subcloned into each multiple cloning site of pIRES vector (Clontech), by electroporation using Nucleofector (Amaxa Biosystems, Koeln, Germany). The {alpha}lA-AR density of stably transfected PC12 cell lines was determined by a saturation binding assay (18). Cell lines of {alpha}lA-AR-, Snapin-{alpha}lA-AR-, Snapin-, and mock-PC12 were selected by G-418 (0.5 mg/ml).

Confocal Laser Scanning Microscopy—PC12 cells stably transfected with FLAG-{alpha}lA-AR and Snapin were fixed with 4% formaldehyde, blocked with 5% bovine serum albumin, and labeled with mouse anti-FLAG and rabbit anti-Snapin antibodies as primary antibodies. Cells were then incubated with TRITC-conjugated anti-mouse IgG and FITC-conjugated anti-rabbit IgG antibodies. They were visualized by laser scanning confocal microscopy (TCS SP2, Leica Microsystems).

To estimate the abundance of Snapin distributed at the plasma membrane or in the intracellular region, we developed a morphometric analysis approach employing confocal images of 8 bit depth in resolution that were taken under the identical conditions (including the laser power, gain and offset of the photomultiplier detector, and the duration and amount of antibodies applied). These images were analyzed by our custommade software, as reported previously (19). More than 20 lines across a cell trajectory in a confocal image were randomly drawn, and pixel intensity units (range in our system, 0-255 arbitrary pixel units) versus the distance from the origin of the line was plotted (see Fig. 3C). The plasma membrane location was identified as the region where the intensity of two consecutive pixels was higher by two standard deviations from the average intensity measured in the same direction just prior to the cell margin. The density of Snapin at the plasma membrane was then estimated as the average pixel intensity measured over a 1-µm distance at each edge of each cell. Then the density of Snapin distributed in the intracellular area was calculated by measuring the average pixel intensity over the mid-point range in between the two 1-µm cell edge regions, as depicted in Fig. 3, C and D.

Measurement of [Ca2+]i and Inositol Phosphate Accumulation Assay—PC12 cells were loaded with 5 µM Fura-2 AM (Invitrogen) for 45 min to 1 h, washed, and then resuspended in Ca2+(+) assay buffer, containing (in mM) NaCl 136.9, KCl 5.4, MgCl2 1.0, CaCl2 1.5, glucose 10, and HEPES 10 (pH 7.4 by NaOH) with 3% fetal bovine serum. [Ca2+]i was measured by fura-2 ratiofluorometry using a CAF-110 fluorescence spectrophotometer (Jasco, Tokyo, Japan) (14). During the measurements, cells were continuously stirred and kept suspended at 37 °C. To assess the intracellular Ca2+ response of the cells in the absence of extracellular Ca2+, buffers were prepared without the addition of Ca2+ either in the absence (nominally, Ca2+-free buffer) or presence of 2 mM EGTA [Ca2+(-) buffer]. Accumulation of inositol phosphates was assayed as reported previously (20).

RNAi of Snapin—To knock down Snapin, three hairpin siRNAs for Snapin were designed and subcloned into pSilencer 4.1-CMV siRNA expression vector (Ambion, Austin, TX). Each {alpha}lA-PC12 cell clone and Snapin-{alpha}lA-PC12 cell clone was transfected with either of the three Snapin siRNA vectors or a vector containing missense siRNA and selected using 0.25 mg/ml hygromycin. One of the knocked down cell lines was used for further study. The sequence of the siRNA for Snapin, scrambled siRNA, and missense siRNA was 5'-TGCTGGATTCGGGAATTTA-3', 5'-AATGGCTTGGATTGGATTC-3', and 5'-GACGAGTTGACTGCGATTG-3', respectively.

Statistical Analyses—Data are expressed as the mean ± S.E. Statistical differences of data were evaluated by ANOVA followed by Dunnett's post-hoc multiple comparison tests, and considered to be significant at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Identification of Snapin as an {alpha}lA-AR-interacting Protein—A yeast two-hybrid screening of a human brain cDNA library with human {alpha}lA-AR (GenBankTM accession number U03866 [GenBank] ) as a bait yielded a number of positive clones that encoded full-length human Snapin (GenBankTM accession number NM012437). Snapin was first identified on synaptic vesicle membranes and has been considered to be involved in the exocytotic process (15, 16, 21). The prepared anti-Snapin antibody (see "Experimental Procedures") detected a band of ~15 kDa in every lane loaded with membrane preparations obtained from Snapin- or mock-transfected PC12 cells and rat cortex (Fig. 1B), and the intensity of the band was higher in the lane loaded with membrane of Snapin-transfected cells than that of mock-transfected cells, showing the reliable specificity of the antibody. The interaction between Snapin and the {alpha}lA-AR was confirmed by co-immunoprecipitation experiments using the following: (i) in vitro synthesized Snapin and {alpha}lA-AR (Fig. 1A); (ii) a stably transfected PC12 cell line expressing both Snapin and the {alpha}lA-AR (designated as Snapin-{alpha}lA-PC12) (Fig. 7E); or (iii) solubilized membrane extracts from rat brain cortex where both Snapin and {alpha}lA-AR are known to be expressed abundantly (Fig. 1C) (15, 22).


Figure 1
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  		FIGURE 1.
Immunoprecipitation and Western blot analyses of rat brain membrane extracts and of recombinantly expressed Snapin and {alpha}lA-AR in solution either alone or in combination. A, in vitro translated [35S]methionine-labeled c-Myc-{alpha}lA-AR (lanes 2 and 5) and HA-Snapin (lanes 1 and 6) and the mixture of both proteins (lanes 3 and 4) were immunoprecipitated (IP) with anti-c-Myc antibody (lanes 1, 3, and 5) or anti-HA antibody (lanes 2, 4, and 6), respectively. The immunoprecipitates were then subjected to PAGE. HA-Snapin (15 kDa) was detected in lanes 3, 4, and 6, and c-Myc-{alpha}lA-AR (50 kDa) in lanes 3-5. B, to confirm the specificity of anti-Snapin antibody, an equal amount (10 µg) of solubilized membrane preparations obtained from Snapin- (left lane) or mock-transfected PC12 cells (middle lane) and rat cortex (right lane) was subjected to SDS-PAGE followed by Western blot (WB) analysis. A band of ~15 kDa was detected in every lane, and the intensity of the band was higher in the lane loaded with membrane of Snapin-transfected cells than that of mock-transfected cells. C, a solubilized membrane preparation from rat cortex was used directly for Western blot analysis (lane 4) or immunoprecipitated with anti-Snapin antibody (lane 1), anti-{alpha}lA-AR antibody (lane 2), or rabbit pan-IgG (lane 3) as a negative control. The immunoprecipitates were analyzed by Western blot analysis using the same anti-receptor and anti-Snapin antisera. Snapin (15 kDa) and {alpha}lA-AR (50 kDa) were co-immunoprecipitated with the counterpart, respectively. The positions of the molecular size markers (kDa) are shown on the left in B and C.

 


Figure 2
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  		FIGURE 2.
Colony-lift filter assay of WT and four C-terminal truncated mutants of {alpha}lA-AR. Either the WT receptor or each of the {alpha}lA-AR mutants shown in the left panel subcloned into pGBKT7 containing GAL4 DNA binding domain was transformed in yeast together with Snapin subcloned in pGADT7 containing GAL4 activation domain. beta-Galactosidase (beta-GAL) activity in each clone was examined as shown in the right panel. Yeast colonies transformed with WT and -399 constructs showed positive beta-galactosidase activity, while others were negative.

 
{alpha}1A-AR via Its C-terminal Sequence Interacts with Snapin—We searched for the domain of {alpha}lA-AR responsible for the interaction with Snapin by a colony-lift filter assay. Four C terminus deletion mutants ({alpha}lA-AR-(1-335), {alpha}lA-AR-(1-347), {alpha}lA-AR-(1-375), and {alpha}lA-AR-(1-399)) and a full-length wild type (WT) of {alpha}lA-AR (in the pGBKT7 vector, as shown in Fig. 2) were co-transformed into a yeast strain with Snapin in the pGADT7 vector, and the beta-galactosidase activity in each clone was examined. Although the mutant {alpha}lA-AR-(1-399) as well as the WT {alpha}lA-AR demonstrated an interaction with Snapin, the other three mutants did not (Fig. 2). These results indicated that the C-terminal 24 amino acids (376-399) were essential for the direct Snapin-receptor interaction.

Dynamic Interaction of Snapin and {alpha}lA-AR upon Activation of the Receptor—To study the dynamic or functional interaction of Snapin and the {alpha}lA-AR further, we used a PC12 cell line, in which we had confirmed the expression of endogenous Snapin by Western blot analysis (Fig. 6A and Fig. 1B) but not the {alpha}lA-AR (absence of radioligand binding and ligand-mediated Ca2+ signaling as shown Fig. 4A). We examined the intracellular localization of these proteins by immunocytochemistry using PC12 cells with or without the transfection of {alpha}lA-AR. In mock-transfected PC12 cells, the anti-Snapin antibody that recognized Snapin reliably on Western blots (Fig. 6A) visualized endogenous Snapin both in the cytoplasm and at the plasma membrane (Fig. 3A, left upper panel). To estimate quantitatively the amount of Snapin distributed at plasma membrane and in the intracellular area, confocal images were subjected to morphometric analysis as outlined under "Experimental Procedures," and the abundance of Snapin was calculated from the average pixel intensity in regions corresponding either to the 1-µm plasma membrane region or over the mid-range of the intracellular domain between the two cell membrane margins (Fig. 3C). As shown by the morphometric analysis data summarized in Fig. 3D, transfection of the {alpha}lA-AR into the PC12 cells resulted in a slight increase in the abundance of Snapin at the plasma membrane as shown qualitatively in Fig. 3A (see arrowheads, lower left panel) and by the quantitative morphometric analysis in Fig. 3D (an increase to 46 ± 5.1 pixels in {alpha}lA-AR transfected cells compared with 31 ± 4.7 in untransfected cells, respectively; compare Mock versus receptor-transfected cells Before agonist treatment in Fig. 3D). Upon activating the {alpha}lA-AR with 100 µM methoxamine (Fig. 3B), the abundance of Snapin in the 1-µm plasma membrane region significantly increased from 46 ± 5.1 to 59 ± 5.4 and 79 ± 6.3 pixel intensity units at 3 and 5 min after agonist activation, respectively (Fig. 3D, black bars on right). As reported previously, the expressed receptor was also mainly localized at the plasma membrane (Fig. 3A, right lower panel) (23). In the {alpha}lA-AR-transfected cells, the recruitment of Snapin to the plasma membrane did not appreciably deplete the intracellular abundance of Snapin (Fig. 3D, compare white versus black bars). The activation-induced recruitment of Snapin to co-localize with the receptor suggested not only that {alpha}lA-AR and Snapin are functionally coupled but that they might be able to interact directly at the molecular level.


Figure 3
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  		FIGURE 3.
Cellular localization of {alpha}lA-AR and Snapin in PC12 cells stimulated or not with methoxamine. A, PC12 cells transfected with mock vector (upper panels) or FLAG-tagged {alpha}lA-AR (lower panels) were immunostained with anti-Snapin (visualized with FITC-conjugated secondary antibody, left panels) or anti-FLAG (visualized with TRITC-conjugated secondary antibody, right panels) and examined by confocal laser scanning microscopy. The arrowheads in the lower left panel show the membrane-localized site of accumulation of Snapin in the receptor-transfected cells. B, PC12 cells transfected with FLAG-tagged {alpha}lA-AR were stimulated with methoxamine (100 µM), fixed by 4% formaldehyde at 3 and 5 min after stimulation, and immunostained with anti-Snapin antibody. Panels show the confocal microscopy images obtained at 3 and 5 min after methoxamine treatment. Magnified images of each micrograph are shown in lower panels to visualize more clearly the accumulation of Snapin at the plasma membrane after receptor activation. C, representative plot of intensity of green pixel units, reflecting the abundance of Snapin along a trajectory across a cell during morphometric analysis. The trajectory across the cell for this plot is indicated as a white arrow in the upper right panel of B. In the pixel plot (C), the lower horizontal straight line of an intensity of 15 shows the mean background pixel intensity. The upper dotted line at 40 pixel intensity units (i.e. mean background intensity plus 2x the background S.D.) shows the threshold value used to determine the beginning of the cell edges. Pixel intensity was averaged over a 1-µm distance at the cell edge and then over a region corresponding to the mid-range of the intracellular domain. Averaged pixel intensity units (± S.E.) for more than 20 trajectories across the cell were expressed as histograms as in D. D, morphometric analysis of the abundance of endogenous Snapin at the 1-µm plasma membrane region and at the mid-range of the intracellular domain before and after the stimulation of {alpha}lA-AR in PC12 cells transfected with mock vector or FLAG-tagged {alpha}lA-AR-transfected cells. Snapin average pixel intensity was measured at the 1-µm plasma membrane region (filled bars) and in the intracellular space (open bars) as described under "Experimental Procedures" and as outlined in C. Snapin at the plasma membrane region (as outlined above) was slightly increased in {alpha}lA-AR-transfected cells relative to mock-transfected cells and was augmented significantly at 3 and 5 min after receptor activation by methoxamine treatment (*, p < 0.05; **, p < 0.01 by ANOVA and post hoc tests), whereas the abundance of intracellular Snapin was not significantly changed.

 
Snapin Augments {alpha}lA-AR-stimulated Ca2+ Influx—Upon activation, the {alpha}1A-AR causes a transient rapid IP3-mediated mobilization of [Ca2+]i followed by a sustained increase in [Ca2+]i from extracellular entry. We hypothesized that Snapin might modulate these two phases of {alpha}1A-AR-triggered increases in [Ca2+]i. In PC12 cells stably transfected with the {alpha}lA-AR ({alpha}lA-PC12), Snapin (Snapin-PC12), or both Snapin and the receptor (Snapin-{alpha}lA-PC12), the resting [Ca2+]i values did not differ. In {alpha}1A-PC12 cells that expressed either basal or enhanced levels of Snapin, methoxamine (100 µM) caused a biphasic increase in [Ca2+]i with an initial transient phase peaking within 15 s and a subsequent tonic phase sustained for at least 3 min (Fig. 4, A and C). The initial transient response was not modified by co-expression of Snapin with the receptor (peak increase in [Ca2+]i: 370 ± 24 nM in {alpha}lA-PC12 and 370 ± 17 nM in Snapin-{alpha}lA-PC12, n = 6). Significantly, in Snapin-{alpha}lA-PC12 cells, the sustained increase in [Ca2+]i was much more prominent, remaining at a plateau instead of decreasing. The sustained Ca2+ level was doubled relative to cells expressing the {alpha}1A-AR along with only endogenous Snapin (180 ± 6nM in {alpha}lA-PC12; 320 ± 27 nM in Snapin overexpressing Snapin-{alpha}lA-PC12 cells at 3 min after stimulation, respectively, n = 6; see Fig. 4, A, C, and D). These Ca2+ responses were completely inhibited by either prazosin (100 nM) or silodosin (30 nM), specific {alpha}l-AR antagonists (data not shown). The levels of {alpha}lA-AR were the same in {alpha}lA-PC12 cells and in Snapin-{alpha}lA-PC12 cells (700 ± 93 and 630 ± 130 fmol/mg membrane protein, respectively). Mock vector-transfected cells or Snapin-PC12 cells that did not possess the {alpha}lA-AR by ligand binding measurements (data not shown) failed completely to respond to methoxamine (Fig. 4A).

The P2Y (P2Y-R) and muscarinic acetylcholine receptors are endogenously expressed and are coupled with Gq/11 in PC12 cells (24, 25). Stimulation of the P2Y-R by 10 µM UTP produced a peak and sustained phase increase in [Ca2+]i, which was the same in cells transfected with the {alpha}lA-AR and Snapin either alone ({alpha}lA- or Snapin-PC12 cells) or in combination (Snapin-{alpha}lA-PC12 cells) (Fig. 4B and data not shown). Similarly, transfection of Snapin did not affect the peak and sustained Ca2+ response caused by activation of the muscarinic acetylcholine receptor by 10 µM carbachol (data not shown). These results suggested that Snapin selectively enhanced the ability of the {alpha}lA-AR to increase intracellular Ca2+.

Snapin Increases [Ca2+]i by Enhancing {alpha}1A-AR-tiggered Ca2+ Entry via ROC Channel—As is well accepted, the tonic sustained increase in intracellular Ca2+ levels can be attributed to the influx of Ca2+ through SOC/ROC channels (2). As shown in Fig. 4, C and D, the tonic sustained phase of elevated intracellular Ca2+ (but not the initial transient phase) in the presence of either endogenous Snapin or in Snapin-transfected cells was strongly attenuated in the absence of extracellular Ca2+ (Ca2+(-) buffer, supplemented with 2 mM EGTA). Furthermore, pretreatment with SK&F 96365 (10 µM, SK&F, a blocker of SOC/ROC channels) also suppressed the sustained increase in [Ca2+]i in cells that expressed the {alpha}lA-AR in the presence of either background or Snapin-supplemented cells (Fig. 4, C and D).

To assess further the mechanism of Ca2+ entry, the following drugs were examined in methoxamine-stimulated cells that expressed the {alpha}lA-AR either alone or in combination with Snapin overexpression. For category I, the following were examined: YM-254890 (1 µM, Gq inhibitor (26)), U-73122 (10 µM, PLC inhibitor), 2-aminoethyl diphenylborinate (100 µM, nonselective inhibitor of Ca2+ channels including ROC/SOC channels (27)), and lanthanide (La3+, 1 µM, known as an inhibitor of ROC/SOC channels and TRPC channels (28)). All of these category I inhibitors reduced the receptor-triggered sustained elevation of [Ca2+]i in {alpha}lA-PC12 cells (Fig. 4E, left) and Snapin-{alpha}lA-PC12 cells (Fig. 4E, right). For category II, the following were examined: voltage-gated Ca2+ channel blockers, nifedipine (2 µM), {omega}-conotoxin GVIA (1 µM), and {omega}-agatoxin TK (0.2 µM) (L-, N-, and P/Q-type Ca2+ channel blockers, respectively) and the ryanodine receptor blocker, dantrolene (10 µM). None of these category II blockers affected the tonic increases in [Ca2+]i (Fig. 4E and data not shown). Based on these results, we hypothesized that the augmentation of the sustained phase of [Ca2+]i accompanying the co-expression of Snapin along with the receptor was due to the enhanced entry of Ca2+ through ROC/SOC channels.

To minimize the effect of Ca2+ release from the intracellular Ca2+ store, cells were first stimulated with methoxamine in nominally Ca2+-free buffer (Ca2+-free buffer, without added EGTA) and then Ca2+ was re-added to the bath. The transient Ca2+ release in cells expressing the {alpha}lA-AR with or without overexpressed Snapin did not differ (Fig. 5A, left-hand part). However, the sustained level of intracellular Ca2+ observed upon replenishing extracellular Ca2+ was much greater in the Snapin-overexpressing cells (p < 0.01, n = 3) compared with cells expressing background levels of Snapin (Fig. 5A, right-hand part). Addition of 1 µM La3+ (Fig. 5A, far right) abolished this Ca2+ influx in both cell lines ({alpha}1A-AR with or without overexpressed Snapin). Neither the mock-transfected cells nor the cells overexpressing Snapin, but not the {alpha}1A-AR, responded in aLa3+-sensitive manner to the re-addition of Ca2+ to the buffer (lower curves, Fig. 5A).

We next turned to a brief analysis of the mechanism of Ca2+ entry (Fig. 5, B-D). First, we examined whether the augmentation of the sustained Ca2+ influx in {alpha}lA-AR cells with or without overexpressed Snapin was caused by the activation of SOC channels induced by store depletion or by the direct activation of ROC channels upon {alpha}1A-AR stimulation. Upon treatment of cells in Ca2+-free medium with DBHQ (10 µM, an inhibitor of the sarco/endoplasmic reticulum Ca2+ pump), the re-addition of Ca2+ evoked La3+-sensitive Ca2+ influx, which is considered to be mediated via SOC channels (29) (Fig. 5B). Prior to receptor activation, the amplitudes of Ca2+ influx were not different between {alpha}lA-PC12 cells and Snapin-{alpha}lA-PC12 cells (Fig. 5, B and D). However, the influx of Ca2+ after activation of {alpha}lA-AR with methoxamine was significantly larger in Snapin-{alpha}lA-PC12 cells than in the {alpha}lA-PC12 cells that express only background Snapin (p < 0.01, n = 3) (Fig. 5, C and D) even when cells were pretreated with DBHQ. These results suggested that Snapin selectively augmented {alpha}lA-AR-mediated ROC influx but not SOC influx.


Figure 4
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  		FIGURE 4.
[Ca2+]i responses in clonal PC12 cells evoked by methoxamine or by UTP. A-D, representative tracings of [Ca2+]i transients in response to methoxamine (100 µM) or UTP (10 µM) in the absence or presence of extracellular Ca2+ and SK&F. Shown are tracings acquired in the normal Ca2+(+) buffer in receptor alone ({alpha}lA-PC12) (black tracing), both the receptor and Snapin (Snapin-{alpha}lA-PC12) (green), Snapin alone (blue), or mock (gray)-transfected cells stimulated with methoxamine (100 µM) (A) or UTP (10 µM) (B). Responses stimulated with methoxamine in {alpha}lA-PC12 (C) and in Snapin-{alpha}lA-PC12 cells (D) were monitored in normal Ca2+(+) assay buffer, Ca2+(-) buffer (purple), and normal buffer with 10 µM SK&F (light blue). E, bars in the histogram show the increases in [Ca2+]i (in nM) from the basal level during the tonic phase of Ca2+ entry (at 3 min) after stimulation of either {alpha}lA-AR or Snapin-{alpha}lA-PC12 cells in the absence or presence of the inhibitors shown below each bar. Values represent the means ± S.E. (error bars: n = 3-7). The two bars on the far left in the data for each cell line show the values in normal Ca2+ assay buffer (control) and in Ca2+-free buffer [Ca(-)], respectively. The increases in [Ca2+]i were also measured upon treatment with the inhibitors, YM-254890 (1 µM), U-73122 (10 µM), SK&F (10 µM), 2-aminoethyl diphenylborinate (2-APB) (100 µM), La3+ (La, 1 µM), and nifedipine (2 µM). Cells were exposed to these agents during the tonic phase of increase in [Ca2+]i (about 2 min after methoxamine stimulation). (# and *, statistically different at the level of p < 0.05 versus control of {alpha}lA-PC12 cells and versus control of Snapin-{alpha}lA-PC12 cells, respectively).

 
Selective Effect of Snapin-{alpha}lA-AR Interaction on Sustained Increases of [Ca2+]i—As determined by colony-lift assays (Fig. 2), the C-terminal {alpha}lA-AR residues 376-399 confer its interaction with Snapin. Whether this interaction is necessary for the augmentation of [Ca2+]i increase was examined using an {alpha}lA-AR with these residues mutated. The mutant with a random sequence ("RWGRKIEPVTRKSDYCVSGFTEIF") substituted for the 24-amino acid residues ({alpha}1A-AR-(S376-399), see supplemental Methods) was transiently transfected into Snapin-PC12 cells. In contrast with cells co-transfected with wild-type {alpha}lA-AR and Snapin, the sustained [Ca2+]i level was significantly lower in the cells co-transfected with this {alpha}1A-AR-(S376-399) mutant (Fig. 5E, black bar). We did not test the complete truncation mutant, since the C-terminal residues of the receptor are believed to be important on their own for regulating intracellular Ca2+ (14). Although the sustained levels of intracellular Ca2+ were lower in the cells expressing the {alpha}1A-AR-(S376-399) mutant (receptor levels equivalent to the wild-type receptor), the initial transient increase in [Ca2+]i was not different between the two cell lines (data not shown). The results demonstrated further a functional consequence of the interaction between Snapin and the {alpha}lA-AR to augment sustained Ca2+ entry.

Knockdown of Snapin by siRNA—To confirm the specific involvement of Snapin further in cell signaling induced by {alpha}1A-AR activation, Snapin mRNA was targeted by RNAi in both Snapin-{alpha}lA-PC12 cells and {alpha}lA-PC12 cells (endogenously expressing Snapin), whereas Snapin levels were markedly reduced (Fig. 6A). Relative to the background PC12 cells expressing the {alpha}1A-AR, Western blot analysis and quantitative densitometric scanning (Fig. 6B) revealed that in the Snapin siRNA-treated cells there was about a 70% decrease in the level of Snapin protein that migrated in the region just above 15 kDa (Fig. 6A, lanes 1 and 2). This result showed the efficacy of the siRNA and served to validate the selectivity of the anti-Snapin antibody used for the studies. In the Snapin-overexpressing {alpha}1A-AR cells, there was about a 2-fold increase in the levels of Snapin, as shown by the Western blot analysis (Fig. 6A, lane 3, compared with lane 1). Snapin siRNA treatment resulted in about a 50% reduction of Snapin in these cells that co-expressed the {alpha}1A-AR (Fig. 6A, lane 4). The cells overexpressing Snapin without the {alpha}1A-AR expressed levels of Snapin that were equivalent to the cells co-expressing Snapin along with the receptor (Fig. 6A, lane 5).

In both the {alpha}lA-PC12 and Snapin-{alpha}lA-PC12 cells treated with Snapin siRNA, the acute transient increases in [Ca2+]i caused by methoxamine were not changed. However, upon treatment with Snapin siRNA, the sustained {alpha}1A-AR-mediated increases in [Ca2+]i were attenuated both in the cells expressing background levels of Snapin and in the cells overexpressing Snapin (Fig. 6, C, D, and G). The net increase of [Ca2+]i 3 min after the {alpha}lA-AR stimulation was 100 ± 11 nM and 180 ± 4in {alpha}lA-PC12 cells transfected with sense and missense siRNA for Snapin, respectively, and 170 ± 18 and 288 ± 40 nM in Snapin-{alpha}lA-PC12 cells transfected with sense and missense siRNA for Snapin, respectively. Knockdown of Snapin also caused a reduction of the receptor-operated Ca2+ influx as monitored by the Ca2+ addition protocol (Fig. 6E). The data indicated that even in the cells expressing low levels of endogenous Snapin, the Snapin-{alpha}1A-AR interaction affected receptor-regulated Ca2+ influx (Fig. 6, C and E). In contrast, Snapin siRNA in Snapin-{alpha}lA-PC12 cells did not affect the acute and sustained increases in [Ca2+]i caused by activation of the P2Y-R by UTP (Fig. 6F). We also designed scrambled siRNA as another control, transfected into both Snapin-{alpha}lA-PC12 cells and {alpha}lA-PC12 cells, and measured the [Ca2+]i response to the methoxamine stimulation. The responses were not significantly different from those in the control cells transfected with missense siRNA (see supplemental Methods and supplemental Fig. 4).


Figure 5
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  		FIGURE 5.
Sustained Ca2+ entry in clonal PC12 cells. A, isolation of the sustained Ca2+ influx invoked by {alpha}lA-AR activation in Snapin-{alpha}lA-PC12 (green), {alpha}lA-PC12 (black), Snapin (blue), or mock (gray)-transfected cells monitored by [Ca2+]i measurement. Each cell clone was stimulated by methoxamine (100 µM) in nominally Ca2+-free buffer, and the transient increase in [Ca2+]i was allowed to return toward base line. Then, 1.5 mM CaCl2 was added to the buffer to promote the influx of extracellular Ca2+. At the plateau of Ca2+ entry, La3+ (1 µM) was then added to the medium, and the level of [Ca2+]i was continually monitored. B and C, representative traces of [Ca2+]i transients and the effect of La3+ in the absence (B) or presence (C) of methoxamine stimulation after DBHQ treatment (10 µM) for 10 min in either {alpha}lA-AR (black) or Snapin-{alpha}lA-PC12 cells (green). D, bars in the histogram show the increases in [Ca2+]i (in nM) from the basal [Ca2+]i level after the addition of CaCl2. The responses in the absence or presence of DBHQ (10 µM) and stimulation of methoxamine (100 µM) in {alpha}lA-AR-(black), Snapin-{alpha}lA-(green), Snapin-(blue), or mock (gray)-transfected PC12 cells are shown in each bar graph. Values represent the means ± S.E. (n = 3-4) for independent experiments done using independently grown preparations of the cell lines; #, statistically significant at p < 0.05 in one-way ANOVA; *, p < 0.01 in two-way ANOVA. E, bars show the average increases in [Ca2+]i from the basal level during the tonic phase of Ca2+ entry in Snapin-PC12 cells transiently transfected with either the WT {alpha}lA-AR or the C-terminal receptor mutant ({alpha}1A-AR-(S376-399)). The [Ca2+]i increase in the WT {alpha}lA-AR transfected cells (closed green bar) was higher than that of the mutant {alpha}lA-AR (closed black bar), and the responses in both cell transfects were abolished by the removal of extracellular Ca2+ (green and black shaded bar for WT {alpha}lA-AR or {alpha}1A-AR-(S376-399), respectively) (n = 3-7 for independent experiments done using separate preparations of Snapin expressing cells transfected with either WT or mutant of {alpha}lA-AR); # and *, statistically significant (p < 0.05) versus WT {alpha}lA-AR-transfected cells (closed green bar) in Ca2+(+) buffer, and versus {alpha}1A-AR-(S376-399) transfected cells (closed black bar) in Ca2+(+) buffer, respectively).

 
Interaction of Snapin with TRPC6—Because Snapin could augment Ca2+ entry ostensibly via ROC channels, we wondered if Snapin might also interact directly or indirectly with such channels. Recently, some transient receptor potential channels, especially TRPC3, TRPC5, and TRPC6, have been shown to be activated by GqPCRs. In Western blot analysis using membrane preparations obtained from PC12 cells and rat cortex, anti-TRPC6 antibody detected a band of ~120 kDa (Fig. 7B), which was absent when the antibody was pre-absorbed with the antigen peptide (Fig. 7C). These results, together with preliminary Western blot analysis, showed a predominant expression of TRPC6 among the three TRPCs in PC12 cells and in rat cortex. A direct interaction between Snapin and TRPC6 was demonstrated by co-immunoprecipitation experiments using in vitro synthesized Snapin and TRPC6 (Fig. 7A). Using an immunoprecipitation-Western blot approach, we found further that TRPC6 co-immunoprecipitated with Snapin from extracts of rat brain cortex (Fig. 7D). The TRPC3, but not the TRPC5, of rat cortex extracts also co-immunoprecipitated with Snapin but in amounts that were much lower that of TRPC6 (data not shown). In keeping with these results with brain tissue extracts, we found that in Snapin-{alpha}lA-PC12 cells, not only did the channel co-immunoprecipitate with Snapin but the activation of the receptor by methoxamine (100 µM) considerably increased the amount of TRPC6 that was found in the anti-Snapin immunoprecipitate (Fig. 7, E and F). The interaction between Snapin and {alpha}lA-AR was also increased by the receptor activation (Fig. 7, E and F). In contrast, prazosin (100 nM), an {alpha}l-AR antagonist, decreased the amounts of TRPC6 and {alpha}lA-AR in the anti-Snapin immunoprecipitate after methoxamine-induced {alpha}lA-AR activation and even in cells that had not been exposed to agonist (Fig. 7, E and F). This result suggested a receptor-mediated interaction with TRPC6 via Snapin. In keeping with our results pointing to increases of the interaction of Snapin with {alpha}lA-AR localized to the cell surface, we hypothesized that the abundance of cell surface TRPC6 accessible to biotinylation at the plasma membrane would also increase upon activating the {alpha}lA-AR. Indeed, in {alpha}lA-AR-Snapin cells surface-labeled with biotin, treatment with methoxamine increased the proportion of biotinylated surface-accessible TRPC6, relative to total cellular TRPC6 (Fig. 7, G and H).


Figure 6
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  		FIGURE 6.
RNAi for Snapin. A, expression level of Snapin was analyzed by Western blotting (WB) in the {alpha}lA-PC12 cell clone (lanes 1 and 2) and in the Snapin-{alpha}lA-PC12 clone (lanes 3 and 4) transfected with the missense siRNA or pSilencer siRNA expression vector for Snapin and in the Snapin-PC12 cells (lane 5). Expression of beta-actin was shown together to confirm the equal protein loading. B, four experiments as shown in A were repeated, and the bands were analyzed by an image densitometer. The bars in the graph show relative expression level of Snapin in each cell clone (n = 4; #, statistically significant at p < 0.05). C-F, tracings show the [Ca2+]i increase induced by methoxamine (100µM, C-E) or UTP (10µM, F) in the {alpha}lA-AR (C and E) and in the Snapin-{alpha}lA-AR PC12 cells (D and F) that were transfected with the siRNA for Snapin (gray) or the missense siRNA (black). G, bars show the net increase of [Ca2+]i in each cell clone 3 min after {alpha}lA-AR stimulation (n = 4-12; #, statistically significant at p < 0.05). E, Ca2+ influx caused by {alpha}lA-AR activation in the {alpha}lA-PC12 cells (black) was suppressed in the cells transfected with siRNA for Snapin (gray). Each cell clone was first stimulated by methoxamine in Ca2+-free buffer, and at about 3 min the buffer was supplemented with added Ca2+ to a final concentration of 1.5 mM. C, D, and F, the augmented [Ca2+]i response induced by {alpha}lA-AR activation was abolished in Snapin knocked down cells (C and D), while that caused by P2Y-R activation remained unchanged (F).

 
Effect of Snapin on {alpha}lA-AR-activated Inositol Phosphate Metabolism—The effects of Snapin on inositol phosphates metabolism were finally examined. Starting at the same basal levels, the increased accumulation of inositol phosphates induced by methoxamine (10 µM) was not significantly different between {alpha}lA-PC12 and Snapin-{alpha}lA-PC12 cells (19 ± 3.3 and 18 ± 0.9 x 103 dpm/2 x 105 cells, respectively). These results showed that, in contrast with the sustained phase in [Ca2+]i response, the {alpha}lA-AR-activated PLC-IP3 signaling system was not affected by co-expression of Snapin. This result was consistent with a lack of effect of Snapin on the initial transient increase in [Ca2+]i, which is due to IP3-mediated Ca2+ release from the ER.


Figure 7
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  		FIGURE 7.
Involvement of TRPC6 in cellular signaling evoked by{alpha}lA-AR activation. A, in vitro translated [35S]methionine-labeled c-Myc-TRPC6 (lanes 2 and 5) and HA-Snapin (lanes 1 and 6), and the mixture of both proteins (lanes 3 and 4) were immunoprecipitated (IP) with anti-c-Myc antibody (lanes 1, 3, and 5) or anti-HA antibody (lanes 2, 4, and 6), respectively. The immunoprecipitates were then subjected to PAGE. HA-Snapin (15 kDa) was detected in lanes 3, 4, and 6 and c-Myc-TRPC6 (120 kDa) in lanes 3-5. The molecular masses estimated for Snapin (15 kDa) and TRPC6 (120 kDa) are also shown. B, anti-TRPC6 antibody detected a band of ~120 kDa in both lanes loaded with equal amounts (20 µg) of solubilized membrane obtained from PC12 cells and rat cortex. The positions of the molecular size markers (kDa) are shown. C, anti-TRPC6 channel antibody was pre-absorbed with an antigen peptide provided with the antibody (Alomone Labs). Membrane protein (10 µg) of PC12 cells was subjected to Western blot (WB) analysis using either pre-absorbed or untreated antibody. The pre-absorbed antibody could not detect a band of TRPC6 (120 kDa) (left lane), whereas the normal untreated antibody could (right lane), and in either lane beta-actin could be similarly detected on the same membrane by anti-beta-actin antibody. D, solubilized rat brain cortex membrane fraction was immunoprecipitated with anti-Snapin antibody. TRPC6 (120 kDa) was strongly enriched in the Snapin co-immunoprecipitate (right), as compared with the crude membrane preparation (left). Positions of the molecular weight markers (kDa) are also shown. E, interaction between Snapin and TRPC6 or {alpha}lA-AR was promoted by receptor activation. The Snapin-{alpha}lA-PC12 cells were stimulated by methoxamine (100 µM) for 10 min with or without 100 nM prazosin, lysed, and immunoprecipitated with anti-Snapin antibody. The immunoprecipitates were analyzed by Western blot analysis. The amounts of TRPC6 and {alpha}lA-AR co-immunoprecipitating with Snapin were significantly increased by {alpha}lA-AR activation and that association was reversed by the receptor antagonist prazosin. F, experiments in E were repeated (n = 3) and analyzed by an image densitometer. The bars show the relative amounts of TRPC6 (left graph) and {alpha}lA-AR (right graph) immunoprecipitated with anti-Snapin-antibody, normalized for the abundance of TRPC6 and {alpha}lA-AR harvested from agonist/antagonist untreated cells. Both co-immunoprecipitated TRPC6 and {alpha}lA-AR were significantly increased upon activation of the receptor by methoxamine (*, at p < 0.05 versus nonactivated cells). Treatment with prazosin significantly reduced the amount of co-immunoprecipitated TRPC6 with or even without methoxamine-induced receptor activation (#, at p < 0.05). G, increase in cell surface TRPC6 in the Snapin-{alpha}lA-PC12 cells upon {alpha}lA-AR activation. Cells were stimulated or not with methoxamine (100 µM) for 10 min. Cell surface proteins on plasma membrane were then biotinylated for 30 min; cells were lysed, and surface proteins were harvested using an avidin-conjugated affinity column. Total and surface TRPC6 with or without treatment by methoxamine were compared by Western blot analyses. As a control, HSP90, a cytoplasmic protein, was also examined by Western blot analyses, to establish by its absence of biotinylation (not shown) that only surface proteins were successfully biotinylated. H, three experiments as shown in G were repeated, and the bands were analyzed by an image densitometer. The bars in the graph show the ratio of surface TRPC6 to total TRPC6 in untreated and methoxamine-treated Snapin-{alpha}lA-PC12 cells (*, significant at p < 0.05 versus untreated cells).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The index finding of our study was obtained with a yeast two-hybrid screening colony-lift assay, showing that Snapin, known as a SNAP-25 binding protein of the SNARE complex, can interact with the C-terminal domain of the {alpha}lA-AR. Co-immunoprecipitation studies using rat cortex tissue, Snapin-{alpha}lA-PC12 cells, or with the in vitro synthesized proteins provided further evidence for an interaction between Snapin and the {alpha}lA-AR. In addition, our data indicate an unexpected interaction between Snapin and TRPC6, using the same methods, thus pointing to a tripartite complex between {alpha}lA-AR, Snapin, and TRPC6. A functional interaction between Snapin and the receptor was established by studying Snapin-mediated increases in the sustained levels of [Ca2+]i triggered by {alpha}lA-AR activation.

The functional consequences of an interaction between Snapin and the {alpha}lA-AR were first examined in a model neuronal PC12 cell system that does not constitutively express the {alpha}lA-AR but does express low endogenous levels of Snapin, as documented by Western blot analysis. Using this model system, along with a morphometric immunohistochemical approach and employing Snapin siRNA, we found that both endogenously expressed and overexpressed Snapin has an impact on {alpha}lA-AR signaling. In either wild-type PC12 cells or those transfected with the {alpha}lA-AR supplemented or not with additional Snapin transfection, Snapin was distributed both at the plasma membrane and intracellularly. However, upon transfecting the PC12 cells with the {alpha}lA-AR, morphometric analysis revealed a modest increase of the abundance of Snapin at the plasma membrane relative to mock-transfected cells. Activation of the receptor with methoxamine resulted in a further time-dependent recruitment of Snapin to the plasma membrane region, relative to the abundance of Snapin in the intracellular compartment, which remained essentially unchanged, presumably because of the much larger amount of intracellular Snapin relative to the amount recruited to the region of the plasma membrane (Fig. 3D). This physical recruitment of Snapin to the plasma membrane was in keeping with its impact on receptor-mediated Ca2+ influx.


Figure 8
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  		FIGURE 8.
A simplified model of a mechanism for the augmentation of Ca2+ influx via TRPC6 caused by activating the Snapin-associated {alpha}1A-AR. Before {alpha}1A-AR activation, Snapin is shown to be pre-coupled to the {alpha}1A-AR, but many of TRPC6 are not inserted into the plasma membrane and do not serve as a Ca2+ entry pathway (left). Upon {alpha}1A-AR activation, the {alpha}1A-AR and Snapin dimer forming a tripartite complex with TRPC6 is shown to facilitate the insertion of TRPC6 into the plasma membrane. Ca2+ influx then ensues via the TRPC6 channels recruited to the plasma membrane (right).

 
TRPC channels are strong candidates to represent the long sought after ROC/SOC channels (2, 30). In the Western blot analysis, TRPC6 was a predominant component detected among the three TRPCs evaluated (TRPC3, TRPC5, and TRPC6) in the PC12 cells and the rat cortex. Upon finding that TRPC6 co-immunoprecipitated with Snapin from homogenates of rat brain cortex, we examined whether {alpha}lA-AR activation might influence the interaction between Snapin and TRPC6 in a cultured cell system. Preliminarily, we confirmed that Snapin overexpression did not cause any change in the expression level of both TRPC6 and Gq proteins in this cell system (see supplemental Fig. 1). The co-immunoprecipitation data suggested that upon activation of the {alpha}lA-AR, the interaction between TRPC6 and Snapin was considerably augmented (Fig. 7, E and F). Moreover, the interaction of Snapin with the {alpha}lA-AR was also increased upon activation of the receptor itself. These increases of TRPC6 and {alpha}lA-AR in the anti-Snapin immunoprecipitate upon activation of the receptor were antagonized by prazosin, an {alpha}l-AR antagonist. It is interesting to note that the interaction of Snapin with TRPC6 in agonist-untreated cells was reduced by the antagonist, prazosin (Fig. 7, E and F). This result can be explained by the inverse agonism of prazosin, which would be expected to reverse a constitutive interaction between the {alpha}lA-AR and its effectors involved in Ca2+ influx in these {alpha}l-AR-overexpressing cells (31, 32). In accord with these results and the localization of {alpha}lA-AR at the cell membrane (Fig. 3), the abundance of TRPC6 on the cell surface was also increased by receptor activation (Fig. 7, G and H). From these results, we suggest that activation of {alpha}lA-AR triggers the following: (i) an increased interaction of Snapin with both the {alpha}lA-AR and TRPC6, (ii) an increase in the redistribution of TRPC6 to the cell surface, and (iii) increased Ca2+ influx via the recruited TRPC6.

The augmentation of Ca2+ influx caused by co-expression of the {alpha}lA-AR along with Snapin (either endogenous or overexpressed) was observed only when the {alpha}lA-AR was activated. The influx of Ca2+ after emptying the Ca2+ store by treatment with DBHQ was not affected by co-expression of Snapin, if the {alpha}lA-AR was not stimulated (Fig. 5B). This result suggests that the enhancement in sustained Ca2+ influx in Snapin-{alpha}lA-PC12 cells may be predominantly caused through {alpha}lA-AR-mediated ROC channels rather than SOC channels (Fig. 5C). This conclusion is consistent with the report that TRPC6 can serve as an {alpha}l-AR-stimulated Ca2+-permeable cation channel in vascular smooth muscle (33). Thus, our results can be summarized by the following model. (i) Snapin interacts directly with the {alpha}lA-AR. (ii) This interaction provides a key link between {alpha}lA-AR cellular signaling and ROC channels (probably TRPC6) upon {alpha}lA-AR activation. (iii) The tripartite receptor-Snapin-TRPC complex transmits the receptor-activated signal by increasing Ca2+ influx (Fig. 8). Further work is clearly warranted to evaluate in more depth the tripartite interaction we propose (receptor-Snapin-TRPC6).

This kind of mechanism and process for Ca2+ entry would be akin to the role of the Homer protein, which interacts both with TRPC channels as well as the IP3 receptor (5, 34). However, in this case, disruption of the Homer-TRPC interaction was reported to result in the store-dependent activation of TRPC1 (35). PLC-{gamma}, which can interact with TRPCs via its PH domain, is another key element in the ROC signaling complex. Patterson et al. (10) have demonstrated that PLC-{gamma} activation in PC12 cells causes an increase in both intracellular Ca2+ release and Ca2+ entry through ROC channel upon activation of Gq-protein-coupled receptors, including the P2Y-R. However, in this study, the entry of Ca2+ induced by activation of either the P2Y-R or the muscarinic acetylcholine receptors was not affected by either Snapin overexpression nor knockdown, suggesting that the Snapin-receptor interaction represents a new ROC signaling mechanism.

Snapin was originally identified as a binding protein of SNAP-25, one of constituents of SNARE complex (15), enhancing the association of synaptotagmin, a potent Ca2+ sensor, with the SNARE complex (15, 16). Recent data suggest further that TRPC translocation or activation depends on members of the SNARE complex, including VAMP (36) and SNAP-25 (37). Although our data suggested the direct coupling of receptor-Snapin-TRPC6 channel, Snapin in the SNARE complex may help the trafficking of TRPC6 triggered by the Ca2+ increase upon activation of the {alpha}1A-AR. In our Snapin-{alpha}lA-PC12 cells, Snapin did co-immunoprecipitate with SNAP-25, and this interaction was enhanced by activation of {alpha}lA-AR.4 Thus, we suggest that the SNARE complex may be involved in the trafficking of TRPC6 to the cell surface, enhancing further the entry of Ca2+ via ROC channels. The involvement of the SNARE complex in this mechanism of Ca2+ influx augmentation and the mechanism(s) whereby the SNARE complex collaborates with the Snapin-{alpha}1A-AR complex linking to TRPC6 remain to be clarified.

In summary, our results suggest that Snapin interacts with both the {alpha}lA-AR and TRPC6 to augment {alpha}lA-AR-operated Ca2+ influx through the TRPC6 channel in response to adrenergic receptor activation. This hypothesis, supported by our data, merits further intensive in-depth evaluation in the immediate future.


   FOOTNOTES
 
* This work was supported in part by grant-in-aid for scientific research from the Japan Society of the Promotion of Science (to F. S. and I. M.) and by the 21st Center of Excellence research program (Medical Science). 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Methods and Figs. 1-4. Back

1 Both authors contributed equally to this work. Back

2 To whom correspondence should be addressed. Tel.: 81-776-61-8328; Fax: 81-776-61-8130; E-mail: muramatu{at}u-fukui.ac.jp.

3 The abbreviations used are: GqPCR, Gq-protein-coupled receptor; {alpha}1A-AR, {alpha}1A-adrenoceptor; ROC, receptor-operated Ca2+;IP3, inositol 1,4,5-triphosphate; SNARE, soluble NSF attachment protein receptor; WT, wild type; siRNA, short interfering RNA; TRPC, transient receptor potential canonical; PLC, phospholipase C; [Ca2+]i, intracellular Ca2+; FITC, fluorescein isothiocyanate; TRITC, tetramethylrhodamine isothiocyanate; ANOVA, analysis of variance; SOC, store-operated Ca2+; RNAi, RNA interference; HA, hemagglutinin; DBHQ, 2,5-di-(t-butyl)-1,4-benzohydroquinone. Back

4 F. Suzuki and I. Muramatsu, unpublished observations. Back


   ACKNOWLEDGMENTS
 
We express great appreciation to Professor M. D. Hollenberg (University of Calgary) for the helpful discussions and comments concerning the writing of our manuscript. We also thank Drs. K. Yamada and T. Taniguchi for much valuable advice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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