A Calmodulin/Inositol 1,4,5-Trisphosphate (IP3) Receptor-binding Region Targets TRPC3 to the Plasma Membrane in a Calmodulin/IP3 Receptor-independent Process*

Barbara J. Wedel, Guillermo Vazquez, Richard R. McKay, Gary St. J. Bird and James W. Putney, Jr. {ddagger}

From the Laboratory of Signal Transduction, NIEHS, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina 27709

Received for publication, April 14, 2003 , and in revised form, May 1, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Conformational coupling with the inositol 1,4,5-trisphosphate (IP3) receptor has been suggested as a possible mechanism of activation of TRPC3 channels and a region in the C terminus of TRPC3 has been shown to interact with the IP3 receptor as well as calmodulin (calmodulin/IP3 receptor-binding (CIRB) region). Here we show that internal deletion of 20 amino acids corresponding to the highly conserved CIRB region results in the loss of diacylglycerol and agonist-mediated channel activation in HEK293 cells. By using confocal microscopy to examine the cellular localization of Topaz fluorescent protein fusion constructs, we demonstrate that this loss in activity is caused by faulty targeting of CIRB-deleted mutants to intracellular compartments. Wild type TRPC3 and mutants lacking a C-terminal predicted coiled coil region downstream of CIRB were targeted to the plasma membrane correctly in HEK293 cells and exhibited TRPC3-mediated calcium entry in response to agonist activation. Mutation of conserved YQ and MKR motifs to alanine within the CIRB region in TRPC3-Topaz, which would be expected to interfere with IP3 receptor and/or calmodulin binding, had no effect on channel function or targeting. Additionally, TRPC3 targets to the plasma membrane of DT40 cells lacking all three IP3 receptors and forms functional ion channels. These findings indicate that the previously identified CIRB region of TRPC3 is involved in its targeting to the plasma membrane by a mechanism that does not involve interaction with IP3 receptors.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium entry in nonexcitable cells is most commonly triggered by the activation of a receptor stimulating phospholipase C to cleave phosphatidylinositol 4,5-bisphosphate into diacylglycerol (DAG),1 which stimulates protein kinase C, and inositol 1,4,5-trisphosphate (IP3), which binds to the IP3 receptor to release calcium from intracellular stores (1). TRPC3, a member of the transient receptor potential family of ion channels, has been shown to be activated by agonist activation of plasma membrane G-protein-coupled receptors, by synthetic diacylglycerols, and by store depletion in some cell types (24). A conformational coupling model physically linking TRPC3 with the IP3 receptor has been suggested, and a region in the cytosolic C terminus of TRPC3 has been shown to co-immunoprecipitate with regions of the IP3 receptor (5, 6). An overlapping region of TRPC3 binds to calmodulin (CaM), which is hypothesized to be tethered to the channel and to be displaced competitively by the activated IP3 receptor; the region that commonly binds both IP3 receptor and calmodulin has been termed the calmodulin/IP3 receptor-binding (CIRB) region (7). However, binding of CaM to the isolated CaM-binding region of TRPC3 is dependent on the calcium concentration, which is inconsistent with a model of CaM being tethered to the channel independently of the calcium concentration. Calmodulin binding to a CIRB homologous region in all members of the TRPC family (TRPC1–7) has been demonstrated (8). A second CaM-binding region has been identified in other TRPs. For TRPC4, CaM binds in a region unique to the splice variant TRPC4{alpha}, which is not present in the splice variant TRPC4{beta} (8, 9). This region has also been shown to interact with the C terminus of IP3 receptors in a yeast two-hybrid screen and glutathione S-transferase pull-down assays (10). Binding to both CaM-binding domains of TRPC4 occurs only above 10 µM Ca2+ with an apparent Kd of 100–200 nM CaM (9). For TRPC1 a second CaM-binding domain has also been reported that overlaps with a predicted coiled coil region common to the C terminus of all TRPCs (11). This second CaM-binding region has been shown to be involved in calcium-dependent inactivation of TRPC1 (11). Our goal was to identify functional roles of the CIRB and other domains of TRPC3 using N- and C-terminal truncation mutants of TRPC3.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Plasmid Construction—TRPC3 (provided by Lutz Birnbaumer, NIEHS, National Institutes of Health), hemagglutinin-tagged at the C terminus via an AscI site in pcDNA3, was used as a template for the construction of truncation mutants (TRPC3N{Delta}1–TRPC3N{Delta}3 and TRPC3C{Delta}4–TRPC3C{Delta}8). For N-terminal truncation mutants (TRPC3N{Delta}1–TRPC3N{Delta}3), the sequence preceding the desired start site was changed to a BamHI site followed by an ATG using the QuikChangeTM site-directed mutagenesis kit. To remove the 5' sequence including the original ATG, cDNAs were digested with BamHI, which has a recognition sequence in the multicloning box of pcDNA3 5' of the insert and at the newly introduced site, and religated. For C-terminal truncation mutants (TRPC3{Delta}C4–TRPC3{Delta}C8), an NheI site was introduced into the sequence (GCTAGC) with the TAG stop codon positioned at the designated end of the coding region. For TRPC3-Topaz fusion constructs (T3T), a TRPC3 construct fused to the brighter YFP version Topaz at the C terminus via an AscI restriction site was used as a template (12). For N-terminal truncation mutants (T3T-N{Delta}0 and T3T-N{Delta}1), the same strategy as described for untagged mutants was used. For C-terminal deletions (T3T-C{Delta}7 and T3T-C{Delta}8), the AscI recognition site 3' of the stop codon was mutated keeping the AscI site fusing TrpC3 to Topaz intact. An AscI site was introduced into the sequence at the desired site of truncation of TrpC3. The mutants were digested with AscI and religated to yield truncated TrpC3-Topaz fusions. For the internal deletion mutant T3T-C{Delta}78 oligonucleotides 5'-cagcattctcaatcagggatccatgtatcagcagataatgaaaag and 5'-cttttcattatctgctgatacatggatccctgattgagaatgctg were used in the mutagenesis reaction with Trp3-Topaz as template.

Cell Culture and Transfection—For the production of pools of cells stably expressing wild type and mutant TRPC3, HEK293 cells were transfected with LipofectAMINE2000® at ~80% confluency according to the manufacturer's instructions. The day after transfection the cells were split into selection medium containing 0.5 mg/ml G418 and grown for 4 weeks in the continued presence of selection medium. In parallel, control cells transfected only with the fluorescent marker pdsRedmito were selected for G418 resistance as above, and pdsRedmito expression was confirmed by their red fluorescence (excitation, 558 nm; emission, 610 nm).

The chicken B lymphocyte cell line DT40 and the mutant variant in which the genes for all three IP3 receptor types were disrupted were obtained from the Institute of Physical and Chemical Research (RIKEN; Cell Bank code RCB1464 and RCB1467). The cells were cultured essentially as described by Sugawara et al. (13). DT40 were transiently transfected by electroporation (14) with the indicated amounts of the human isoform of TRPC3 or deletion variants or its vector (pcDNA3), along with EYFP-C1 vector (Clontech) as a marker for transfection. The cells were co-transfected with the human M5 muscarinic receptor (50 µg/ml, in pcDNA3). The cells were assayed 17–25 h post-transfection. The fluorescence measurements were performed under the conditions indicated with single enhanced yellow fluorescent protein (EYFP)-positive cells, selected by their yellow/green fluorescence (excitation, 485 nm; emission, 520 nm). Under the conditions of measurement, EYFP expression did not contribute significant fluorescence.

Flow Cytometry—Pools of G418-resistant cells expressing wild type or mutant T3T were subjected to flow cytometric analysis to enrich for cells with high expression levels of fluorescent protein fusions. The cells were treated with trypsin and analyzed on a FACSVantageSE flow cytometer equipped with Cell Quest software (Becton-Dickinson, San Jose, CA). Fluorescence was assayed using an excitation wavelength of 488 nm and an emission wavelength of 530 nm.

Calcium Measurement—Intracellular calcium concentration was measured with a real time fluorescence plate reader system (FLIPR-384; Molecular Devices, Sunnyvale, CA); the cells were plated in poly-D-lysine-coated, black-walled 96-well plates at ~30–40% confluency and incubated overnight at 37 °C to allow attachment of the cells. The cells were loaded for 90 min at 37 °C with 4 µM Fluo4-AM and washed twice with nominally calcium free buffer (20 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 11.1 mM glucose, pH 7.4). GdCl3, methacholine, and CaCl2 at final concentrations of 1 µM, 100 µM, and 1.8 mM, respectively, were added at the time points indicated in the figure. For measurement of 1-oleoyl-2-acetyl-sn-glycerol (OAG) activation, the cells were loaded with Fluo4-AM as described above and washed twice with calcium containing buffer (20 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 0.8 mM MgCl2, 11.1 mM glucose, pH 7.4, 1.8 mM Ca2+). OAG (final concentration, 100 µM) in calcium-containing buffer was added at the indicated times. The fluorescent dye was excited with an argon laser at 488 nm, and the resultant emitted light (540 nm) was detected by a cooled CCD camera. When representative traces are shown, they are averages of multiple wells within an experiment, and each experiment was repeated at least three times with similar results.

For fluorescence measurements of DT40 cells, the fluorescence intensity of multiple Fura-2-loaded EYFP-positive cells or EYFP-negative control cells was monitored with a CCD camera-based imaging system (Universal Imaging) mounted on a Zeiss Axiovert 35 inverted microscope equipped with a Zeiss 40x (1.3 NA) fluor objective. A Sutter Instruments filter changer enabled alternative excitation at 340 and 380 nm, whereas the emission fluorescence was monitored at 510 nm with a Paultek Imaging camera (model PC-20) equipped with a GenII-Sys intensifier (Dage-MTI, Inc.). The images of multiple cells collected at each excitation wavelength were processed using the MetaFluor software (Universal Imaging Corp., West Chester, PA) to provide ratios of Fura-2 fluorescence from excitation at 340 nm to that from excitation at 380 nm (F340/F380).

Confocal Microscopy—The fluorescence images were acquired with a Zeiss LSM510 confocal laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY) with an argon laser and excitation at 488 nm through a 100x (oil immersion) objective lens (optical slice thickness, 0.5 µm). The level of expression of TRPC3-Topaz and thus the brightness of the images in DT40 cells were considerably less than in HEK293 cells. Therefore, all of the images have been subjected to a software-driven equalization procedure, scaling gray scale pixel values between the same minimum (black) and maximum (white) values. This allows for maximal contrast and maximal spatial information, which is required in the current study, but precludes quantitative comparisons between different images.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The initial goal of the present studies was to define domains of TRPC3 that are required for channel activation by truncating the cytosolic N and C termini of TRPC3. We generated a series of deletion mutants (mutants N{Delta}1–3 and C{Delta}4–8; Fig. 1), all of which retained the six predicted transmembrane domains (TM1–TM6) and a putative pore region between TM5 and TM6. In N{Delta}1 four predicted ankyrin repeats were deleted (amino acids 1–199), whereas in N{Delta}2 an adjacent predicted coiled coil region was additionally deleted (amino acids 1–330). Mutant N{Delta}3 lacked virtually all of the cytosolic N terminus (amino acids 1–367). The cytoplasmic C terminus includes the Trp signature motif (EWKFAR), a highly conserved proline-rich motif (TLPXPF) and a region that was shown in pull-down assays to be CIRB. This CIRB site is followed by a second predicted coiled coil region. For C-terminal truncations amino acids 690, 730, 759, 775, or 790–844 were deleted, resulting in a TRPC3 lacking one or more of these domains (C{Delta}4–8; Fig. 1). All of the mutants were stably expressed in HEK293 cells and tested in FLIPR (see "Materials and Methods") for agonist-dependent Ca2+ entry in the presence of 1 µM Gd3+, which blocks the endogenous, store-operated channels (15, 16). In contrast to control cells expressing only the fluorescent marker pdsRedmito, wild type TRPC3-expressing cells showed significant Gd3+-insensitive Ca2+ entry (Fig. 2). All N-terminal mutants (N{Delta}1–3) lacked Ca2+ entry in response to 100 µM methacholine, suggesting a crucial role for ankyrin repeats in the proper folding, targeting, or anchoring of TRPC3 (Fig. 2). Of the C-terminal truncation mutants C{Delta}8, which includes the CIRB site, retained agonist dependent Ca2+ entry, whereas C{Delta}7, which excludes this region, was inactive (Fig. 3).



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FIG. 1.
Schematic presentation of the TRPC3 structure. TRPC3 contains six predicted transmembrane domains (TM1–TM6) and a putative pore region between TM5 and TM6. The cytosolic N terminus consists of four ankyrin repeats (AK1–AK4), a predicted coiled coil region (CC), and a hydrophobic region, possibly a reentry loop. The cytoplasmic C terminus includes the TRP signature motif (EWKFAR), a highly conserved proline-rich motif (PP), the CIRB region, and a predicted coiled coil region (CC). The position of truncation in the N-terminal mutants N{Delta}1–3 and the C-terminal mutants C{Delta}4–8 is indicated in the structure.

 


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FIG. 2.
Agonist-induced Ca2+ entry in TRPC3 N-terminal truncation mutants. HEK cells stably expressing pDsRedmito (control), TRPC3, or N-terminal truncation mutants N{Delta}1, N{Delta}2, or N{Delta}3 were seeded in 96-well plates and loaded with Fluo4-AM to measure [Ca2+]i signals. Ca2+ release in response to 100 µM methacholine in the absence of extracellular Ca2+ and Ca2+ entry upon restoration of 1.8 mM extracellular Ca2+ was assessed in the presence of 1 µM Gd3+, which blocks endogenous channels. The change in fluorescence (cps, counts/s) is plotted as a function of time. Shown is a representative experiment.

 


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FIG. 3.
Agonist-induced Ca2+ entry in TRPC3 C-terminal truncation mutants. HEK cells stably expressing pcDNA3, TRPC3, or C-terminal truncation mutants C{Delta}4–8 were assayed in a FLIPR as described under "Materials and Methods" and as in the legend to Fig. 1. The light emitted by the fluorescent dye (cps) is recorded as a measure for [Ca2+]i. Ca2+ release in response to 100 µM methacholine in the absence of extracellular Ca2+ and Ca2+ entry upon restoration of 1.8 mM extracellular Ca2+ were assessed in the presence of 1 µM Gd3+, which blocks endogenous channels. Shown is a representative experiment.

 

To examine expression and targeting of the truncated proteins, we constructed a subset of mutants in a TRPC3-Topaz fusion construct (T3T-N{Delta}1, T3T-C{Delta}7, and T3T-C{Delta}8; Fig. 4, top panel). We also constructed two additional deletion mutants. A N-terminal splice variant of TRPC6 had previously been shown to selectively lose OAG but not agonist-stimulated Ca2+ entry (Ref. 17 and see "Discussion"). Thus, we constructed a N-terminal deletion of the first 27 amino acids upstream of the first ankyrin repeat (T3T-N{Delta}0). We also constructed a C-terminal deletion lacking most of the CIRB region but retaining the downstream coiled coil sequence (amino acids 775–789, T3TC{Delta}78); a similar TRPC3 construct lacking the CIRB region had previously been demonstrated to have increased current activity following exposure to a TRPC3-interacting IP3 receptor peptide (7). We selected pools of cells stably expressing T3T or T3T mutants with G418 and enriched for cells with high expression levels using flow cytometry of G418-resistant cell populations (18). The cells were tested for Gd3+-insensitive Ca2+ entry in response to agonist stimulation and Ca2+ entry in response to OAG using a FLIPR. As shown in Fig. 5, Ca2+ entry of the truncated Topaz fusion constructs corresponded to Ca2+ entry seen with their corresponding untagged counterparts. T3T-expressing control cells exhibited Gd3+-insensitive Ca2+ entry in response to methacholine activation, as did T3T-C{Delta}8, whereas deletion constructs missing the N-terminal ankyrin repeats (T3T-N{Delta}1) or the coiled coil and CIRB region (T3TC{Delta}7) showed no Ca2+ entry. The conservative N-terminal deletion mutant, T3T-N{Delta}0, showed normal or partially reduced entry, whereas the CIRB deletion, T3T-C{Delta}78, was inactive. The latter deletion is similar in length to the active T3T-C{Delta}8, indicating that it is the specific sequence of amino acids in the C{Delta}78 (CIRB) region that is important for activity. All of the mutants that showed Gd3+-insensitive Ca2+ entry in response to methacholine could also be activated by OAG (Fig. 6).



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FIG. 4.
Structure of Topaz-tagged truncation mutants of TRPC3 and alignment of C-Terminal Sequences of Trp channels. Top panel, structural features of TRPC3 C-terminally tagged with Topaz are as described in the legend to Fig. 1. The region(s) deleted in the respective truncation mutant are indicated in the structure. Bottom panel, C-terminal sequences including the CIRB region and putative coiled coil region of TRPC1–7 are shown in alignment. The C14 peptide has been shown by Zhang et al. (7) to co-precipitate with the IP3 receptor and Ca2+/CaM, whereas the C8 peptide interacts solely with Ca2+/CaM. The positions of truncation for TRPC3 mutants C{Delta}6–8 are indicated with arrows. Amino acids mutated in T3T-YQ/AA and T3T-MKR/AAA are gray and marked with dots.

 


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FIG. 5.
Agonist-induced Ca2+ entry in Topaz-tagged TRPC3 truncation mutants. HEK cells stably expressing pcDNA3, T3T, or T3T truncation mutants were assayed in a FLIPR as described under "Materials and Methods" to determine Ca2+ release and entry in response to treatment with 100 µM methacholine. The protocol is as described in the legend to Fig. 3. The measurements were performed in the presence of 1 µM Gd3+ to block endogenous channels. Shown is a representative experiment.

 


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FIG. 6.
OAG-activated Ca2+ entry in Topaz-tagged TRPC3 truncation mutants. HEK cells stably expressing pcDNA3, T3T, or T3T truncation mutants were assayed in a FLIPR. 100 µM OAG was added to cells in the continued presence of 1.8 mM Ca2+. A pcDNA3 control trace obtained in the same FLIPR experiment as for the truncation mutants is repeated in each panel. Shown is a representative experiment.

 

To determine whether mutants that showed no Ca2+ entry were properly targeted to the plasma membrane, we examined the cellular localization of the respective Topaz fusion proteins by confocal microscopy (Fig. 7). As previously reported for a GFP fusion of TRPC3 (18), wild type T3T was mostly localized in punctate regions in the plasma membrane. There was additional staining in intracellular compartments, possibly in the region of the endoplasmic reticulum (ER) and Golgi apparatus, which may represent newly synthesized protein (Fig. 7). A similar pattern was observed for the conservative and physiologically active deletion mutants, T3T-N{Delta}0 and T3T-C{Delta}8, although the labeling appears more uniform and less punctate. The physiologically inactive N-terminal truncation mutant T3T-N{Delta}1 and C-terminal truncation mutants T3T-C{Delta}7 and T3T-C{Delta}78 appeared to be predominantly located in intracellular compartments with no detectable labeling of the plasma membrane (Fig. 7).



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FIG. 7.
Confocal images of HEK cells stably expressing Topaz-tagged TRPC3 truncation mutants. HEK cells stably expressing pcDNA3, T3T, or T3T truncation mutants were analyzed by confocal microscopy. Plasma membrane localization of fusion constructs is indicated with an arrow.

 

Because the CIRB region appeared to be crucial for plasma membrane targeting of T3T, we also mutated conserved amino acids within the overlapping IP3 receptor/CaM-binding domain to see whether mutations within this region (T3T-YQ/AA and T3T-MKR/AAA; Fig. 4, bottom panel) could also cause loss of plasma membrane targeting as well as agonist and OAG-induced Ca2+ entry seen with deletion of the entire region (Fig. 4, bottom panel). Mutation to alanine of the corresponding residues in L-type Ca2+ channels was shown to reduce both CaM binding and Ca2+-dependent inactivation (19). All of the T3T mutants were expressed in HEK293 cells, and G418-resistant cells with high expression levels were selected using flow cytometry. However, these mutations within the CIRB region (T3T-YQ/AA and T3T-MKR/AAA) did not reduce Gd3+-insensitive Ca2+ entry in response to methacholine or OAG as compared with wild type T3T (Fig. 8, A and B). Consistent with this phenotype T3T-YQ/AA was detectable throughout the plasma membrane with some protein in cytosolic compartments, possibly ER/Golgi (Fig. 8C).



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FIG. 8.
Mutations within the CIRB region have no effect on agonist/OAG-induced Ca2+ entry and subcellular localization of TRPC3. HEK cells expressing T3T constructs mutated within the CIRB region (T3T-YQ/AA and T3T-MKR/AAA; see Fig. 4) were tested in a FLIPR for Gd3+-insensitive Ca2+ entry in response to methacholinemediated (A) and OAG-mediated (B) Ca2+ entry as described under "Materials and Methods." C, confocal images of T3T and T3T-YQ/AA expressing HEK cells show plasma membrane expression as indicated by arrows.

 

The findings to this point demonstrate that deletions of the CIRB region block function of TRPC3 by preventing proper trafficking of the protein to the plasma membrane. Because this region has been shown to interact with IP3 receptors (20), we considered the possibility that this interaction with IP3 receptors is involved in proper targeting of TRPC3. To examine the possible role of IP3 receptors in targeting of TRPC3, we made use of a DT40 chicken B-lymphocyte cell line lacking all three types of IP3 receptors (DT40-KO). To be able to compare phenotypes of T3T and deletion mutants between HEK cells and DT40-KO cells, we first set out to optimize transfection levels of T3T in wild type DT40 cells, which are by experience harder to transfect than HEK cells, aiming to reach similar expression levels as in HEK cells. We accomplished this by two different strategies: first by increasing the amount of plasmid used for transfection from 10 to 100 µg/ml cells as described recently by Vazquez et al. (21) and second by lowering the incubation temperature after transfection to 30 °C. For DT40 cells grown at 37 °C, no TRPC3-Topaz fluorescence was detectable when applying the settings used for HEK cells in Figs. 7 and 8C (optical slice thickness, 0.5 µm); rather, fluorescent images could be obtained only when the optical slice thickness was maximized, such that the resulting images were not confocal (data not shown). Confocal images of T3T expressing DT40 cells grown at 30 °C, however, could be obtained using the same settings used for HEK cells (Fig. 9A) and show a punctate pattern in the plasma membrane as seen before in HEK cells. Previous functional studies on TRPC3 expressing cells have suggested that IP3 receptors are not required for agonist induced Ca2+ entry in this cell line (21, 22). Consistent with the functional data, IP3 receptors also do not appear to be required for plasma membrane targeting of T3T because we observe plasma membrane expression of the Topaz-labeled protein in both wild type and DT40-KO cells (Fig. 9A). As for HEK293 cells, deletion of the CIRB region (C{Delta}7) resulted in the loss of the distinct punctate pattern of plasma membrane localization (Fig. 9A). For functional studies DT40-KO cells were transfected with cDNAs encoding the M5 muscarinic receptor under the control of the chicken {beta}-actin promoter, EYFP as a transfection marker, and the respective T3T construct (Fig. 9B). DT4-KO cells are known to respond to phospholipase C-coupled receptors with generation of IP3, but they do not generate phospholipase C-linked cytosolic Ca2+ signals (13). The addition of carbachol to DT40-KO cells expressing T3T and M5 receptors presumably causes formation of IP3 and DAG but no release of Ca2+ from stores because of the lack all three types of IP3 receptors. Despite this lack of IP3 receptors, T3T is able to function in a receptor-operated mode as revealed by significant entry of Ba2+ (Fig. 9B). When T3T-C{Delta}7 and T3T-C{Delta}8 were expressed in DT40-KO cells, we obtained the same phenotypes as in HEK293 cells; whereas the predicted C-terminal coiled coil region could be deleted in T3T without loss of agonist induced entry, additional deletion of the CIRB region results in loss of function, a function that is independent of the presence of IP3 receptors.



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FIG. 9.
T3T and T3T-C{Delta}7 expression in wild type or IP3 receptor knockout DT40 cells, and carbachol-induced Ba2+ entry. A, confocal images of wild-type (WT) or IP3 receptor knockout (IP3R-KO) DT40 cells transfected with 100 µg OF T3T or T3T-C{Delta}7. The cells were cultured at 30 °C to enhance expression, as described under "Results." B, Ba2+ entry responses of cell transfected with vector (Control), T3T, T3T-C{Delta}7, or T3T-C{Delta}8, together with the M5 muscarinic receptor. At the times indicated 300 µM carbachol followed by 2 mM Ba2+ was added to cells incubated in the nominal absence of Ca2+. Shown are the averages of traces of 15–20 EYFP-positive or -negative (control) cells.

 

Because the CIRB region of TRPC3 has been shown to bind to both the IP3 receptor and CaM, we next set out to test the role of CaM in targeting of TRPC3. We therefore overexpressed T3T in DT40-KO cells together with wild type CaM or a dominant negative CaM mutant (CaMEF1–4mut), in which all four EF hand motifs are mutated (7). We observed no difference in plasma membrane targeting of T3T in CaM versus CaMEF1–4mut expressing cells (not shown). Thus, in aggregate, our data demonstrate that CIRB (or elements contained therein) is necessary for proper targeting of TRPC3 to the plasma membrane, but neither CaM nor the IP3 receptor, the binding partners so far shown to interact with this region, appear to play a role in this regard.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In recent years several genetic diseases have been linked to mutations within ion channel genes (23). Some of theses mutations interfere with proper trafficking of the channel to the plasma membrane, for example, the most common mutation of the cystic fibrosis transport regulator, {Delta}F508 (24, 25). There seems to be an important trafficking checkpoint in the ER; membrane proteins such as channels/channel subunits may contain an ER retention signal, which is masked by another protein or a part of the same protein. Truncation of a channel or lack of a corresponding subunit can unmask the ER retention signal and thus prevents nonfunctional or improperly assembled channels from reaching the plasma membrane (26). This has recently been demonstrated for a variety of channels including the L-type calcium channel, HERG channel, KATP channel, N-methyl-D-aspartate receptor, and cyclic-nucleotidegated channel (2731). In this study we show that deletion of the CIRB region in TRPC3 (mutants T3T-C{Delta}78 and T3T-C{Delta}7) results in trapping of the channel in intracellular compartments possibly ER/Golgi. A predicted C-terminal coiled coil region, which is also conserved in all TRPC channels, is not required for targeting or for OAG and agonist-dependent Ca2+ entry. It is interesting to note that C-terminal truncation of TRPC1 up to the start of the transmembrane domains results in functional ion channels that are regulated by store depletion (32). It is possible that truncation of the CIRB region in TRPC3 results in exposure of an upstream ER retention signal keeping the channels from trafficking to the plasma membrane. A dibasic motif, either KK or RR, located close to the C terminus has been identified as an ER retention signal for several misassembled membrane proteins (33). The ER retention signal appears to be RKR for KATP, RGR for HERG, and RRR for the NMDA receptor. We considered a RRRR sequence (R4) in TRPC3 downstream of the transmembrane domains, upstream of the CIRB region as a candidate ER retention sequence. However, mutation of R4 to A4 in T3T-C{Delta}7 T3T did not rescue plasma membrane targeting (data not shown). More extensive studies will be necessary to identify a putative ER retention signal. Mutation of the ER retention signal would allow the functional study of mutants that would otherwise be trapped in the ER/Golgi and would provide a means to exclude defective trafficking as a reason for lack of function. It is intriguing that the TRP-related protein, PKD2, appears to be localized in the ER, whereas a PKD2 mutant lacking 34 C-terminal amino acids is now detectable in the plasma membrane (34).

Calmodulin plays a crucial role in the activation, inactivation, or modulation of a variety of ion channels (35). CaM has been shown to bind to the CIRB region of TRPC1–7 with different affinities (8), although its effects on channel activity appear to vary among different isoforms. In this study we show that deletion of the CIRB region in TRPC3 (mutants T3T-C{Delta}78 and T3T-C{Delta}7) results in trapping of the channel in intracellular compartments possibly ER/Golgi. Interestingly, a TRPC1 mutant lacking the CIRB region did not alter Ca2+-dependent feedback inhibition of SOC in a human submandibular cell line, and from the data presented it is not clear whether there is a phenotype associated with this mutant (11). It is therefore tempting to speculate that this mutant does not target properly to the plasma membrane as is the case for the corresponding Topaz-tagged TRPC3 mutant T3T-C{Delta}78 (Fig. 7). We considered the possibility that CaM binding to CIRB was a prerequisite for targeting TRPC3 to the plasma membrane because it has been shown for Ca2+-activated K+ channels that CaM regulates the trafficking and surface expression of these ion channels (36). To test this hypothesis, we applied two strategies; we mutated conserved residues within the CIRB region that might be expected to interfere with CaM and/or IP3 receptor binding (T3TYQ/AA and T3T-MKR/AAA; Fig. 8), and we co-expressed T3T with a dominant negative CaM mutant to see whether we could detect an effect on T3T targeting (see "Results"). However, neither of these strategies disrupted plasma membrane targeting of T3T.

A recent study showed that a coiled coil region in the N terminus of TRPC1, but not a region containing the ankyrin repeats, was able to homodimerize based on a yeast two-hybrid screen (37). Thus, the function of ankyrin repeats in TRPC channels is unclear. As we show here ankyrin repeats appear to be required for targeting of TRPC3 to the plasma membrane (T3T-N{Delta}1), whereas N-terminal deletion of TRPC3 up to the ankyrin repeats yields functional and plasma membrane-targeted ion channels (T3T-N{Delta}0). A similar trafficking defect has been described for a TRPC6 mutant lacking 131 N-terminal amino acid residues that include the first ankyrin repeat (38). Ankyrin repeats are found in a wide spectrum of proteins, including plant potassium channels, TRPC, and vanilloid TRP subfamilies, and are thought to mediate protein-protein interactions (39). However, ankyrin repeats are able to accommodate a variety of target molecules, making it hard to predict a possible binding partner.

It has been shown that members of the TRPC 3/6/7 subfamily as well as the Drosophila isoforms TRP and TRP-like receptor can be activated by the diacylglycerol analog OAG (40, 41). The exact mechanism of this activation is not known, but OAG may mimic the effects of phospholipase C-induced generation of DAG. Recently Zhang and Saffen (17) discovered a splice variant of TRPC6, TRPC6B, which appeared to be activated in response to agonist but not OAG activation. The TRPC6A splice variant that contains 54 additional amino acids at the N terminus was activated by both OAG and receptor stimulation. These authors (17) concluded that these 54 amino acids are crucial for OAG activation. However, none of the other TRP isoforms that are OAG-activated possess this extended N terminus. In agreement with that, Jung et al. (42) have recently reported OAG activation for the respective TRPC6 splice variant. Similarly, we show here that the N terminus of TRPC3 can be truncated up to the start of the ankyrin repeats without loss of proper targeting, OAG activation, or receptor activation (Figs. 5, 6, 7, see mutant T3T-N{Delta}0). It remains unknown whether DAG and OAG exert their effect by acting on the TRPC3 channel directly. The well described DAG target protein kinase C does not seem to be involved in activation, because protein kinase C inhibitors do not block OAG activation of TRPC3 or TRPC6 (40, 43). However, protein kinase C is not the only effector of DAG, and several newly described targets of DAG should be investigated as possible intermediates between DAG and TRPC3 channel activation (44, 45).

Conformational coupling has been suggested as a mechanism of activation of TRPC3, and a N-terminal, IP3-binding fragment of the IP3 receptor has been shown to activate TRPC3 channels (5). The CIRB region of TRPC3 was subsequently identified as a region that not only interacts with CaM but also with two sequences within the cytoplasmic N terminus of the IP3 receptor (7, 8). However, TRPC3-mediated Ba2+ entry into DT40 lacking all three types of IP3 receptors has been reported (21, 22), excluding an absolute requirement for the IP3 receptor in TRPC3 activation. We show here that T3T targets to the plasma membrane of DT40-KO cells, which suggests that the dependence of plasma membrane targeting of TRPC3 on the CIRB region is unrelated to the interaction with the IP3 receptor. We also demonstrate that the truncation mutants T3T-C{Delta}7 and T3T-C{Delta}8 exhibit comparable phenotypes when expressed in HEK cells or DT40-KO cells. However, we cannot rule out the possibility that ryanodine receptors can partially assume the function of IP3 receptors in DT40-KO cells. However, the sequences of the IP3 receptor, which have been shown to interact with TRPC3 do not appear to be conserved in ryanodine receptors. In this context it is also interesting to note that the L-type Ca2+ channel and the ryanodine receptor can directly interact via their respective calmodulin-binding regions (46).


    FOOTNOTES
 
* 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

{ddagger} To whom correspondence should be addressed: NIEHS, P.O. Box 12233, Research Triangle Park, NC 27709. Tel.: 919-541-1420; Fax: 919-541-7879; E-mail: Putney{at}niehs.nih.gov.

1 The abbreviations used are: DAG, diacylglycerol; TRPC, canonical transient receptor potential; ER, endoplasmic reticulum; IP3, inositol 1,4,5-trisphosphate; OAG, 1-oleoyl-2-acetyl-sn-glycerol; CIRB, calmodulin/IP3 receptor-binding; CaM, calmodulin; EYFP, enhanced yellow fluorescent protein. Back


    ACKNOWLEDGMENTS
 
We are grateful to Jeff Reece for help with confocal microscopy and to Carl Bortner for assistance with flow cytometry. We thank Drs. Mariel Birnbaumer and David Armstrong for critically reading the manuscript and Dr. Lutz Birnbaumer for providing CaM expression constructs. We are indebted to Rebecca Boyles for technical assistance.



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 MATERIALS AND METHODS
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 DISCUSSION
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