A Pathogenic C Terminus-truncated Polycystin-2 Mutant Enhances Receptor-activated Ca2+ Entry via Association with TRPC3 and TRPC7*

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca2+ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca2+ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca2+ influx that may lead to dysregulated cell growth in ADPKD.

Mutations in PKD2 gene result in autosomal dominant polycystic kidney disease (ADPKD). PKD2 encodes polycystin-2 (TRPP2), which is a homologue of transient receptor potential (TRP) cation channel proteins. Here we identify a novel PKD2 mutation that generates a C-terminal tail-truncated TRPP2 mutant 697fsX with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 from a family showing mild ADPKD symptoms. When recombinantly expressed in HEK293 cells, wild-type (WT) TRPP2 localized at the endoplasmic reticulum (ER) membrane significantly enhanced Ca 2؉ release from the ER upon muscarinic acetylcholine receptor (mAChR) stimulation. In contrast, 697fsX, which showed a predominant plasma membrane localization characteristic of TRPP2 mutants with C terminus deletion, prominently increased mAChR-activated Ca 2؉ influx in cells expressing TRPC3 or TRPC7. Coimmunoprecipitation, pulldown assay, and cross-linking experiments revealed a physical association between 697fsX and TRPC3 or TRPC7. 697fsX but not WT TRPP2 elicited a depolarizing shift of reversal potentials and an enhancement of single-channel conductance indicative of altered ion-permeating pore properties of mAChR-activated currents. Importantly, in kidney epithelial LLC-PK1 cells the recombinant 679fsX construct was codistributed with native TRPC3 proteins at the apical membrane area, but the WT construct was distributed in the basolateral membrane and adjacent intracellular areas. Our results suggest that heteromeric cation channels comprised of the TRPP2 mutant and the TRPC3 or TRPC7 protein induce enhanced receptor-activated Ca 2؉ influx that may lead to dysregulated cell growth in ADPKD.
Autosomal dominant polycystic kidney disease (ADPKD) 3 is a genetically heterogeneous Mendelian inheritance disorder affecting ϳ1 in 1000 live births (1). ADPKD is characterized clinically by progressive formation and enlargement of renal cysts that demonstrate abnormalities in cell growth, fluid secretion, and extracellular matrix with other common complications (2). Linkage analyses have shown that either PKD1 or PKD2 loci are responsible for almost all ADPKD pedigrees. Nearly 85% of ADPKD pedigrees have been linked to PKD1, and ϳ15% have been linked to PKD2 (2,3). In PKD2 cases end-stage renal disease develops at a mean age of 10 -15 years later than in PKD1 cases, although heterogeneity in clinical phenotype is seen among PKD2 mutations (4).
The PKD2 gene, consisting of 15 exons, encodes a 968-amino acid integral transmembrane protein polycystin-2 (PC2, TRPP2) (5). Recent reports suggested that the C terminus of TRPP2 interacts with the coiled-coil domain of the PKD1 gene product, polycystin-1 (PC1) (6). TRPP2 has homology to polycystin-L (TRPP3) (7), another member of the polycystin superfamily that has been shown to conduct Ca 2ϩ -permeable cation currents (8 -10), and to a family of transient receptor potential (TRP) channels as well as voltage-gated Ca 2ϩ channels, which raises the possibility that TRPP2 mediates transmembrane Ca 2ϩ fluxes (3,5) as a member of the TRP subfamily, TRP"P". Invertebrate and vertebrate TRP homologues of the so-called "canonical" TRPC subfamily are characterized by activation induced upon stimulation of phospholipase C-coupled receptors (11). TRPC channels have been originally proposed as store-operated channels activated by Ca 2ϩ depletion of stores, whereas closely related TRPC homologues, TRPC3, TRPC6, and TRPC7, showed activation sensitivity to the membranedelimited action of diacylglycerol (12)(13)(14). Notably, an Orai family distinct from TRPs had recently emerged as a major molecular entity for store-operated channel subtypes (15).
Thus, members of the TRPC family, which form homomeric or heteromeric channels different in their function and regulation (16), are currently the best candidates for receptor stimulationactivated Ca 2ϩ entry channels.
Different types of PKD2 mutations include nonsense mutations and frameshift-inducing deletions/insertions that result in truncation of the TRPP2 protein sequence (17,18). The physiological function of TRPP2 and pathogenesis of TRPP2 mutations have been explained by seemingly conflicting hypotheses, particularly with regard to the truncation mutations that generate aberrant TRPP2 proteins lacking the C-terminal tail (6, 19 -29). Hanaoka et al. (6) suggested that PC1 and TRPP2 coassemble at the plasma membrane (PM) to produce a new channel and regulate renal tubular morphology and function. Nauli et al. (20) proposed that PC1 and TRPP2 form mechanosensitive channels in the primary cilium of kidney cells. Naturally occurring pathogenic mutations of TRPP2, which disrupt their associations through their C-terminal tails (30), result in the defect in translocation of TRPP2 to PM. In contrast, a role of TRPP2 as a subunit of intracellular channels with the endoplasmic reticulum (ER)-targeting sequence, whose deletion induces trafficking to PM in pathogenic PKD2 mutants, has been suggested (26,27). Another possibility is that TRPP2 functions appropriately both as Ca 2ϩ release channels in ER and, under certain defined conditions, as PM Ca 2ϩ entry channels (31). However, it is still unclear how the C terminus-truncated TRPP2 mutant proteins behave after being mistranslocated from ER to the PM independently of the interaction with PC1. Thus, the physiological function of normal and pathogenic mutant TRPP2 as well as its operating subcellular site is yet to be established.
In the present study we have identified a novel PKD2 gene mutation (2092delA) that generates a TRPP2 product (697fsX) with a frameshift resulting in an aberrant 17-amino acid addition after glutamic acid residue 697 (Glu 697 ) at the C terminus in a Japanese family. The recombinant TRPP2 mutant 697fsX was examined for subcellular localization as well as molecular and functional properties in HEK293 cells. 697fsX localized at the PM elicited a physical association with the TRPC3 or TRPC7 protein and muscarinic acetylcholine receptor (mAChR)-activated Ca 2ϩ influx in HEK293 cells coexpressing TRPC3 or TRPC7, whereas wild-type (WT) TRPP2 localized at the ER significantly enhanced mAChR-activated Ca 2ϩ release. In polarized kidney epithelial LLC-PK1 cells, confocal image analysis revealed codistribution of native TRPC3 with transfected 697fsX in the apical membrane but not with WT TRPP2 distributed in the basolateral membrane area. These observations suggest dual impacts of PKD2 mutations producing TRPP2 proteins deleted with the C-terminal tail in the pathogenesis of ADPKD.

EXPERIMENTAL PROCEDURES
Patients-After informed consent had been obtained, the proband diagnosed with polycystic kidney disease and his living family members were assessed by abdominal ultrasonography and clinical evaluation including routine laboratory tests of renal and liver function. The criteria for the diagnosis of ADPKD were based on the studies of Bear et al. (32). Individuals were considered to be affected if they were found to have at least one cyst in each kidney and at least two cysts in one kidney. Abdominal computed tomography imaging was performed using Hi Speed Advantage (GE Medical Systems).
Linkage Analysis-Genomic DNAs were extracted from peripheral blood leukocytes of the family members from whom informed consent had been obtained, according to standard procedures (33). Linkage analysis was performed using four CA-repeat markers for the PKD1 locus (D16S521, D16S3024, D16S3027, D16S423) and four for the PKD2 locus (D4S2964, D4S1534, D4S414, D4S1572) as described elsewhere (34). AmpliTaq gold DNA polymerase (PerkinElmer Life Sciences) and a universal touch-down thermal cycling program with GeneAmp PCR System Model 9600 (PE Applied Biosystems) was applied. Amplified products, distinguishable by their fluorescent wavelength and size, were loaded onto a 30-cm-long non-denaturing 10% polyacrylamide gel. After gel electrophoresis for 3 h at 25 watts, the data were analyzed with Genescan software (PerkinElmer Life Sciences) to obtain exact genosizes (35). Logarithm of odds scores were calculated with the M-Link program of the Linkage package Version 5.2 (36).
PKD2 Gene Sequencing-PCR of exons 1-15 was performed using AmpliTaq (PerkinElmer Life Sciences) or LATaq (Takara) as recommended by the manufacturer. The specific primers, annealing temperatures, and cycling conditions were as described before (37). The amplified PCR products containing exons after purification by Microcon-PCR (Millipore) were sequenced in both directions on an automatic fluorescence sequencer (ABM Prism 310 DNA sequencer, Applied Biosystems) using the Dye terminator thermal cycle sequence kit (Amersham Biosciences).
Fluorescent Measurement of [Ca 2ϩ ] i Changes-After 36 h of transfection using SuperFect, cells were plated onto poly-L-lysine-coated glass coverslips and subjected to measurement 6 -16 h after plating on the coverslips. The cells on coverslips were loaded with fura-2 in Dulbecco's modified Eagle's medium containing 1 M fura-2/AM (Dojindo Laboratories), 10% fetal bovine serum, 30 units/ml penicillin, and 30 g/ml streptomycin at 37°C for 40 min and washed with HEPES buffer saline containing 107 mM NaCl, 6 mM KCl, 1.2 mM MgSO 4 , 2 mM CaCl 2 , 11.5 mM glucose, and 20 mM HEPES (the pH was adjusted to 7.4 with NaOH). The coverslips were then plated in a perfusion chamber mounted on the stage of the microscope. Fluorescence images of the cells were recorded and analyzed with a video image analysis system (ARGUS CA-20 or AQUA-COSMOS; Hamamatsu Photonics) as described previously (38). All ratio data were calculated to [Ca 2ϩ ] i using an in vivo calibration method (38). The later phase of [Ca 2ϩ ] i was measured at 700 s.
Confocal Visualization of Fluorescent Fusion Constructs and Immunofluorescent Cell Staining-After 36 h of transfection using SuperFect, HEK293 cells were plated onto poly-L-lysinecoated glass base dishes (IWAKI) and subjected to measurement 6 -16 h after plating on the dishes.
LLC-PK1 cells stably expressing EGFP-WT or EGFP-697fsX were grown on Transwell filters (24 mm, 0.4-mm pore polycarbonate, Corning) for 5-7 days, and media were changed every 2 days. Cells were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100. The cells were incubated with primary antibodies followed by subsequent incubation with the anti-rabbit Cy3-conjugated secondary antibody. Primary antibodies were anti-TRPC3 (38) and monoclonal acetylated ␣-tubulin (6-11B-1, Sigma). Fluorescence images were acquired with a confocal laser-scanning microscope (Olympus FV500) using the 488-nm line of an argon laser for excitation and a 505-525-nm band-pass filter for emission (EGFP) or the 543-nm line of a HeNe laser for excitation and a 560-nm longpass filter for emission (DsRed monomer or Cy3). The specimens were viewed at high magnification using plan oil objectives (60ϫ, 1.40 numerical aperture, Olympus).
Glutathione S-Transferase (GST) Pulldown-cDNAs for TRPC3 fragments and GST were subcloned into the pET23 vector (Novagen). All GST fusion proteins were expressed in Escherichia coli (Rosetta strain, Novagen) and affinity-purified using glutathione-Sepharose 4B beads (GE Healthcare). Cell lysate from HEK293 cells transiently expressing EGFP-679fsX was incubated with glutathione-Sepharose beads bound with GST fusion TRPC3 subfragments for 1 h, then the beads were washed with RIPA buffer at 4°C. The proteins retained on the beads were characterized by Western blotting using living colors polyclonal antibody.
Purification of TRPC3 Complex and Chemical Cross-linking-After 48 h of transfection using the calcium phosphate precipitation method, HEK293 cells were homogenized with a Potter Teflon homogenizer in phosphate buffer saline at 4°C. Cell debris was eliminated from the homogenate by centrifuging at 10,000 ϫ g for 15 min. The supernatant was recentrifuged at 100,000 ϫ g for 1 h to sediment membrane fractions.
The membrane fraction was solubilized in phosphate-based RIPA buffer (pH 8.0) containing 0.1% SDS, 0.5% sodium deoxycholate, 1% Nonidet P-40, 150 mM NaCl, 50 mM sodium phosphate, 1 mM phenylmethylsulfonyl fluoride, and 10 g/l leupeptin. After centrifuging at 100,000 ϫ g for 30 min, the supernatant was incubated with anti-FLAG M2 affinity agarose (Sigma). The beads were washed twice with 10 bed volumes of phosphate-based RIPA buffer. Bound proteins were eluted with the buffer containing 100 g/ml FLAG peptide (Sigma). The eluate was concentrated with a Microcon centrifuge filter unit YM-100 (Millipore). For chemical cross-linking, glutaraldehyde was then added to the final concentration of 12 mM at 4°C for 1 h. The cross-linking reaction was terminated by incubation with an equal volume of SDS sample buffer 25°C for 1 h, and the product was analyzed by Western blotting. A protein sample treated similarly, but without glutaraldehyde, was prepared as a control.
Electrophysiology-After cotransfection with pCI-neo-TRPC3 and pEGFP-C plasmids containing the cDNA for WT or 697fsX TRPP2, coverslips with cells were placed in dishes containing the solutions. Currents were recorded at room temperature (22-25°C) using a whole-cell mode of the patch clamp technique with an EPC-9 patch clamp amplifier (HEKA Elektronik) or Axopatch 200B (Axon Instruments) as described previously (14). Patch pipettes were made from borosilicate glass capillaries (1.5-mm outer diameter; Hilgenberg) using a model P-87 Flaming-Brown micropipette puller (Sutter Instrument Co.). Pipette resistance ranged from 2 to 6 megaohms when filled with the pipette solutions described below. The series resistance was electronically compensated to Ͼ50%. Currents were sampled at 1 or 20 kHz after low pass filtering at 2.9 or 5 kHz in the experiments. Data acquisition was performed using the PULSE program (Version 7.5, HEKA Elektronik) or the Clampex program (Version 10.2, Axon Instruments). Wholecell currents were recorded in an external solution that contained 140 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM HEPES, and 10 mM glucose (the pH was adjusted to 7.4 with NaOH). Current-voltage (I-V) relationships were obtained using a 500-ms positive voltage ramp from Ϫ120 to ϩ100 mV. For the experiment to determine permeation selectivity, currents were recorded in external solutions that contained 140 mM CsCl, 10 mM HEPES, 10 mM glucose, and 20 mM mannitol (the pH was adjusted to 7.4 with CsOH) for Cs ϩ external solutions, 140 mM NaCl, 10 mM HEPES, 10 mM glucose, and 26 mM mannitol (the pH was adjusted to 7.4 with NaOH) for Na ϩ external solutions, and 50 mM CaCl 2 , 40 mM HCl, 10 mM HEPES, 10 mM glucose, and 75 mM mannitol (the pH was adjusted to 7.4 with NMDG) for Ca 2ϩ external solution. The pipette solution contained 95 mM cesium aspartate, 40 mM CsCl, 4 mM MgCl 2 , 5 mM EGTA, 2 mM Na 2 ATP, 5 mM HEPES, and 8 mM creatine phosphate (the pH was adjusted to 7.2 with CsOH). The single-channel amplitude and zero-current level were determined by eye using cursors. The open probability (NP O ; N is the number of channels in the patch, and Po is the single-channel open probability) recorded from cell-attached mode was calculated for a series of 50-ms test pulses to Ϫ160 or ϩ140 mV. The NPo of single-channels was calculated by averaging NPo for 100 sweeps in the patches containing active channels. Single-channel currents were recorded in an external solution that contained 143 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 2 mM KH 2 PO 4 , 10 mM HEPES, and 6 mM glucose (the pH was adjusted to 7.4 with KOH). The pipette solution contained 140 mM NaCl, 10 mM HEPES, and 10 mM glucose (the pH was adjusted to 7.4 with NaOH). Test solution was topically applied using the so-called "Y-tube" fast solution exchange device.
Statistical Analysis-All data are expressed as the mean Ϯ S.E. The statistical analyses were performed using Student's t test.

RESULTS
Haplotype Analysis of PKD1 and PKD2 Gene-The male proband suffered from intracranial subarachnoidal hemorrhage from a ruptured cerebrovascular aneurysm, requiring neurosurgery, at the age of 65. Closer medical examination of the proband including abdominal computed tomography scanning revealed multiple cysts in both kidneys and liver ( Fig. 1) with normal renal function, leading to a diagnosis of polycystic kidney disease. As indicated in Fig. 2, abdominal ultrasonography and laboratory tests of his living family members clarified that five family members including the proband had polycystic kidney disease according to the Bear et al. criteria (32). Liver cysts were observed in all five affected members. Some had hematuria, and the proband had a ruptured cerebral aneurysm, whereas there was no known family history of end stage renal disease.
By haplotype analysis with microsatellite markers shown in Fig. 2A, all affected members shared the haplotype against PKD2 indicated in Fig. 2B (see the shaded numbers), but not PKD1, strongly suggesting that the ADPKD phenotype of the pedigree is positively linked to PKD2 locus. Maximum logarithm of odds score against PKD2 was 0.95 at D4S1572 followed by 0.93 at D4S1534 and 0.90 at D4S414 when ϭ 0 (Table 1), which were close to the theoretical maximum values obtained for this pedigree. The logarithm of odds scores for linkage between chromosome 16p markers and ADPKD were not informative.
The Novel PKD2 Mutation Generates an Aberrant 17 Amino Acid Sequence after Glu 397 of TRPP2-DNA sequence analysis of the 15 exons in the PKD2 gene undertaken in the proband revealed heterozygous single deletion at exon 10 (2092delA) (Fig. 3). This novel mutation caused a frameshift in the coding nucleotide sequence and is predicted to result in the addition of aberrant 17 amino acid residues WNSQILSERATIKLWSN after Glu 697 of TRPP2 (697fsX). The sequence examination of the PKD2 gene for his family members confirmed that 2092delA was cosegregated with the disease phenotype.
WT TRPP2 but Not 697fsX Enhances Ca 2ϩ Release from ER-When recombinantly expressed in HEK293 cells, the 697fsX TRPP2 mutant was predominantly localized in the PM area, in contrast to the WT TRPP2 distributed intracellularly in ER (Fig. 4A). The M 3 receptor has been reported as an endoge- Top, typical intrahepatic cysts (black arrows) and probable peribiliary cysts (black arrowheads) are seen. Bottom, computed tomography scan obtained inferior to A depicts multiple cysts in the bilateral kidneys, a large cyst on the left (white arrow), and the multiple small cysts on the right (white arrowheads). L, liver; S, spleen. nous mAChR subtype in HEK293 cells (40). Upon stimulation of endogenous mAChR by 30 M carbachol (CCh), cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) rises were significantly enhanced by WT TRPP2 expression in HEK293 cells, whereas CCh-induced [Ca 2ϩ ] i rises in 697fsX-expressing cells were indistinguishable from those in vector-transfected control HEK293 cells (Fig. 4B). This enhancement of mAChR-activated Ca 2ϩ responses by WT is attributable to increased Ca 2ϩ release from stores, as [Ca 2ϩ ] i rises elicited by CCh stimulation after omis-sion of external Ca 2ϩ were enhanced significantly by WT but not by 697fsX (Fig. 4C). In contrast, Ca 2ϩ entry, observed as [Ca 2ϩ ] i , rises which are elicited by readministration of external Ca 2ϩ under constant CCh stimulation, was enhanced slightly but significantly by 697fsX but not by WT. Interestingly, thapsigargin-induced Ca 2ϩ entry was indistinguishable between control and TRPP2-expressing cells (Fig. 4D). When high K ϩ (30 mM)-containing solution was employed to establish depolarizing experimental conditions, enhancements of Ca 2ϩ release and Ca 2ϩ entry, respectively, by WT and 697fsX were still observed, suggesting that the effects of constructs are not due to membrane potential changes (supplemental Fig. 1). Thus, WT and 697fsX exert contrasting impacts on mAChRactivated [Ca 2ϩ ] i rises; WT may enhance Ca 2ϩ release induced by inositol 1,4,5-trisphosphate, whereas 697fsX may increase Ca 2ϩ entry, activated via mechanisms presumably independent of store depletion. In HEK293 cells reverse transcription-PCR analysis showed endogenous expression of PC1, TRPP2, TRPC1, TRPC3, TRPC4, and TRPC6 (supplemental Fig. 2), which may contribute to the enhancement of Ca 2ϩ release by WT and Ca 2ϩ influx by 697fsX mutant.
Association of 697fsX with TRPC3 or TPRC7 Enhances CChinduced Ca 2ϩ Influx-To characterize targets of action of 697fsX in enhancing receptor-activated Ca 2ϩ entry, TRPP2 constructs were tested in HEK293 cells coexpressing TRPC homologues, which have been reported as receptor-activated Ca 2ϩ -permeable channels coupled to phospholipase C activation and inositol 1,4,5-trisphosphate production (12)(13)(14). The enhancing effect of 697fsX on CCh-induced Ca 2ϩ responses was significantly pronounced by TRPC3 or TRPC7 coexpression (Fig. 5, A and B), resulting in significant augmentation of sustained [Ca 2ϩ ] i rises attributable to Ca 2ϩ entry, observed in the later phase (Fig. 5A) or after readdition of external Ca 2ϩ (Fig. 5C). The 697fsX effect is not due to membrane potential changes, because augmentation of Ca 2ϩ influx by coexpressing 697fsX with TRPC3 was observed under high K ϩ (30 mM) conditions (supplemental Fig. 3). The 697fsX effect is not attributable to protein expression levels of TRPCs, because protein expression levels were comparable between 697fsX-sensitive and -insensitive TRPCs when FLAG-or EGFP-tagged constructs were assessed by Western blotting analysis (supplemental Fig. 4). As observed above in Fig. 4B, WT TRPP2 enhanced peak [Ca 2ϩ ] i rises regardless of the presence of external Ca 2ϩ in TRPC3-expressing cells, supporting enhancement of inositol 1,4,5-trisphosphate-induced Ca 2ϩ release by WT TRPP2 (Fig.  5, A and C).
697fsX but Not WT TRPP2 Interacts with TRPC3 or TPRC7-We used immunoprecipitation to test whether 697fsX forms a protein complex with TRPC3 or TRPC7. HEK293 cells coexpressing TRPP2 and TRPC constructs were solubilized in Nonidet P-40/deoxycholate/SDS-based stringent RIPA lysis buffer or Nonidet P-40/Triton X-100-based high salt IP-500 buffer (41). Western blotting analysis indicated that the TRPC3-EGFP and TRPC7-EGFP constructs were coimmunoprecipitated with FLAG-697fsX but not with FLAG-WT TRPP2 by anti-FLAG antibody under both conditions (Fig. 6A, supplemental  Fig. 5). In contrast to TRPC3 and TRPC7, TRPC1 showed coimmunoprecipitation with both WT (41) and the 697fsX mutant.  In vitro pulldown assays using GST fusion constructs identified the amino acid residues 659 -753 carrying the TRPC EWKFAR motif as a 697fsX-interacting domain in the TRPC3 protein (Fig. 6B, supplemental Fig. 6). In addition, the previous reported C terminus-truncated mutant R742X (5) also showed coimmunoprecipitation with TRPC3 (supplemental Fig. 7), excluding the possibility that aberrant amino acid sequences attached at the C-terminal end of truncated mutants play important roles in associating with TRPC3 and TRPC7. To further confirm protein multimerization of TRPC3 with 697fsX, a chemical cross-linking method was employed. From the membranes of HEK293 cells transiently expressing TRPC3-FLAG and EGFP-697fsX, protein complexes were solubilized with phosphate-based RIPA buffer and purified using immunoaffinity techniques. Protein complexes including TRPC3-FLAG were trapped to anti-FLAG agarose and were competitively eluted with 100 g/ml FLAG. Then the purified proteins were crosslinked by glutaraldehyde, and the product was analyzed by Western blotting. The band of TRPC3 was shifted to an upward position by the cross-linking, which corresponds well with the size of the tetramer (Fig. 6C, left) (42). Importantly, a protein band of cross-linked EGFP-697fsX copurified with TRPC3-FLAG using anti-FLAG agarose was similar in size to the cross-linked TRPC3 (Fig. 6C, right). Although exact protein stoichiometry of complex formation was not determined, it can be estimated that the content of 697fsX is low compared with that of TRPC3, as efficiency of crosslinking of EGFP-697fsX with TRPC3-FLAG (Fig. 6C, right) is not as high as that between TRPC3-FLAG proteins (Fig. 6C, left). These results suggest that TRPC3 and 697fsX form heteromeric protein complex. Furthermore, confocal images demonstrated overlapping localization of EGFP-697fsX with coexpressed TRPC3-DsRed monomer near the PM area (Fig. 6D). However, WT showed no significant colocalization with coexpressed TRPC3. In polarized kidney epithelial LLC-PK1 cells, native TRPC3 was colocalized with transfected EGFP-697fsX in the apical membrane area but not with WT TRPP2 distributed in the basolateral membrane and adjacent intracellular areas (Fig. 7). The results suggest that 697fsX localized at the PM enhances receptor-activated Ca 2ϩ influx through physical association with TRPC3 or TRPC7. Interestingly, WT TRPP2 and the C-terminal-truncated 697fsX mutant were distributed in cilia in polarized LLC-PK1 as previously reported for TRPP2 WT and the L703X mutant (supplemental Fig. 8) (43).
697fsX Modifies Ion Permeation Properties of TRPC3-To directly investigate the functional impacts of 697fsX on TRPC3-mediated Ca 2ϩ entry, ionic currents were examined using the whole-cell mode of the patch clamp method (Fig. 8). At a holding potential (V h ) of Ϫ60 mV, 697fsX but not WT TRPP2 significantly augmented inward currents evoked by stimulation with 60 M CCh in TRPC3-expressing cells (TRPC3 plus WT, 2.5 Ϯ 0.8 pA/picofarad (pF), n ϭ 6; TRPC3 plus 697fsX, 7.3 Ϯ 1.0 pA/pF, n ϭ 16; TRPC3 plus vector, 3.7 Ϯ 0.8 pA/pF, n ϭ 5) (Fig. 8, A and B), consistent with the above [Ca 2ϩ ] i measurements. I-V relationships showed a prominent rectification at depolarizing potentials as previously reported for receptor-activated TRPC3 currents (Fig. 8C) (44). Currents induced by 1-oleoyl-2-acetyl-sn-glycerol, the membrane-per-meable analogue of the physiological TRPC3 activation trigger diacylglycerol, were augmented in HEK293 cells coexpressing TRPC3 with 697fsX compared with cells expressing TRPC3 with WT or vector control (supplemental Fig. 9). As indicated in Fig. 8C, 697fsX significantly shifted reversal potential toward depolarizing potentials compared with WT and vector control. To quantitatively evaluate the cation selectivity of cation channels formed in HEK293 cells, extracellular Cs ϩ was replaced with Na ϩ or Ca 2ϩ . The ratio of permeability of different ions to that of intracellular Cs ϩ was calculated from changes in reversal potential brought about by ion replacement using equations derived from the Goldman-Hodgkin-Katz equations for the  biionic conditions (14). The values of P Na /P Cs and P Ca /P Cs were 0.93 Ϯ 0.01 and 1.08 Ϯ 0.06 for TRPC3 plus WT (n ϭ 7), 0.93 Ϯ 0.01 and 1.10 Ϯ 0.09 for TRPC3 plus vector (n ϭ 6), and 0.97 Ϯ 0.01 and 1.15 Ϯ 0.08 for TRPC3 plus 697fsX (n ϭ 7, p Ͻ 0.05 versus WT and vector for P Na /P Cs ). The data are indicative of an increase in permeation selectivity to Na ϩ for TRPC3 plus 697fsX.

DISCUSSION
Coimmunoprecipitation, GST pulldown, and cross-linking experiments suggest that the C terminus-truncated TRPP2 mutant 697fsX undergoes physical association with TRPC3 and TRPC7. In electrophysiological recordings, coexpression of 697fsX but not that of WT TRPP2 caused a depolarizing shift of reversal potentials and an increased single-channel amplitude of mAChR-induced currents in TRPC3-expressing cells. These altered ion permeation properties of TRPC3mediated cation currents after coexpression of 697fsX suggest that TRPC3 and 697fsX form heteromultimeric cation channels in which both TRPC3 and 697fsX function as poreforming subunits, and pore-lining residues are different from those of homomultimeric TRPC3 channels. Consistent with this notion, HEK293 cells coexpressing 697fsX with WT TRPC3 and those with E632Q TRPC3 mutant showed single-channel currents with different amplitudes, excluding a possibility that TRPC3 proteins, which form channels independently of 697fsX proteins, support co-translocation of 697fsX via indirect mechanisms, for example, by sharing the same membrane microdomain.
Previously, the TRPP2 C-terminal tail containing the coiledcoil domain 772-796 was shown to be responsible for the assembly of TRPP2 with PC1 using the C-terminal tail-truncated mutant R742X (6). Because the coiled-coil domain is also deleted from the TRPP2 sequence in the 697fsX mutant, 697fsX can be incapable of forming a complex with PC1. In LLC-PK1 cells, native TRPC3 proteins were colocalized with transfected 697fsX in the apical membrane area but not with WT TRPP2 distributed in the basolateral membrane and adjacent intracellular areas (Fig. 7). Therefore, in native tissues of ADPKD patients with 697fsX, a deficiency of PC1 interaction can lead to the formation of heteromultimeric TRPP2/TRPC3 channels with ion permeation properties different from those of TRPC3containing channels in normal tissues. Interestingly, cation influx activity is increased when 697fsX is coexpressed with TRPC3. It is, therefore, possible that 697fsX enhances trafficking of TRPC3 channels through protein multimerization, as previously reported for TRPV4 associated with TRPP2 (47). Other TRPP2-interacting proteins may also participate in trafficking of TRPP2 proteins (48 -51).
TRPP2 abundantly expressed in ER membrane has been suggested to act as a Ca 2ϩ release channel of intracellular stores (26) and to functionally interact with inositol 1,4,5-trisphosphate receptor-induced Ca 2ϩ release (52). TRPP2 is also reported to form protein complexes with PC1 or TRPC1 to induce Ca 2ϩ influx at the PM (6,41). However, because our data clearly indicate that overexpression of TRPP2 potentiates receptor-activated Ca 2ϩ release but not Ca 2ϩ influx in HEK293 cells, TRPP2 should mainly function as a Ca 2ϩ release channel. In this study we detected endogenous expression of PC1, TRPP2, TRPC1, TRPC3, TRPC4, and TRPC6 in HEK293 cells (supplemental Fig. 2). Interestingly, our data suggest a higher expression level of TRPP2 compared with those of PC1 and TRPC1, further suggesting that endogenous PC1 and TRPC1 are fully associated with endogenous TRPP2 and that PC1 and TRPC1 are not freely accessible for recombinant TRPP2. With regard to the augmentation of Ca 2ϩ influx in HEK293 cells recombinantly expressing 697fsX, endogenous TRPC3 but not TRPC7 was detected in HEK293 cells. Other TRPCs such as TRPC1, TRPC4, and TRPC6 are also endogenously expressed in HEK293 cells but fail to show enhancement of Ca 2ϩ influx by 697fsX when recombinantly expressed (Fig. 5B). Therefore, endogenous TRPC3 may interact with 697fsX at the PM.
The present study reveals that the TRPP2 C terminus-truncating mutations exert a dual pathogenic impact at different subcellular sites, the ER and PM, on receptor-induced [Ca 2ϩ ] i mobilization. At the ER, the potentiation effect of WT TRPP2 on receptor-activated Ca 2ϩ release from ER was abolished by the 697fsX mutation in HEK293 cells. This is consistent with the previous report of Cai et al. (27), which suggested that the C terminus-truncated mutation (R742X) abolishes retention of TRPP2 at the ER membrane, where TRPP2 regulates Ca 2ϩ release (26). At the PM, physical and functional association of 697fsX TRPP2 proteins with TRPC3 or TRPC7 prominently enhanced receptor-activated Ca 2ϩ entry into HEK293 cells. This finding clarifies the interaction target of C terminus-truncated TRPP2 proteins and their activation mechanism as ion channels, which were unresolved issues. Previous reports only described the disrupted assembly of TRPP2 with PC1 and consequent PM mistranslocation of TRPP2 by the R742X mutation (6) as well as the abnormal spontaneous [Ca 2ϩ ] i -independent cation current via the R742X TRPP2 mutant (29). Furthermore, compared with the proposed loss of Ca 2ϩ influx by ablation of the association between PC1 and TRPP2s, the enhanced Ca 2ϩ influx via complexation with TRPC3 or TRPC7 proteins is a gain of function more likely consistent with the dominant phenotype of the PKD mutations. Thus, the obtained data may provide an important clue to address the discrepancy between seemingly conflicting hypotheses with regard to the subcellular sites of the pathogenic action of TRPP2 C terminus mutants (6,26).
The present study also implies the biological context in which the C-terminal-truncated mutant channels function. The observed enhancement of receptor-induced Ca 2ϩ entry via association of 697fsX with TRPC3 supports the contention that 697fsX induces dysregulated cell growth and death in ADPKD, as TRPC3 channels are indeed activated upon receptors such as B cell (38) and T cell receptors (53), and brain-derived neurotrophic factor receptors (54,55) linked to cell growth and survival, and play an important role in cardiac hypertrophy (56 -59). Importantly, ADPKD is a systemic disorder with a variety of other manifestations including liver cysts, cerebral aneurysms, and various cardiac valvular abnormalities. Wide expression of TRPC3, TRPC7, and TRPP2 in various tissues including kidney (60 -62) suggests that the pathogenic association between C terminal-truncated TRPP2s and TRPC3 or TRPC7 underlies the ADPKD abnormalities observed in multiple tissues (1). In normal tissues native TRPP2 and TRPC3 showed an interesting contrast in subcellular localization, with TRPP2 concentrated in the basolateral membrane and TRPC3 localized in the apical domain of rat tubular cells (61)(62)(63)(64)(65). Our observation (Fig. 7) is consistent with this localization pattern. In addition, basolateral localization of TRPC1 in MDCK cells (64) and its physical interaction with TRPP2 have been reported (41). Notably, during preparation of this manuscript, it has been reported that the heteromeric channels comprised of TRPP2 and TRPC1 are activated in response to receptor stimulation (66). It is, therefore, speculated that C-terminal truncations disrupt the PC1-TRPP2 assembly as well as the formation of heteromultimeric TRPC1/TRPP2 channels in the basolateral membrane (65). The heteromultimerization may consequently induce mislocalization of TRPP2 proteins and the formation of heteromultimeric TRPC3/TRPP2 channels in the apical membrane, finally leading to transduction of pathogenic signals in ADPKD tissues. Taking into consideration that TRPC3 and TRPC7 are different from TRPC1 in molecular and functional properties (11), a conclusion based on TRPC1 should not be directly extrapolated to TRPC3 and TRPC7. This is consistent with an idea that TRPP2 mutants associated with TRPC3 and TRPC7 indeed play unique pathophysiological roles in signal transduction. We can raise a different possibility that unknown regulatory mechanisms, which suppress the action of the TRPP2 C terminus, may induce translocation to the apical membrane and subsequent heteromultimerization with WT TRPP2 and TRPC3 also in normal tissues under certain cellular conditions. In this scenario it is conceivable that the 697fsX mutant is constantly present at the apical membrane to transmit constitutively active aberrant signals in ADPKD tissues.
At the cellular level ADPKD inherited in a dominant manner has been explained by a recessive mechanism, leading to the complete loss of function through somatic mutations in the normal PKD2 allele (the two-hit model). However, TRPP2 (PC2) is frequently observed in renal cystic epithelium of human ADPKD, raising the possibility that deregulated activation of PKD2 may be associated with the cystogenesis of human ADPKD (67,68). Furthermore, PKD2-overexpressing transgenic mice showed a development of typical renal cysts and an increase of proliferation and apoptosis, which are reflective of the human ADPKD phenotype (68). The transgenic mice also showed up-regulation of B-Raf/mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK)/ERK sequential signaling, a possible molecular mechanism of cystogenesis (68). Strikingly, TRPC3-mediated Ca 2ϩ influx has been shown to play essential roles in ERK activation in various cell types (38,69,70). Therefore, formation of heteromeric channels comprised of 697fsX and TRPC3 and possible up-regula-tion of ERK activity through Ca 2ϩ regulation may be sufficient to trigger renal cystogenesis.
ADPKD is often considered a disease of adults, but it is clear that it already begins in childhood. Renal cysts in children with ADPKD have been associated with a wide clinical spectrum ranging from a total absence of symptoms to massive renal enlargement, hypertension, oliguria, and pulmonary hypoplasia in newborns (71). In this context the seemingly delayed onset symptoms shown by the 697fsX family can be categorized as a manifestation of a milder type of polycystic kidney disease. Interestingly, abnormal [Ca 2ϩ ] i regulation associated with PKD2 haploinsufficiency has been related to vascular abnormalities (72). Based on this report, we can speculate that 697fsX restores the dosage reduction due to haploinsufficiency; despite mislocalization of 697fsX-containing channels, 697fsX-mediated Ca 2ϩ entry may incompletely but significantly compensate for the net reduction in [Ca 2ϩ ] i mobilization by supplementing partial Ca 2ϩ entry and Ca 2ϩ release via TRPP2, derived from the remaining normal allele in heterozygous persons. In contrast, those mutants truncated at the middle or on the N-terminal side of the transmembrane core may be inactive as functional Ca 2ϩ entry channels and may act as dominant negative forms for gene products of the normal PKD2 allele or for TRPC3 to worsen gene dosage insufficiency. Further studies are required to establish the relationship between mutant properties and the severity of ADPKD.