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Am J Physiol Heart Circ Physiol 292: H874-H883, 2007. First published September 29, 2006; doi:10.1152/ajpheart.00785.2006
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TRPC3 channels colocalize with Na+/Ca2+ exchanger and Na+ pump in axial component of transverse-axial tubular system of rat ventricle

Monu Goel,2 Cheng-Di Zuo,2 William G. Sinkins,2 and William P. Schilling1,2

1Rammelkamp Center for Education and Research, MetroHealth Medical Center and 2Department of Physiology and Biophysics, Case Western Reserve University School of Medicine, Cleveland, Ohio

Submitted 21 July 2006 ; accepted in final form 22 September 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Transient receptor potential canonical (TRPC) proteins form Ca2+-permeable, nonselective cation channels activated after stimulation of G protein-coupled membrane receptors linked to phospholipase C (PLC). Although the PLC/inositol phosphate signaling pathway is known to exist in heart, expression and subcellular distribution of TRPC channel proteins in ventricular myocardium have not been evaluated. Of the six members of the TRPC channel family examined here, only TRPC3 was found by Western blot analysis of membrane proteins from rodent or canine ventricle. Likewise, only TRPC3 was observed in immunofluorescence analysis of thin sections from rat ventricle. TRPC3 was also the only family member observed in neonatal rat ventricular myocytes in culture. In longitudinal sections of rat ventricle, TRPC3 was predominantly localized to the intercalated disk region of the myocyte. However, transverse sections through heart muscle or single isolated adult myocytes revealed TRPC3-specific labeling in a vast network of intracellular membranes, where it colocalized with the Na+-K+-ATPase (NKA) pump and the Na+/Ca2+ exchanger (NCX) but not with the ryanodine receptor or the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump. Reciprocal immunoprecipitation assays from rat or canine ventricle showed that TRPC3 associates with NKA and NCX but not with the plasmalemmal Ca2+-ATPase pump. Immunoprecipitations from Sf9 insect cells heterologously expressing TRPC3, NKA, and NCX in various combinations revealed that NKA and NCX interact and that TRPC3 and NCX interact, but that TRPC3 does not directly associate with NKA. Together, these results suggest that TRPC3 is localized in the ventricular myocyte to the axial component of the transverse-axial tubular system, where it exists in a signaling complex that includes NCX and NKA.

ventricular myocytes; Ca2+ channels; immunohistochemistry; immunoprecipitation

THE CALCIUM ION is a ubiquitous intracellular second messenger in the cardiovascular system, and periodic coordinated Ca2+ fluxes are vital for normal operation of the heart. In cardiac muscle, channels that allow Ca2+ to move from the extracellular space or from intracellular storage pools into the cytosol are found in specialized regions of the membrane (for recent review see Ref. 3). Likewise, the pumps and transporters that remove Ca2+ from the cytosol are located in distinct regions of the myocyte. Thus on a beat-to-beat basis there is a continuous vectorial flow of Ca2+ from regions of influx to the contractile elements to regions of efflux. This process is carefully balanced and tightly regulated at the level of the myocyte by hormones, neurotransmitters, and paracrine factors that ultimately control cardiac contractility. The receptors for these factors also appear to be localized in specific signaling domains, and, likewise, the downstream effectors may be tethered to specific sites within the cell (14). Thus it is not sufficient to simply understand the structure, function, and regulation of the individual transport proteins, but rather a detailed knowledge of the location and juxtaposition of the various proteins is essential to our understanding of cardiac Ca2+ signaling.

A variety of hormones regulate cardiac contractility by interaction with specific G protein-coupled membrane receptors (GPCRs). In particular, activation of {alpha}1-adrenergic, endothelin-1, P2Y purinergic, or angiotensin II receptors increases cardiac contractility. Although it is well established that these GPCRs are linked to phospholipase C (PLC) and the subsequent generation of inositol 1,4,5-trisphosphate and diacylglycerol (32), the mechanism(s) responsible for the increase in contractility and the Ca2+ transport processes involved remains incompletely understood. In this regard, Ca2+ influx may be mediated by the recently discovered transient receptor potential (TRP) family of ion channels (19). There are 32 members of the mammalian TRP superfamily, which can be divided into at least 6 distinct subfamilies (33). Of these, the TRP canonical (TRPC) subfamily has seven members, designated TRPC1–TRPC7, that are activated by stimulation of GPCRs linked to PLC. In humans, TRPC2 is a pseudogene (31). The TRPC proteins are known to form Ca2+-permeable, nonselective cation channels and, as such, are believed to play an important role in Ca2+ signal transduction in nerve, smooth muscle, and most nonexcitable cell types (19). The presence of TRPC channels in cardiac myocytes and their role in regulating contractile force have not been examined. Unfortunately, functional studies of native TRPC channels have been hampered by the lack of specific pharmacological tools and by the realization that most tissues and cell types express multiple TRPC proteins that may form heteromultimeric channel structures. Furthermore, TRPC channels are thought to exist as part of large macromolecular signaling complexes ("signalplexes"), and their mechanism of activation and regulation may depend on the association with other signalplex components (1).

Given the structural complexity of the cardiac myocyte and the clear segregation of channels, pumps, transporters, and receptors to distinct domains of the sarcolemma and sarcoplasmic reticulum (SR), the first steps toward understanding the role of TRPC channels in cardiac function are to 1) determine which channels are present in the ventricular myocyte, 2) evaluate their subcellular localization and distribution, and 3) determine whether the channels interact with other proteins known to play a role in excitation-contraction coupling in the heart. Toward this end, we have developed two affinity-purified, polyclonal antibodies specific for each TRPC channel subtype (10, 11). Previous studies have shown that these antibodies are useful for evaluation of TRPC channels in the central and peripheral nervous systems and in the kidney (4, 8, 10, 11). In the present study, these antibodies have been used for immunoprecipitation and Western blot analysis and for immunohistochemical evaluation of the TRPC signalplex in ventricular myocytes. The results show that TRPC3 is the only detectable member of the TRPC channel family in rat ventricle. Furthermore, TRPC3 appears to be localized to the axial component of the transverse-axial tubular system (TATS), where it exists in a complex that includes the Na+-K+-ATPase (NKA) pump and the Na+/Ca2+ exchanger (NCX). This places the TRPC3 signalplex near sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pumps present on the longitudinal component of the SR, a position that could directly impact SR Ca2+ load and contractility.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. We previously described (10, 11) the generation, purification, and characterization of two affinity-purified, rabbit polyclonal antibodies specific for each TRPC channel protein. These antibody preparations have been designated {alpha}A-TRPCx and {alpha}B-TRPCx. Commercial antibodies used were from the following sources: secondary antibodies were mouse Alexa 488-, 594-, and 697-conjugated anti-rabbit, anti-mouse, and anti-goat IgG, respectively (Molecular Probes catalog nos. A11034 [GenBank] , A11032, and A11058 [GenBank] , respectively); mouse anti-NKA ({alpha}1-subunit) and mouse anti-plasmalemmal Ca2+-ATPase (PMCA) (Upstate Biotechnology catalog nos. 05-369 and 05-625, respectively); mouse anti-NCX (Swant catalog no. R3F1); and mouse anti-ryanodine receptor (RyR) and anti-SERCA (Affinity BioReagents catalog nos. MA3-916 and MA3-912, respectively).

Isolation of adult rat cardiac myocytes. All experimental protocols involving the use of animals were approved by and performed in compliance with guidelines of the Case Western Reserve University Institutional Animal Care and Use Committee. Hearts removed from adult Sprague-Dawley rats were mounted on a Langendorf apparatus and subjected to retrograde perfusion at room temperature with oxygenated Tyrode solution containing (mM) 130 NaCl, 5.4 KCl, 0.4 NaH2PO4, 1.4 MgCl2·6H2O, 0.5 CaCl2, 10 HEPES, 10 glucose, 20 taurine, and 10 creatine (pH to 7.3 with NaOH). When the coronary circulation had cleared of blood, perfusion was continued with Ca2+-free Tyrode solution (in which CaCl2 had been replaced with 0.1 mM EGTA) for 4 min, followed by perfusion for a further 20 min with Tyrode solution containing 0.8 mg/ml collagenase (type II; Worthington Biochemical; Lakewood, NJ) and 0.05 mM CaCl2. The ventricles were then perfused for 10 min with high-K+ (HK) solution containing (mM) 40 KCl, 1.4 MgSO4·7H2O, 20 KH2PO4, 20 taurine, 10 creatine, 0.5 EGTA, 10 HEPES, 10 glucose, and 50 L-glutamic acid. The ventricles were then excised from the heart, and tissue pieces were passed through a nylon mesh filter. Ventricular myocytes were collected in the eluate and resuspended again in HK solution. The myocytes were used immediately for immunohistochemical analysis.

Isolation and culture of neonatal rat cardiac myocytes. Hearts isolated from rat pups were minced and placed in ice cold Hanks' balanced salt solution (HBSS) containing 0.5 mg/ml of trypsin at 4°C overnight (~18 h). On day 2, tissue was washed three times with HBSS to remove trypsin. Collagenase was slowly added to the minced tissue and incubated at 37°C for 30–45 min with occasional shaking. Tissue was triturated to disperse the cells and subsequently centrifuged for 5 min at 2,000 rpm. The cell pellet was resuspended in L15 culture medium (Worthington Scientific) containing 10% fetal bovine serum. The cells were plated in 35-mm dishes and incubated at 37°C. After 24 h, the medium was replaced with serum-free medium.

Preparation of rat heart lysates. Heart ventricles isolated from adult Sprague-Dawley rats were minced and suspended in lysis buffer containing 150 mM NaCl, 10 mM Tris-Cl (pH 7.5), and 0.1% deoxycholate. The tissue suspension was homogenized on ice with a Brinkman PT10/35 Polytron fitted with a 10-mm generator (3 x 10-s pulses at a power setting of 5). Homogenates/lysates were centrifuged at 6,000 g for 10 min at 4°C to remove tissue debris. The resulting supernatant was incubated at 4°C for 30 min and subsequently subjected to centrifugation at 200,000 g for 60 min at 4°C to remove unsolubilized membrane fragments. The resulting supernatants/lysates were used immediately for immunoprecipitation experiments.

Isolation of canine cardiac sarcolemmal preparation. A highly enriched sarcolemma preparation was isolated from dog heart ventricle as previously described (23, 30). In this procedure, a pregradient microsomal fraction is layered over 24% sucrose and the final sarcolemma preparation is obtained after a high-speed centrifugation step. The final sarcolemma preparation contains ~60% sealed right side out, 22% sealed inside out, and 18% leaky vesicles and is highly enriched in sarcolemmal markers including ouabain-sensitive Na+ pumps, voltage-gated Ca2+ channels, muscarinic receptors, and NCX (2, 13, 23, 30).

Immunoprecipitations and immunoblots. Tissue, cell, and membrane lysates were precleared by adding control IgG together with protein A/G agarose beads for 1 h at 4°C. Precleared lysates were incubated with the primary antibodies, and the immunocomplexes were captured by incubation with protein A/G agarose beads at 4°C for 12 h. Beads were pelleted, washed four times with lysis buffer, resuspended in 100 µl of 2x SDS sample buffer, and boiled for 3 min. Proteins were separated by SDS-PAGE and electrotransferred to polyvinylidene difluoride membrane (100 V for 1 h) in Tris-glycine buffer. Blots were probed with the indicated primary antibody and detected, after incubation with horseradish peroxidase-conjugated secondary antibody, by SuperSignal West Pico chemiluminescent substrate (Pierce). Figures 1–8 show representative results from experiments repeated at least three times.


Figure 1
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  		Fig. 1. Localization of transient receptor potential canonical (TRPC)3 channels in rat ventricle. A: membrane lysates, prepared from rat heart ventricle as described in METHODS, were subjected to immunoprecipitation followed by Western blot analysis. Antibodies used for immunoprecipitation (IP) and immunoblotting (Blot) are indicated below each lane. Numbers on right indicate molecular mass (in kDa). B and C: fluorescence confocal images of thin sections from rat ventricle labeled with the {alpha}A-TRPC3 antibody; longitudinal (B) and transverse (C) sections are shown. D: fluorescence image of a thin section from rat ventricle labeled with the {alpha}A-TRPC3 antibody plus the immunizing peptide; blue reflects DAPI staining of the nuclei. E and F: fluorescence confocal image of isolated adult rat ventricular myocytes labeled with the {alpha}A-TRPC3 antibody; intact myocytes (E) and a transverse thin section (F) are shown. G: fluorescence confocal image of cultured neonatal rat cardiac myocytes labeled with the {alpha}A-TRPC3 antibody. In this and all subsequent figures, identical results were obtained with the {alpha}B-TRPC3 antibody.

 

Figure 8
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  		Fig. 8. Coimmunoprecipitation of TRPC3 with NKA and NCX from Sf9 insect cells. Sf9 insect cells were coinfected with the recombinant baculoviruses indicated below each column of gels. Whole cell lysates were subjected to immunoprecipitation using the primary antibody listed at the top of each lane, and the antibody used for immunoblot is given on the left of each row.

 
Immunofluorescence. Frozen sections (6 µm) from adult rat heart ventricles were fixed in paraformaldehyde for 30 min. For frozen transverse sections of myocytes, isolated adult myocytes were mixed with tissue freezing medium (Fisher) and immediately frozen in liquid nitrogen. Frozen myocyte sections (8 µm) were fixed in paraformaldehyde for 30 min. The sections were briefly rinsed in phosphate-buffered saline (PBS) and subsequently incubated with blocking solution containing 3% IgG-free BSA (Vector laboratories), 10% normal donkey serum, and 0.1% Triton X-100 for 1 h at room temperature. Sections were incubated with the indicated primary antibody overnight at 4°C. After being washed three times for 5 min with PBS at room temperature, the sections were incubated with Alexa 488- or Alexa 594-conjugated anti-rabbit or anti-mouse IgG for 1 h at room temperature. Sections were washed three times with PBS for 5 min and mounted with Vectashield mounting medium (Vector Laboratories). Confocal images were acquired with a Leica TCS SP2 confocal microscope. All of the images shown are x-y scans obtained with a x100 oil immersion objective (1.4 numerical aperture). For longitudinal sections of rat ventricle, the scan mode was 512 x 512, zoom factor of ~3, which yields a x-y pixel size of ~100 x 100 nm. For transverse sections of rat ventricle, the scan mode was 1,024 x 1,024, zoom factor of ~2, which yields a x-y pixel size of ~80 x 80 nm. For transverse sections of isolated adult myocytes, the scan mode was 1,024 x 1,024, zoom factor of ~3, which yields a x-y pixel size of ~55 x 55 nm.

Culture of Sf9 insect cells. Spodoptera frugiperda (Sf9) cells were obtained from American Type Culture Collection and cultured as previously described (21) with Grace's Insect Medium supplemented with 2% lactalbumin hydrolysate, 2% yeastolate solution, 2 mM L-glutamine, 10% heat-inactivated fetal bovine serum, and 1% penicillin-streptomycin-neomycin solution (GIBCO).

Generation of recombinant baculovirus. The cDNAs for mouse TRPC3, rat {alpha}1-NKA, and rat NCX (a generous gift from John Reeves, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, NJ) were subcloned into baculovirus transfer vector pVL1393 as previously described (10). Recombinant baculoviruses were produced with the BaculoGold Transfection Kit (Pharmingen, San Diego, CA) as described in the instructions provided by the manufacturer. Recombinant viruses were amplified to obtain a high-titer viral stock solution and were stored at 4°C under sterile conditions until use.

Infection of Sf9 insect cells with recombinant baculovirus. Sf9 cells in Grace's Insect Medium were plated onto 100-mm plastic tissue culture dishes at a density of ~105 cell/cm2. After incubation for 30 min, an aliquot of viral stock was added (multiplicity of infection was ~10), and the cells were maintained at 27°C in a humidified air atmosphere. For coinfections, viral stocks were simultaneously added to the Sf9 cells in a 1-to-1 ratio. Unless otherwise indicated, cells were used at 28 h after infection.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification and subcellular localization of TRPC channels in rat cardiac myocytes. As seen in Fig. 1A, Western blots of immunoprecipitated proteins from rat ventricle with either the {alpha}A-TRPC3 or {alpha}B-TRPC3 antibody revealed a single protein with a molecular mass appropriate for TRPC3 (~97 kDa). In longitudinal sections through the ventricle, TRPC3 immunoreactivity was predominantly seen at the intercalated disk region of the individual myocytes (Fig. 1B). Likewise, images of isolated adult rat cardiomyocytes showed prominent staining at the intercalated disk and sparse, nonstriated labeling over the majority of the cell surface (Fig. 1E). However, in transverse sections through the rat ventricle (Fig. 1C) and through isolated myocytes (Fig. 1F), specific TRPC3 labeling was observed in what appeared to be an extensive network of intracellular membrane structures. In cultured rat neonatal myocytes, TRPC3 labeling was seen in or near the plasmalemma and intracellularly, where TRPC3 immunoreactivity was arranged along stress/contractile fibers running the length of the cells (Fig. 1G). No immunofluorescence was detected when the primary antibody was preincubated with the immunizing peptide (Fig. 1D) or when the sections were incubated with only the secondary antibody (not shown).

To determine whether additional TRPC family members are expressed in ventricle, tissue lysates were subjected to immunoprecipitation using either the {alpha}A-TRPCx or {alpha}B-TRPCx antibodies and the resulting proteins in the immunocomplexes were separated by electrophoresis and subjected to Western blot analysis. TRPC1, -C4, -C5, -C6, and -C7 were undetectable with either antibody. Likewise, no specific immunofluorescence labeling was observed in thin cryosections from rat heart or in neonatal rat cardiomyocytes in culture with either {alpha}A-TRPCx or {alpha}B-TRPCx of TRPC1, -C4, -C5, -C6, and -C7 (data not shown). Thus these channel proteins are below the limit of detection with our present antibody preparations. To further confirm this result, a highly enriched cardiac sarcolemmal preparation was obtained from canine ventricle (23). Only TRPC3 and several specific sarcolemmal markers including NKA, NCX, and PMCA were readily detectable on Western blot analysis (Fig. 2). Likewise, TRPC3 was the only TRPC channel protein detectable by immunoblot of sarcolemmal proteins immunoprecipitated with either {alpha}A-TRPCx or {alpha}B-TRPCx antibody (not shown). These results suggest that TRPC3 is the primary TRPC family member expressed in ventricular tissue.


Figure 2
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  		Fig. 2. Presence of TRPC3 channel proteins in a highly enriched sarcolemma preparation from canine heart ventricle. Proteins from a detergent lysate of a highly enriched sarcolemma preparation isolated from canine ventricle were separated by SDS-PAGE (10 µg protein/lane) and subjected to Western blot analysis as described in METHODS. Antibodies used for immunoblotting are indicated above each lane. The same results were obtained on gels in which 50 µg of protein was loaded per lane and with either the {alpha}A-TRPCx or {alpha}B-TRPCx antibodies for the immunoblot. NCX, Na+/Ca2+ exchanger; NKA, N+-K+-ATPase; PMCA, plasmalemmal Ca2+-ATPase.

 
To confirm that TRPC3 was present in plasmalemma, thin sections from rat ventricle were colabeled with {alpha}A/B-TRPC3 and with antibodies to NKA (Fig. 3) and NCX (Fig. 4). Both NKA and NCX were seen at the intercalated disk region and in the peripheral plasmalemma. Additionally, NKA and NCX immunoreactivity was observed intracellularly in transverse sections through the ventricle and through isolated myocytes. As seen in the merged images in Figs. 3 and 4, intense colocalization of TRPC3 and the NKA and NCX proteins was observed at the intercalated disk region and in the intracellular membrane network. Interestingly, the peripheral sarcolemma, which stained positively for both NKA and NCX, was essentially devoid of TRPC3 as indicated by the red outline surrounding each cardiac cell in merged images of transverse sections through the ventricle and isolated myocytes (Figs. 3 and 4). These results provide additional support for the hypothesis that TRPC3 is localized to specific plasma membrane structures deep within the muscle cell.


Figure 3
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  		Fig. 3. Colocalization of TRPC3 with NKA pump in rat ventricle. Fluorescence confocal images of thin sections from rat ventricle colabeled with {alpha}A-TRPC3 (green, left) and anti-NKA antibodies (red, center) are shown; merged images are shown on right. Longitudinal (A) and transverse (B) sections through the rat ventricle and a transverse section through a single isolated adult myocyte (C) are shown. Note that the myocytes are outlined in red, indicating that TRPC3 does not colocalize with NKA in the peripheral sarcolemma.

 

Figure 4
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  		Fig. 4. Colocalization of TRPC3 with NCX in rat ventricle. Fluorescence confocal images of thin sections from rat ventricle colabeled with {alpha}A-TRPC3 (green, left) and anti-NCX antibodies (red, center) are shown; merged images are shown on right. Longitudinal (A) and transverse (B) sections through the rat ventricle and a transverse section through a single isolated adult myocyte (C) are shown. Note that the myocytes are outlined in red, indicating that TRPC3 does not colocalize with NCX in the peripheral sarcolemma.

 
To determine the relationship between TRPC3 and the SR, the distribution of TRPC3 was compared with that of both RyR and SERCA pumps. Labeling of longitudinal sections through the rat ventricle with anti-RyR antibody showed the typical striated labeling indicative of distribution near the transverse tubular membrane (Fig. 5A). There was little evidence in merged images for colocalization of RyR with TRPC3 either at the intercalated disk region or in the transverse tubule component when examined in either longitudinal or transverse sections. However, high-magnification images showed that in most areas of the cell TRPC3 and RyR exist in close proximity and in a few regions overlap is observed (Fig. 5). In contrast, no evidence for colocalization of TRPC3 with SERCA was seen in either longitudinal or transverse sections (Fig. 6). It should be noted that several transverse sections through ventricle also included centrally located dark regions when stained for either RyR (Fig. 5) or SERCA pumps (Fig. 6). As indicated by DAPI staining, these dark regions reflect a section through the nucleus (not shown). Interestingly, only SERCA is seen in the membrane surrounding the nucleus (Fig. 6). Thus it would appear that TRPC3 and RyR are excluded from the nuclear membrane.


Figure 5
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  		Fig. 5. Localization of TRPC3 and the ryanodine receptor (RyR) in rat ventricle. Fluorescence confocal images of thin sections from rat ventricle colabeled with {alpha}A-TRPC3 (green, left) and anti-RyR antibodies (red, center) are shown; merged images are shown on right. Longitudinal (A) and transverse (B) sections through the rat ventricle and a transverse section through a single isolated adult myocyte (C) are shown.

 

Figure 6
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  		Fig. 6. Localization of TRPC3 and the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump in rat ventricle. Fluorescence confocal images of thin sections from rat ventricle colabeled with {alpha}A-TRPC3 (green, left) and anti-RyR antibodies (red, center) are shown; merged images are shown on right. Longitudinal (A) and transverse (B) sections through the rat ventricle and a transverse section through a single isolated adult myocyte (C) are shown.

 
Localization of TRPC3, NKA, and NCX to the same membrane structure within the cardiac myocyte suggests that these proteins may interact. To test this hypothesis, reciprocal immunoprecipitations were performed from rat ventricular lysates (Fig. 7). TRPC3 coimmunoprecipitated with NKA and NCX, but not with PMCA. TRPC3 also coimmunoprecipitated with NKA and NCX from the canine cardiac sarcolemma preparation (not shown). It is important to note that before immunoprecipitation the ventricular lysates were subjected to a high-speed centrifugation step (200,000 g x 60 min) to remove unsolubilized membrane fragments and small vesicles that may form during the isolation and lysis procedure. Thus the coimmunoprecipitation of these three proteins suggests that they strongly interact. A small fraction of the total RyR found in the immunocomplexes also coimmunoprecipitated with TRPC3 (Fig. 7), consistent with the minor amount of colocalization seen in the immunohistochemical analysis. Interestingly, RyR did not coimmunoprecipitate with either NCX or NKA antibodies (Fig. 7). This result suggests that the TRPC3-RyR complex is not associated with NCX or NKA. Thus there may be two distinct pools of TRPC3 in the myocyte.


Figure 7
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  		Fig. 7. Coimmunoprecipitation of TRPC3 with NKA and NCX from rat ventricle. Membrane lysates prepared from rat heart ventricle as described in METHODS were subjected to immunoprecipitation followed by Western blot analysis. Antibodies used for immunoprecipitation are indicated above each lane, and antibodies used for immunoblot are indicated on left.

 
Previous studies in brain and kidney showed that TRPC6, but not TRPC3, coimmunoprecipitates with NKA (9). Thus the finding that TRPC3 associates with NKA in ventricular muscle was somewhat surprising. To determine whether the interaction of TRPC3 and NKA was direct, TRPC3, NKA, and NCX were heterologously expressed in various combinations in Sf9 insect cells using recombinant baculovirus. As seen in Fig. 8, TRPC3 coimmunoprecipitated with NCX and NKA coimmunoprecipitated with NCX, but TRPC3 only coimmunoprecipitated with NKA when coexpressed along with NCX. These results suggest that the presence of NKA in the TRPC3 signaling complex may reflect interaction between the NKA and NCX proteins.


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we examined the distribution and subcellular localization of TRPC channels in rat ventricular myocytes. The only TRPC channel protein observed by either immunoblot or immunohistochemical analysis was TRPC3. Likewise, TRPC3 was the only family member found in neonatal rat ventricular myocytes or in a highly enriched sarcolemmal preparation isolated from canine ventricle. Previous studies have shown that all of the antibody preparations used in the present study are able to immunoprecipitate the appropriate channel protein from either tissue or cultured cell lysates (9–11, 26). However, immunoprecipitations from adult or neonatal cardiac myocytes, or from canine sarcolemma lysates, again showed no evidence for TRPC1, TRPC4, TRPC5, TRPC6, or TRPC7. One note of caution is that we cannot eliminate the possibility that ventricular myocytes express splice variant forms of these channels lacking the epitopes used to generate our antibody preparations, but it seems unlikely that the amino acids for both {alpha}A-TRPCx and {alpha}B-TRPCx would be absent. Together the results suggest that these channel proteins are below the limit of detection with our present antibody preparations. We also failed to detect any of these channel proteins after immunoprecipitation with either {alpha}A-TRPC3 or {alpha}B-TRPC3 antibody preparations. This result suggests that TRPC3 does not associate with other members of the TRPC channel subfamily. Thus it seems likely that TRPC3 channels in heart are homotetrameric. Using commercially available TRPC antibodies, one study reported expression of TRPC4 and TRPC5 in rat neonatal cardiac myocytes (25), and another recent study reported the presence of TRPC1, TRPC3, TRPC4, and TRPC5, but no TRPC6 or TRPC7, in isolated adult mouse cardiac myocytes (20). The reasons for the discrepancy with the present study is unknown but may be related to the antibodies used. In this regard, Flockerzi et al. (6) showed that the TRPC4 antibody from Alomone recognizes a band of appropriate molecular mass on Western blot analysis of proteins from TRPC4-knockout mice. Thus results obtained with this antibody should be viewed with caution.

In longitudinal sections of rat ventricle and in enzymatically dissociated adult ventricular myocytes, TRPC3 immunoreactivity was predominantly found at the intercalated disk region. Although both the SERCA pump and the RyR channel appear to be excluded from this region, the NKA and NCX proteins were also found in high concentration at the intercalated disk, where they colocalized with the TRPC3 protein. Previous studies have shown that transport proteins such as NCX (7, 15) and the voltage-gated Na+ channel (17, 18) are localized to the disk region, but the physiological implications of this distribution are at present unclear. Since gap junction channels are sensitive to cytosolic Ca2+ (29), the presence of TRPC3 channels in this region may provide a mechanism by which stimulation of GPCRs linked to PLC could regulate cell-to-cell communication and alter action potential propagation and conduction velocity. The role of TRPC3 channels in the regulation of gap junction function remains to be investigated.

Because of the unique size and shape of the cardiac myocyte, a system of transverse tubules (t tubules) has evolved in ventricular muscle to transmit the electrical signal to areas deep within the cell, enabling essentially simultaneous contraction of all regions of the myocyte. The t tubules are specialized invaginations of the surface membrane that occur near the z line of the sarcomere and form a complex system of branching tubules with both transverse and axial elements, i.e., the transverse-axial tubular system, or TATS (27). As shown by localization studies, the TATS can be subdivided into three domains: 1) a region that contains the voltage-gated Ca2+ channel that interacts with the RyR in the lateral cisternae of the SR forming the dyad structure, 2) a region containing the voltage-gated Na+ channel, and 3) a region containing NCX (24). It is estimated that ~60% of the t tubule membrane is near the z line and that 40% exists between the z lines, i.e., 40% is axial (27). In transverse sections through rat ventricle or through isolated adult myocytes, TRPC3 immunoreactivity was found in a vast network of intracellular membranes. Although there was some evidence from both immunohistochemical analysis and immunoprecipitation assays that a small fraction of the TRPC3 channels associate with RyR, for the most part TRPC3 did not colocalize with either RyR or SERCA pumps. Thus TRPC3 is not present in either the longitudinal component or the terminal cisternae of the SR. In sharp contrast, TRPC3 colocalized with the NKA and NCX proteins in these intracellular membranes. It was quite obvious, however, that NKA and NCX immunoreactivity was present in the peripheral sarcolemma, a region that did not label with either the {alpha}A-TRPC3 or {alpha}B-TRPC3 antibody preparations. This result suggests that TRPC3 is exclusively present in the axial component of the TATS and at the intercalated disk region. The protein and lipid composition of the axial element of the TATS, and indeed the role of this tubular segment in regulation of contractility, are essentially unknown. It is interesting to note, however, that the axial component of the TATS is in close proximity to the longitudinal component of the SR. This region of the SR is enriched in SERCA pumps and is the primary site of Ca2+ reuptake into the storage site. Thus the localization of Ca2+-permeable channels to the axial component of the TATS could greatly influence the amount of Ca2+ within the SR and, hence, contractility. Furthermore, recent studies have shown that there is a dramatic remodeling of the TATS during heart failure with an increase in the axial component (28). It will be interesting to evaluate the expression and distribution of TRPC3 in various models of heart failure.

The colocalization of TRPC3 with the NKA and NCX proteins in the ventricular myocyte suggested a close physical association. To test this hypothesis we performed reciprocal immunoprecipitation assays. TRPC3 coimmunoprecipitated with both proteins from lysates obtained from rat ventricle, neonatal rat myocytes, and the highly enriched canine sarcolemmal preparation. Likewise, when TRPC3 was coexpressed in Sf9 insect cells with both NCX and NKA, each of these proteins was found in the immune complexes. However, when individually coexpressed with either NKA or NCX, TRPC3 only immunoprecipitated with NCX. Likewise, NKA only immunoprecipitated with NCX. Although it is possible that protein-protein interactions may be different in Sf9 insect cells because of differences in posttranslational modification or targeting in this expression system versus mammalian cells, we recently showed (9) that TRPC3 does not coimmunoprecipitate with NKA from rat kidney lysates. Together, our results suggest that TRPC3 interacts with NKA in the ventricular myocyte indirectly via NCX. A physical association of NCX and NKA was previously reported in both heart (5) and brain (16), and both physical and functional interaction of the NCX with TRPC3 have been reported in HEK cells heterologously expressing TRPC3 channel protein (22). In that study, Na+ entry via the TRPC3 channel fueled Ca2+ entry into the cell via reverse-mode NCX activity. Thus it seems likely that TRPC3, NCX, and NKA form part of a signalplex in the ventricular myocyte that may act in a coordinated fashion to regulate Ca2+ entry in response to activation of PLC. The position of this signalplex near the SERCA pumps present in the longitudinal SR would greatly impact SR Ca2+ loading. Whether Ca2+ entry following activation of PLC occurs via TRPC3 directly, or indirectly via reverse-mode NCX activity, remains to be determined, but the close proximity of NKA and NCX is consistent with the well-established involvement of these proteins in the mechanism of cardiac glycoside-induced positive inotropy. Furthermore, the close proximity of these three transport proteins suggests that the positive inotropic effects of {alpha}1-adrenergic, endothelin-1, or angiotensin II receptor stimulation may be dramatically enhanced in the presence of cardiac glycosides, but to our knowledge this experiment has never been reported.


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This study was supported by National Institute of General Medical Sciences Grant GM-52019.


   ACKNOWLEDGMENTS
 
We thank Dr. Diana L. Kunze and Pat Glazebrook for helpful discussion.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. P. Schilling, Rammelkamp Center for Education and Research, Rm. R-322, MetroHealth Medical Ctr., 2500 MetroHealth Dr., Cleveland OH 44109-1998 (e-mail: wschilling{at}metrohealth.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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  1. Ambudkar IS. Ca2+ signaling microdomains: platforms for the assembly and regulation of TRPC channels. Trends Pharmacol Sci 27: 25–32, 2006.[CrossRef][Medline]
  2. Bartschat DK, Lindenmayer GE. Calcium movements promoted by vesicles in a highly enriched sarcolemma preparation from canine ventricle. J Biol Chem 255: 9626–9634, 1980.[Abstract/Free Full Text]
  3. Bers DM. Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002.[CrossRef][Medline]
  4. Buniel MC, Schilling WP, Kunze DL. Distribution of transient receptor potential channels in the rat carotid chemosensory pathway. J Comp Neurol 464: 404–413, 2003.[CrossRef][ISI][Medline]
  5. Dostanic I, Schultz J, Lorenz JN, Lingrel JB. The {alpha}1 isoform of Na, K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart. J Biol Chem 279: 54053–54061, 2004.[Abstract/Free Full Text]
  6. Flockerzi V, Jung C, Aberle T, Meissner M, Freichel M, Philipp SE, Nastainczyk W, Maurer P, Zimmermann R. Specific detection and semi-quantitative analysis of TRPC4 protein expression by antibodies. Pflügers Arch 451: 81–86, 2005.[CrossRef][ISI][Medline]
  7. Frank JS, Mottino G, Reid D, Molday RS, Philipson KD. Distribution of the Na+-Ca2+ exchange protein in mammalian cardiac myocytes: an immunofluorescence and immunocolloidal gold-labeling study. J Cell Biol 117: 337–345, 1992.[Abstract/Free Full Text]
  8. Glazebrook PA, Schilling WP, Kunze DL. TRPC channels as signal transducers. Pflügers Arch 451: 125–130, 2005.[CrossRef][ISI][Medline]
  9. Goel M, Sinkins WG, Keightley A, Kinter M, Schilling WP. Proteomic analysis of TRPC5- and TRPC6-binding partners reveals interaction with the plasmalemmal Na+/K+-ATPase. Pflügers Arch 451: 87–98, 2005.[CrossRef][ISI][Medline]
  10. Goel M, Sinkins WG, Schilling WP. Selective association of TRPC channel subunits in rat brain synaptosomes. J Biol Chem 277: 48303–48310, 2002.[Abstract/Free Full Text]
  11. Goel M, Sinkins WG, Zou CD, Estacion M, Schilling WP. Identification and localization of TRPC channels in the rat kidney. Am J Physiol Renal Physiol 290: F1241–F1252, 2006.[Abstract/Free Full Text]
  12. Hungerford RT, Lindenmayer GE, Schilling WP, VanAlstyne E. The effects of membrane potential on sodium-dependent calcium transport in cardiac sarcolemma vesicles. In: Electrogenic Transport: Fundamental Principles and Physiological Implications, edited by Blaustein MP and Lieberman ML. New York: Raven, 1984, p. 239–251.
  13. Insel PA, Head BP, Ostrom RS, Patel HH, Swaney JS, Tang CM, Roth DM. Caveolae and lipid rafts: G protein-coupled receptor signaling microdomains in cardiac myocytes. Ann NY Acad Sci 1047: 166–172, 2005.[Abstract/Free Full Text]
  14. Kieval RS, Bloch RJ, Lindenmayer GE, Ambesi A, Lederer WJ. Immunofluorescence localization of the Na-Ca exchanger in heart cells. Am J Physiol Cell Physiol 263: C545–C550, 1992.[Abstract/Free Full Text]
  15. Lencesova L, O'Neill A, Resneck WG, Bloch RJ, Blaustein MP. Plasma membrane-cytoskeletal-endoplasmic reticulum complexes in neurons and astrocytes. J Biol Chem 279: 2885–2893, 2004.[Abstract/Free Full Text]
  16. Maier SKG, Westenbroek RE, McCormick KA, Curtis R, Scheuer T, Catterall WA. Distinct subcellular localization of different sodium channel {alpha} and beta subunits in single ventricular myocytes from mouse heart. Circulation 109: 1421–1427, 2004.
  17. Maier SKG, Westenbroek RE, Schenkman KA, Feigl EO, Scheuer T, Catterall WA. An unexpected role for the brain-type sodium channels in coupling of cell surface depolarization to contraction in the heart. Proc Natl Acad Sci USA 99: 4073–4078, 2002.[Abstract/Free Full Text]
  18. Montell C. The TRP superfamily of cation channels. Sci STKE 2005: re3, 2005.[Abstract/Free Full Text]
  19. Nakayama H, Wilkins BJ, Bodi I, Molkentin JD. Calcineurin-dependent cardiomyopathy is activated by TRPC in the adult mouse heart. FASEB J 20: 1660–1670, 2006.[Abstract/Free Full Text]
  20. O'Reilly DR, Miller LK, Luckow VA. Baculovirus Expression Vectors: A Laboratory Manual. New York: Freeman, 1992.
  21. Rosker C, Graziani A, Lukas M, Eder P, Zhu MX, Romanin C, Groschner K. Ca2+ signaling by TRPC3 involves Na+ entry and local coupling to the Na+/Ca2+ exchanger. J Biol Chem 279: 13696–13704, 2004.[Abstract/Free Full Text]
  22. Schilling WP, Zaher MC, Rampe D. Effect of inorganic calcium channel blockers on dihydropyridine binding to cardiac sarcolemma. Mol Pharmacol 37: 80–89, 1990.[Abstract]
  23. Scriven DR, Dan P, Moore EDW. Distribution of proteins implicated in excitation-contraction coupling in rat ventricular myocytes. Biophys J 79: 2682–2691, 2000.
  24. Seth M, Sumbilla C, Mullen SP, Lewis D, Klein MG, Hussain A, Soboloff J, Gill DL, Inesi G. Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes. Proc Natl Acad Sci USA 101: 16683–16688, 2004.[Abstract/Free Full Text]
  25. Sinkins WG, Goel M, Estacion M, Schilling WP. Association of immunophilins with mammalian TRPC channels. J Biol Chem 279: 34521–34529, 2004.[Abstract/Free Full Text]
  26. Soeller C, Cannell MB. Examination of the transverse tubular system in living cardiac rat myocytes by 2-photon microscopy and digital image-processing techniques. Circ Res 84: 266–275, 1999.[Abstract/Free Full Text]
  27. Song LS, Sobie EA, McCulle S, Lederer WJ, Balke CW, Cheng H. Orphaned ryanodine receptors in the failing heart. Proc Natl Acad Sci USA 103: 4305–4310, 2006.[Abstract/Free Full Text]
  28. Spray DC, Burt JM. Structure-activity relations of the cardiac gap junction channel. Am J Physiol Cell Physiol 258: C195–C205, 1990.[Abstract/Free Full Text]
  29. VanAlstyne E, Burch RM, Knickelbein RG, Hungerford RT, Gower EJ, Webb JG, Poe SL, Lindenmayer GE. Isolation of sealed vesicles highly enriched with sarcolemma markers from canine ventricle. Biochim Biophys Acta 602: 131–143, 1980.[Medline]
  30. Wes PD, Chevesich J, Jeromin A, Rosenberg C, Stetten G, Montell C. TRPC1, a human homolog of a Drosophila store-operated channel. Proc Natl Acad Sci USA 92: 9652–9656, 1995.[Abstract/Free Full Text]
  31. Woodcock EA, Matkovich SJ. Ins(1,4,5)P3 receptors and inositol phosphates in the heart—evolutionary artefacts or active signal transducers? Pharmacol Ther 107: 240–251, 2005.[CrossRef][ISI][Medline]
  32. Yu FH, Catterall WA. The VGL-chanome: a protein superfamily specialized for electrical signaling and ionic homeostasis. Sci STKE 2004: re15, 2004.[Abstract/Free Full Text]



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