Department of Physiology and Biophysics, University of Nebraska Medical Center, Omaha, Nebraska 68198-5850
Submitted 4 February 2004 ; accepted in final form 18 March 2004
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ABSTRACT |
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canonical transient receptor potential; TRPC4-; TRPC4-
; TRPC1; fura 2; glomerulus
Glomerular mesangial cells, which are smooth muscle-like cells involved in regulating the filtration surface area, contract in response to vasoactive agonists such as angiotensin II, which induces a G protein-coupled and IP3-mediated release of stored Ca2+ (26). The first evidence for SOC in (rat) mesangial cells was provided by Menè et al. (18), who used fura 2 fluorescence techniques. More recently, with the use of patch-clamp techniques and fura 2 ratiometry, investigators at our laboratory (14, 15) confirmed those initial studies and showed that cultured human mesangial cells possess SOC with high Ca2+/Na+ selectivity and single-channel conductance of 2 pS with Ba2+ as the charge carrier.
Several studies support the notion that the canonical subfamily of the transient receptor potential (TRPC) family of proteins contains members that form Ca2+-selective and -nonselective cation channels in a variety of mammalian cells (23, 28, 29, 31, 47, 49). The purpose of this study was to identify which among TRPC1TRPC7 contribute to SOC in mouse mesangial cells (MMC). Whereas mRNA and protein level expression are evident for both TRPC1 and TRPC4 in cultured MMC, immunostaining experiments showed that TRPC4 is the only member localized in the plasma membrane and that it is abundantly expressed in the glomeruli of mouse renal tissue sections.
Several splice variants of TRPC4 have been revealed by RT-PCR in a variety of species (19, 33, 35). However, the most abundantly described transcripts have been designated TRPC4- and TRPC4-
, which have an 84-amino acid deletion in the cytosolic COOH terminus (19). It was previously determined that TRPC4-
transcripts are expressed in rat kidney (33) and that both TRPC4-
and TRPC4-
are expressed in the human kidney (19). We therefore performed RT-PCR to distinguish transcripts of TRPC4-
and TRPC4-
in MMC. Functional studies with the use of fura 2 ratiometry and antisense were used to determine the role of TRPC4-
as a SOC in MMC.
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MATERIALS AND METHODS |
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RT-PCR. Total RNA was isolated from MMC, and primers were used to determine the presence of TPRC1TRPC7 by RT-PCR as previously described (40, 46). In brief, 1 µg of total RNA, first isolated from MMC with Trizol reagent (GIBCO-BRL, Grand Island, NY), was reverse transcribed with the use of reverse transcriptase (Promega, Madison, WI). The primer sequences for TRPC1TRPC7 were published previously (46). The primers used for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were forward 5'-CCCTTCATTGACCTCAACTACATGG-3' and reverse 5'-GAGGGGCCATCCACAGTCTTCTG-3'. The annealing temperatures were as follows: TRPC1, TRPC4, and TRPC5, 60°C for 1 min; TRPC2 and TRPC3, 54°C for 1 min; and TRPC6 and TRPC7, 58°C for 1 min. For all experiments, the denaturing temperature was 94°C and the extension temperature was 72°C, and 3540 PCR cycles were performed. The PCR products were visualized on 1.5% agarose gel containing ethidium bromide under UV light.
To distinguish splice variants TRPC4- and TRPC4-
, isolated mRNA was amplified by PCR with the use of a forward primer (5'-CATTTCTAGCTTCCGCTTCG-3') paired with a reverse primer (5'-TCTTCCACCACCACCTTCTC-3'). Annealing was performed at 60°C for 1 min. All other cycle conditions were as described above.
Western blotting. For the detection of proteins by Western blot analysis, MMC proteins were collected and isolated according to standard protocols by sonication and centrifugation. Protein concentration was assayed with the use of Bio-Rad protein assay dye (Bio-Rad, Hercules, CA), and proteins were loaded onto a 12.5% polyacrylamide gel after being boiled in Laemmli sample buffer. After electrophoresis, proteins were transferred to a polyvinylidene difluoride nitrocellulose membrane blocked in 5% milk in Tris-buffered saline containing Tween 20 (TBST; 0.1% Tween 20) before exposure to the primary antibody (TRPC1, 1:200, or TRPC4, 1:100; Santa Cruz Biotechnology, Santa Cruz, CA). After being washed in TBST, the membranes were exposed to a horseradish peroxidase (HRP)-conjugated secondary antibody and washed once again, and then the signal was visualized with the use of SuperSignal West Femto substrate (Pierce, Rockford, IL). Images were captured with a UVP bioimaging system (EpiChemi II Darkroom; UVP, Upland, CA) and saved as digital image files.
Immunocytochemical staining. For immunocytochemical staining, MMC were plated on glass coverslips, fixed with 4% paraformaldehyde for 10 min at 23°C, and then washed briefly in PBS followed by methanol (10 min at 23°C). Next, the cells were incubated with PBS containing 1% BSA for 1 h at 23°C, incubated with primary antibodies overnight at 4°C, and then washed with PBS three times for 5 min each. After this first incubation, the MMC were incubated for 1 h at 23°C with the secondary antibodies. For TRPC1, the primary antibody used was rabbit anti-human TRPC1 (1:50), commercially available from Santa Cruz Biotechnology. The secondary antibody was goat anti-rabbit IgG (1:400) conjugated with Alexa Fluor 594 (Molecular Probes, Eugene, OR). For TRPC4, the primary antibody used was rabbit polyclonal anti-TRPC4 (1:100; Sigma) or goat polyclonal anti-TRPC4 (1:50; Santa Cruz Biotechnology). The secondary antibody for TRPC4 was Alexa 594-conjugated donkey anti-rabbit IgG or Alexa 488-conjugated donkey anti-goat (1:400; Molecular Probes). The negative control was IgG incubated in the absence of the primary antibody but at the same concentration. Samples were viewed with an Olympus BX50 microscope. Images were captured with a digital camera (Princeton Scientific Instruments, Monmouth Junction, NJ), and the final layout of images was created with Adobe Photoshop software.
Fura 2 ratiometry. For fura 2 ratiometric measurement of Ca2+, cells were grown to confluence, passed onto 22 x 22-mm glass coverslips (Fisher Scientific, Pittsburgh, PA), and studied within 48 h at 23°C. Measurements of intracellular Ca2+ concentration ([Ca2+]i) in MMC were obtained by fura 2 and dual excitation wavelength fluorescence microscopy as previously described (4, 7). In brief, after loading with fura 2, the glass coverslips with MMC were placed into a perfusion chamber (Warner RC-20H; Warner Instruments, Hamden, CT) and then mounted on the stage of a Nikon Diaphot 300 inverted microscope. With light provided by a DeltaScan dual monochromator system (Photon Technology International, Lawrenceville, NJ), the cells were illuminated alternately at 340- and 380-nm wavelengths (3-nm bandwidths). An adjustable optical sampling window was positioned within the light path before detection with a photon-counting photomultiplier to monitor fluorescence emission (510 nm, 20-nm bandpass) from a single cell. Background-corrected data were collected at a rate of 5 points/s and then stored and processed with the use of the FeliX software package (Photon Technology International). Calibration of the fura 2 signal was performed according to established methods (4, 7). Cells were loaded with fura 2 by incubation for 60 min (23°C) in physiological salt solution (135 mM NaCl, 5 mM KCl, 2 mM MgCl2, 1 mM CaCl2 , and 10 mM HEPES) containing 7 µM fura 2-acetoxymethyl ester, 0.09 g/dl DMSO, and 0.018 g/dl Pluronic F-127 (Molecular Probes). For all experiments, the initial bathing solution contained (in mM) 135 NaCl, 5 KCl, 10 HEPES, and 1 CaCl2. Free [Ca2+] was reduced to <10 nM by the addition of EGTA.
SOC activity was measured as previously described by investigators at our laboratory (15) and by others (1). In brief, after stable baseline [Ca2+]i was obtained, 1 µM thapsigargin was applied to the bathing solution containing 1 mM Ca2+. After an initial rapid rise and a subsequent plateau phase, the bath [Ca2+] was reduced to <10 nM by the addition of EGTA. When [Ca2+]i declined to the lowest level, 1 mM Ca2+ was returned to the bath. Once added, thapsigargin was present throughout the experiment. The difference in [Ca2+]i ([Ca2+]i) in response to the return of 1 mM Ca2+ to the external solution was used as the measurement of SOC.
TRPC4 antisense.
To determine the function of TRPC4 in MMC, the cells were pretreated for 48 h with TRPC4 antisense or scrambled oligonucleotides (4 nM). The antisense and scrambled sequences were TTTGTAATAGAACTGAGCCAT and CATTAGGATGCGTAACTTATA, respectively (corresponding to GenBank accession no. NM_016984). [Ca2+]i values were determined by following the same procedures as described above. The differences in
[Ca2+]i among the three groups (control, antisense, and scrambled) were compared by performing ANOVA plus the Student-Newman-Keuls test. Significance was established as P < 0.05.
Immunohistochemistry. For histological analyses, kidneys were removed from 8-wk-old mice (C57BL/6). Coronal cross sections containing the hilus were removed from kidneys, fixed in neutral buffered formalin, embedded in paraffin, and mounted on glass slides. For immunohistochemical analysis, 5-µm sections of paraffin-embedded tissue were deparaffinized in two changes of xylene for 10 min each. The sections were rehydrated in a series of graded alcohol solutions (100, 90, 85, 50, and 30%) for 2 min each. Antigen was retrieved by continuous boiling in 10 mM citrate buffer (pH 6.0) for 20 min. The sections were blocked with 3% H2O2 for 15 min followed by 1% BSA for 15 min at 23°C. After blocking solution was removed, the specimens were covered and incubated in a mixture of goat anti-human TRPC4 antibody (1:50, 1% BSA/PBS; Santa Cruz Biotechnology) overnight at 4°C. The slides were then washed in PBS three times for 5 min each. Each section was then covered and incubated for 1 h at 23°C with sufficient HRP-conjugated donkey anti-goat IgG (1:200; Santa Cruz Biotechnology). The slides were washed again in PBS three times for 5 min each. Each section was then covered with sufficient solution (liquid DAB substrate kit; Zymed Laboratories, South San Francisco, CA) at 23°C for 2 min in the dark and then counterstained with hematoxylin. Samples were viewed with an Olympus BX50 microscope. Images were captured with a Princeton Scientific Instruments digital camera and manipulated with the use of Adobe Photoshop software.
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RESULTS |
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Immunocytochemical localization of TRPC1 and TRPC4.
The localization of TRPC1 and TRPC4 in MMC was determined by immunocytochemistry with the use of anti-TRPC1 and anti-TRPC4, both of which were purchased from Santa Cruz Biotechnology. The specificities of TRPC1 and TRPC4 antibodies were established by Western blot analysis of MMC proteins. As shown in Fig. 2A, two distinct bands, at 90 and 50 kDa, were detected with anti-TRPC1. The molecular mass of 90 kDa is in the range (8095 kDa) previously reported for TRPC1 protein (2, 9, 24, 32, 44, 47, 51). The 50-kDa band, demonstrated previously with the use of a variety of TRPC1 antibodies, has been identified as the heavy chain of IgG (24). Figure 2B shows a Western blot for TRPC4 using MMC proteins in which a single band of 115 kDa was detected. This size is in agreement with previous reports regarding TRPC4 (29, 39, 43).
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DISCUSSION |
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Expression of TRPC isoforms in MMC. Because mesangial cells have a smooth muscle-like phenotype, it is not surprising that TRPC1, previously identified in smooth muscle (24, 37, 38, 51), was also found in MMC. However, TRPC1 was predominately expressed in the cytoplasm of MMC and therefore would not serve as a component of SOC, which occurs in membranes. Because the cytoplasmic localization of TRPC1 was a surprising result, we used three different TRPC1 antibodies (sc-20110, Santa Cruz Biotechnology; ACC-010, Alomone Laboratories; a gift from Dr. L. Tsiokas) and seven fixation methods to ensure the accuracy of this result. In each experiment, we found the localization of TRPC1 to be intracellular. The localization of TRPC1 in the cytoplasm is not understood and is somewhat contradictory to studies showing that TRPC1 is endogenously localized in the plasma membranes of some cells. For example, TRPC1 associates with caveolin-1 in the plasma membrane region of human submandibular glands (13, 41) and sperm cells (42). In the MMC used in this study, we did not find clear evidence of plasma membrane localization of TRPC1; however, we are unable to conclude that TRPC1 is not in the plasma membrane. It remains possible that TRPC1 is localized to the plasma membrane but is not resolvable by our methods. Interestingly, Philipp et al. (29) also found transcripts of both TRPC1 and TRPC4 in adrenal cells. That TRPC4 antisense reduced ICRAC in adrenal cells led to the conclusion that TRPC4 was at least one of the proteins forming SOC. However, it was not determined whether TRPC1 was also a component of ICRAC in the Philipp et al. study.
Function of TRPC4 in native cells. In the absence of pharmacological inhibitors of specific isoforms of TRPC, antisense knockdown technology has been used extensively to study the function of TRPC channels in native environments. Antisense methods provided the first compelling evidence that TRP homologs were involved in SOC activity (52). After the Zhu et al. (52) study, Wu et al. (50) used antisense to show that human TRPC1 is a functional component of SOC in human embryonic kidney (HEK)-293 cells. TRPC1 antisense oligonucleotides successfully inhibited SOC in endothelial cells (3) and in salivary glands (12). Patch-clamp experiments showed that TRPC4 antisense silenced SOC in bovine adrenal cortical cells (29). Thus antisense is a proven method of establishing the SOC function of specific isoforms of TRPC in their native environment.
By performing fura 2 ratiometry in the present study, we demonstrated that TRPC4 antisense reduced SOC by 83% (from 213 to 36 nM), a value close to the 20 µM La3+ reduction that inhibited SOC by 93% [also determined with fura 2 techniques (15)]. Thus TRPC4-antisense appears to be effective in silencing the SOC response to releasing Ca2+ stores with thapsigargin. That TRPC4 antisense did not affect expression of TRPC1, which has 35% amino acid identity with TRPC4, demonstrates the specificity of this particular knockdown strategy.
RNA transcripts of TRPC4 were previously described in a variety of tissues, including brain, kidney, lung, heart, testis, ovary, and adrenal glands (6, 20, 33). Consistent with our results, transcripts of TRPC4 have been discovered in rat (33) and human (19) kidney. However, a study of bovine adrenal cells may have been the first to describe a functional role for TRPC4 in its native environment (29). More recent studies with TRPC4 knockout mice showed that TRPC4, although not necessary for survival, has an important role in regulating blood flow and vascular permeability (5, 40). Freichel et al. (5) demonstrated that TRPC4/ expressed with impaired agonist-dependent vasorelaxation and concluded that TRPC4 forms the SOC channels necessary for Ca2+ stimulation of nitric oxide synthase in endothelial cells. More recently, Tiruppathi et al. (40) showed that lung vascular endothelial cells of TRPC4/ mice lack the thrombin-induced endothelial cell retraction response. These studies show that in some cells, TRPC4 is a crucial component not only of maintaining intracellular Ca2+ stores but also of Ca2+-regulated physiological responses.
Properties of mesangial TRPC4 channels. The membrane-spanning structure of TRPC channels is similar to the molecules that form tetrameric ion-selective channels in the plasma membranes of cells. Therefore, it has been concluded that TRPC isoforms can form either heterotetrameric or homotetrameric channels. Because only TRPC4 was localized in the plasma membrane of MMC, our study results imply that the mesangial SOC is a homotetramer formed by only TRPC4. However, we cannot rule out the possibility that other membrane-associated TRPCs exist that were not detected with our methods. Clearly, further studies are necessary to clarify the role of TRPC4 in store-operated Ca2+ entry. Although several studies have reported SOC currents at the whole-cell level, few studies have reported single-channel currents. Our (15) previous results showed that thapsigargin-induced SOC was expressed in cultured human mesangial cells as a small, La3+-sensitive, Ca2+-selective, 2-pS channel with Ba2+ as the current carrier. The present study shows that the same channels, when examined with fura 2 methods, are silenced by TRPC4 antisense.
In agreement with our findings, previous studies showed that bovine TRPC4 (formerly CCE1), which is closely homologous to mTRPC4-, was Ca2+ selective and activated by store depletion when heterologously expressed in HEK-293 or Chinese hamster ovary cells (27, 48). However, our findings are in disagreement with the described properties in many studies which heterologously overexpress TRPC4 (34, 35). It was shown that TRPC4 channels, presumably expressed as homotetramers, formed receptor-operated, nonselective cation channels activated independently of IP3 or store depletion (17, 30, 34). The single-channel conductance of these nonselective TRPC4 channels was much higher (41 pS) (34) than we found in our previous study (15). It was therefore suggested that TRPC4 did not form ICRAC, which was originally described as Ca2+ selective, inwardly rectifying, and having a small single-channel conductance (8, 16). However, Philipp et al. (29) showed that the endogenous TRPC4 in adrenal cells is a Ca2+-selective SOC. These data are consistent with studies showing that TRPC4/ mice lack a highly Ca2+-selective, store-dependent current in endothelial cells (5, 40). This large discrepancy between the overexpressed and the endogenous TRPC4 is not surprising in light of a recent study by Vazquez et al. (45), who found that TRPC3 functioned as a SOC when expressed at low levels in chicken DT40 B lymphocytes. However, when expressed at higher levels, TRPC3 no longer formed a SOC but could be activated by receptor-coupled phospholipase C. Thus the native TRPC4 may be interacting with another endogenous protein that influences its properties. It is now known that several types of channels have accessory subunits that influence the biophysical, pharmacological, and regulatory properties of the pore-forming subunit (11, 22). It will be interesting to determine whether TRPC channels are similarly influenced by accessory subunits.
Splice variants of TRPC4.
Although there are multiple splice variants of TRPC4 (30), isoforms TRPC4- and TRPC4-
have been shown to be the most abundantly expressed in tissues (19, 34, 35). Northern blot analysis revealed an abundance of both TRPC4-
and TRPC4-
RNA in human renal tissue (19). Satoh et al. (33) also showed that TRPC4-
transcripts were specifically expressed in rat kidney. Because Satoh et al. found both TRPC4-
and TRPC4-
in rat brain, we used mouse brain as a positive control. Our results revealed amplified transcripts consistent with TRPC-
but not TRPC-
expression in MMC. That TRPC4-
forms the SOC channel in MMC is consistent with a study by Mery et al. (19), who showed, using the yeast two-hybrid method, a strong interaction between human TRPC4-
(but not TRPC4-
) and the intracellular Ca2+ release channel (IP3R). An association between TRP and IP3R is the sine qua non of the conformational coupling hypothesis regarding activation of SOC (10).
In summary, we have found that both TRPC1 and TRPC4 are expressed in MMC. TRPC4 was clearly localized in the plasma membrane of glomerular MMC, where it may form a homotetrameric SOC. Recent studies with TRPC4/ mice revealed important roles for TRPC4 SOC in the normal function of cardiovascular and pulmonary systems. It therefore will be interesting to examine the renal function of TRPC4/ mice at the cellular and integrative levels.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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