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J Biol Chem, Vol. 274, Issue 39, 27359-27370, September 24, 1999

Molecular and Functional Characterization of a Novel Mouse Transient Receptor Potential Protein Homologue TRP7
Ca²⁺-PERMEABLE CATION CHANNEL THAT IS CONSTITUTIVELY ACTIVATED AND ENHANCED BY STIMULATION OF G PROTEIN-COUPLED RECEPTOR^*

Takaharu Okada, Ryuji Inoue^§, Kazuto Yamazaki^¶, Akito Maeda, Tomohiro Kurosaki, Tohru Yamakuni^**, Isao Tanaka^¶, Shunichi Shimizu, Kazuhiro Ikenaka^§§, Keiji Imoto, and Yasuo Mori^¶¶

From the Laboratories of  Humoral Information and ^§§ Neural Information, Department of Information Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, the ^§ Department of Pharmacology, Faculty of Medicine, Kyushu University, Fukuoka 812-8582, the ^¶ Tsukuba Research Laboratories, Eisai Co., Ltd., Tsukuba 300-2635, the  Department of Molecular Genetics, Institute for Liver Research, Kanasai Medical University, Moriguchi 570-8506, the ^** Mitsubishi Kasei Institute of Life Sciences, Machida 194-8511, and the  Department of Pathophysiology, School of Pharmaceutical Sciences, Showa University, Tokyo 142-8555, Japan

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Characterization of mammalian homologues of Drosophila transient receptor potential protein (TRP) is an important clue to understand molecular mechanisms underlying Ca²⁺ influx activated in response to stimulation of G_q protein-coupled receptors in vertebrate cells. Here we have isolated cDNA encoding a novel seventh mammalian TRP homologue, TRP7, from mouse brain. TRP7 showed abundant RNA expression in the heart, lung, and eye and moderate expression in the brain, spleen, and testis. TRP7 recombinantly expressed in human embryonic kidney cells exhibited distinctive functional features, compared with other TRP homologues. Basal influx activity accompanied by reduction in Ca²⁺ release from internal stores was characteristic of TRP7-expressing cells but was by far less significant in cells expressing TRP3, which is structurally the closest to TRP7 in the TRP family. TRP7 induced Ca²⁺ influx in response to ATP receptor stimulation at ATP concentrations lower than those necessary for activation of TRP3 and for Ca²⁺ release from the intracellular store, which suggests that the TRP7 channel is activated independently of Ca²⁺ release. In fact, TRP7 expression did not affect capacitative Ca²⁺ entry induced by thapsigargin, whereas TRP7 greatly potentiated Mn²⁺ influx induced by diacylglycerols without involvement of protein kinase C. Nystatin-perforated and conventional whole-cell patch clamp recordings from TRP7-expressing cells demonstrated the constitutively activated and ATP-enhanced inward cation currents, both of which were initially blocked and then subsequently facilitated by extracellular Ca²⁺ at a physiological concentration. Impairment of TRP7 currents by internal perfusion of the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid revealed an essential role of intracellular Ca²⁺ in activation of TRP7, and their potent activation by the diacylglycerol analogue suggests that the TRP7 channel is a new member of diacylglycerol-activated cation channels. Relative permeabilities indicate that TRP7 is slightly selective to divalent cations. Thus, our findings reveal an interesting correspondence of TRP7 to the background and receptor stimulation-induced cation currents in various native systems.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Receptor stimulation by hormones, neurotransmitters, autacoids, Ca²⁺, and antigens leads to phospholipase C activation via G_q protein or protein kinases. It has been recognized that subsequent hydrolysis of phosphoinositides is linked to Ca²⁺ influx across the plasma membrane (1). Diverse ion channels activated by various triggers have been reported to be responsible for this type of Ca²⁺ influx (1). Among members of the group, recent attention was particularly directed to capacitative Ca²⁺ entry (CCE¹; in other words, Ca²⁺ release-activated current or store-operated channel) that is activated through Ca²⁺ release from the intracellular Ca²⁺ store, endoplasmic reticulum, induced by inositol 1,4,5-trisphosphate (IP₃), and consequent depletion of Ca²⁺ from the store (2-8). In addition, other plasma membrane ion channels directly activated by second messengers such as Ca²⁺, IP₃, inositol 1,3,4,5-tetraphosphate, and arachidonic acid metabolites are also categorized as Ca²⁺-permeable channels activated in response to receptor stimulation (1, 9-13).

Drosophila transient receptor potential protein (TRP), which was discovered through the genetic studies of the Drosophila visual transduction mutation (14), and the invertebrate and vertebrate TRP homologues so far have been the only molecular entities functionally characterized as channels responsible for Ca²⁺ influx induced by activation of G_q-coupled receptors (15-25). TRP homologues were originally hypothesized to encode channels responsible for CCE, and some lines of supportive evidence for the hypothesis were presented from cDNA expression experiments on TRP subtypes (21-23, 26-31). Recently, however, store-independent activation of Ca²⁺ influx and cation currents mediated by TRP members were reported (24, 25, 32-36). Especially, as for Drosophila TRP and TRP-like (TRPL), polyunsaturated fatty acids, such as arachidonic acid and linolenic acid, were shown to activate them in both native and recombinant systems (35). This store-independent activation of TRP and TRPL is consistent with the observation that IP₃ receptor is not essential for receptor potentials generated by TRP and TRPL in Drosophila photoreceptors (37). As for vertebrates, six members of mammalian TRP homologues (TRP1-6) were so far identified, and three TRP homologues TRP3, TRP5, and TRP6 have been reported to exhibit store-independent activities in recombinant expression systems (24, 25, 33, 34, 36). Most recently, TRP3 and TRP6 have been reported to be activated by diacylglycerols, whereas TRP5 was unresponsive to them (36).

In Drosophila photoreceptors, two homologues, Drosophila TRP and TRPL, are responsible for the light-sensitive conductances (15, 17, 35, 38). By contrast, in native mammalian cells, cationic currents induced by receptor stimulation have yet to be resolved into components mediated by individual TRP homologues and other unidentified entities. Here we found a novel member of the mammalian TRP family, TRP7, from mouse brain. TRP7 was widely distributed, being the most abundant in the heart, lung, and eye. Functional properties of TRP7 recombinantly expressed in human embryonic kidney (HEK) cells were distinct from those of TRP3, which is structurally the most homologous to TRP7 in the TRP family. TRP7 was constitutively activated to the level much higher than that of TRP3. Ca²⁺ influx mediated by TRP7 was enhanced by ATP receptor stimulation at the concentrations of ATP lower than those necessary for Ca²⁺ release from the store and Ca²⁺ influx via TRP3. The experiments also showed an essential involvement of diacylglycerol and intracellular Ca²⁺ in activation of TRP7. In addition, the content of the intracellular Ca²⁺ store was reduced by TRP7 expression but not by TRP3 expression. Nystatin-perforated patch clamp recordings from TRP7-expressing cells demonstrated the constitutive and ATP-enhanced nonselective cation currents, which were suppressed by a physiological concentration of extracellular Ca²⁺. Our findings demonstrate that TRP7 encodes a cation channel with distinctive functional properties among mammalian TRP homologues, suggesting that TRP7 is responsible for spontaneous and receptor stimulation-induced non-selective cationic currents known for important physiological roles in native tissues.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

cDNA Cloning and Sequence Determination-- To search for a novel mouse TRP homologue, a cDNA library was screened with the cDNA encoding mouse TRP2 as a probe, which was obtained by reverse transcriptase-PCR amplification from the mouse (BALB/c or 129/SvJ) brain poly(A)⁺ RNA using a pair of specific oligonucleotide primers T2-1 (5'-GACGACATGATCCGGTTCATGTTC-3') and T2-2 (5'-CTCGATCTTCTGGAAGGAGTTGGTG-3') (22), SuperScript II RNase H reverse transcriptase (Life Technologies, Inc.), and LA-Taq DNA polymerase (Takara, Otsu, Japan). Screening of the oligo(dT)-primed, size-selected (>1 kb) adult mouse (129/SvJ or BALB/c) brain cDNA library constructed in the Uni-ZAP XR vector (Stratagene, La Jolla, CA) using the TRP2 cDNA probe yielded clone a11. Sequence comparison with the known mammalian TRP homologues (TRP1-6) revealed that a11 carries the cDNA (94 to 2071) of a novel TRP homologue designated as TRP7. To obtain the entire coding sequence of the mouse TRP7 cDNA, rapid amplification of 3'-cDNA ends (3'-RACE) was performed using the specific primer T7-7 (5'-CGTGCTGTATGGGGTTTATAATGTCACC-3'), the template cDNAs constructed from C57BL/6J (B6) mouse brain poly(A)⁺ RNA, and the Marathon cDNA Amplification kit (CLONTECH, Palo Alto, CA). The resulting ~1.2-kb cDNA fragment was subcloned into pBluescript SK(+) (Stratagene) to a yield clone p3RACE7 (1950-3085 followed by a poly(dA) tract). Through comparison with other TRP homologues, it was suggested that the region of cDNA (residues 781-1128) was absent in a11 due to alternative splicing. The region was amplified by reverse transcriptase-PCR from adult mouse brain total RNA using alfalfa mosaic virus reverse transcriptase (Takara), LA-Taq DNA polymerase (Takara), and the specific primers (5'-GACTACTTCTGCAAGTGCAATGAGTGC-3' and 5'-TTCCACAAGTGTAGCACGTACTCCC-3'). The resulting 0.25-0.7-kb cDNA fragments were subcloned into pGEM-T Easy vector (Promega, Madison, WI) to yield p7, p7, p7, and p7 carrying the ~0.70, ~0.52, ~0.33, and ~0.25-kb spliced variants, respectively, of TRP7 cDNA. The insert of p7 corresponds to the spliced form of the cDNA carried by a11.

Northern Blot Analysis-- RNA blot hybridization analysis was carried out using total RNA (20 µg) from various tissues of 2-month-old B6 mice. The probe used to detect TRP7 RNA was the ~0.44-kb SacI/PvuII fragment from p3RACE7. Random Primer DNA Labeling Kit version 2 (Takara) was used to ³²P label the probe. Hybridization was performed at 42 °C in 50% formamide, 5× 5 SSC, 50 mM sodium phosphate buffer (pH 7.0), 0.1% SDS, 0.1% polyvinylpyrrolidone, 0.1% Ficoll 400 (Amerham Pharmacia Biotech), 0.1% bovine serum albumin, and 0.2 mg/ml sonicated herring sperm DNA, as described previously (25).

In Situ Hybridization-- Brains were dissected from anesthetized 8-week-old B6 mice after perfusion with 4% paraformaldehyde in 0.1 M sodium phosphate buffer and were postfixed overnight at 4 °C in the same fixative and subsequently dehydrated, embedded in paraffin, sliced at 8 µm, and mounted onto silicon-coated glass slides.

The recombinant pBluescript SK(+) plasmid, pTRP7-25, containing the TRP7 cDNA fragment amplified from a11 using the primers Ma19 (5'-CGTTAAGACTCTGCCCAA-3') and Ma16 (5'-ACATCCTGCACGTACTGG-3'), was linearized by digesting the EcoRI or HindIII site (on vector) and transcribed using T3 or T7 MAXIscript RNA polymerase (Ambion, Austin, TX) with DIG RNA Labeling Mix (Roche Molecular Biochemicals) for synthesis of the sense or antisense digoxigenin (DIG)-labeled RNA probes.

Sections were partially digested by 0.8% pepsin in 0.2 N HCl and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) after de-paraffinization and rehydration. Hybridization was performed for 12-16 h at 50 °C with 400 ng/ml antisense or sense probes in 10 mM Tris-HCl (pH 7.6), 600 mM NaCl, 0.25% SDS, 1 mM EDTA (pH 8.0), 0.02% polyvinylpyrrolidone, 0.02% Ficoll 400, 0.02% bovine serum albumin, 0.2 mg/ml yeast tRNA, 10% dextran sulfate, and 50% formamide. The sections were washed in 2× SSC with 50% formamide at 50 °C for 30 min, treated with 10 µg/ml RNase A in 10 mM Tris-HCl (pH 7.6), 1 mM EDTA (pH 8.0), and 500 mM NaCl to remove free probes, and washed at increasingly higher stringencies up to final condition of 0.2× SSC at 50 °C for 20 min.

Washed slides were incubated in 1.5% blocking reagent (Roche Molecular Biochemicals) in 100 mM Tris-HCl (pH 7.5), and 150 mM NaCl (DIG buffer) at room temperature for 1 h. Immunochemical detection of the hybridized probes was performed using alkaline phosphatase-conjugated anti-DIG antibody (Roche Molecular Biochemicals) at 500 times dilution in DIG buffer for 60 min. The alkaline phosphatase activity was visualized by 12-16 h incubation with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate alkaline phosphatase detection system (Roche Molecular Biochemicals) in 100 mM Tris-HCl (pH 9.5), 50 mM MgCl₂, and 100 mM NaCl under light protection.

Recombinant Expression in HEK Cells-- Expression plasmids for TRP7 were constructed as follows. A PCR product amplified from the clone a11 using a sense primer (5'-GGGTCGACGGGTTTTTATTTTTAATTTTCTTTCAAATACTTCCACCATGTTGGGGAGCAACACC-3'), designed to contain the untranslated leader sequence from the alfalfa mosaic virus (39), a consensus sequence for translation initiation (40), and nucleotide residues 1-18 of TRP7, and an antisense primer (5'-AGTGACCTCCAAGTGCTC-3') was digested with SalI and ApaLI. Another PCR product amplified from p3RACE7 using T7-7 (see above) and an antisense primer (5'-ATGCGGCCGCTTTGGAATGCTGTTAGAC-3') was digested with NcoI and NotI. The resulting fragments were ligated with respective ApaLI-(701)/NcoI-(1999) fragments from p7, p7, and p7, and the 5.5-kb SalI/NotI fragment from pCI-neo (Promega, Madison, WI) to yield pCI-neo-mTRP7, pCI-neo-mTRP7, and pCI-neo-mTRP7, respectively. The TRP3 expression plasmid was constructed as follows. A PCR product amplified from the clone g-8 (41) using a sense primer (5'-CAGCGGCCGCGGGTTTTTATTTTTAATTTTCTTTCAAATACTGCCACCATGCGTGACAAGGGCCG-3'), designed to contain the untranslated leader sequence from alfalfa mosaic virus (39), a consensus translation initiation sequence (40), and nucleotide residues 1-17 (41), and an antisense primer (5'-GATGGCTAGCAGCAGCGCATCACC-3') was digested with NotI. Another PCR product amplified from the clone m20 (41) using a sense primer (5'-CGTTCCAAACTTTGGCTCTCCTACTTCGATG-3') and an antisense primer (5'-CAGCGGCCGCTTGACTGCAGGTGGCTGCCTCAC-3') was digested with PflMI and NotI. The resulting fragments were ligated with the NheI-(286)/PflMI-(2040) fragment and pCI-neo linearized with NotI to yield pCI-neo-mTRP3.

HEK293 cells (American Type Culture Collection, Manassas, VA) were co-transfected with H3-CD8 containing the cDNA of the T-cell antigen CD8 (42) and one of pCI-neo-mTRP7, pCI-neo-mTRP7, pCI-neo-mTRP7, pCI-neo-mTRP3, and the vector pCI-neo. Transfection was carried out using SuperFect Transfection Reagent (Qiagen, Hilden, Germany). Cells were trypsinized, diluted with Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 30 units/ml penicillin, and 30 µg/ml streptomycin, and plated onto glass coverslips 18 h after transfection. Then cells were subjected to measurements 18-42 h after plating on the coverslips. TRP homologue-expressing cells were selected through detection of CD8 co-expression using polystyrene microspheres precoated with antibody to CD8 (Dynabeads M-450 CD8; Dynal, Oslo, Norway).

Measurement of Changes in [Ca²⁺]_i-- Cells on coverslips were loaded with fura-2 by incubation in Dulbecco's modified Eagle's medium containing 5 µM fura-2/AM (Dojindo Laboratories, Kumamoto, Japan) and 10% fetal bovine serum at 37 °C for 30 min and washed with the HEPES-buffered saline (HBS) containing (in mM) 107 NaCl, 6 KCl, 1.2 MgSO₄, 2 CaCl₂, 1.2 KH₂PO₄, 11.5 glucose, 20 HEPES, adjusted to pH 7.4 with NaOH. The coverslips were then placed 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-20/CA, Hamamatsu Photonics, Hamamatsu, Japan). The fura-2 fluorescence at an emission wavelength of 510 nm (bandwidth, 20 nm) was observed at room temperature by exciting fura-2 alternately at 340 and 380 nm (bandwidth, 11 nm). The 340:380 nm ratio images were obtained on a pixel by pixel basis and were converted to Ca²⁺ concentrations by in vitro calibration. The calibration procedure was performed according to Ueda and Okada (43). All the reagents, except LaCl₃ and GdCl₃, dissolved in water, ethanol, or dimethyl sulfoxide were diluted to their final concentrations in HBS or Ca²⁺-free HBS containing (in mM) 107 NaCl, 6 KCl, 1.2 MgSO₄, 1.2 KH₂PO₄, 0.5 EGTA, 11.5 glucose, 20 HEPES, adjusted to pH 7.4 with NaOH, and applied to the cells by perfusion. LaCl₃ and GdCl₃ (100 mM in water) were diluted in HBS or Ca²⁺-free HBS from which KH₂PO₄ was omitted. Data were accumulated under each condition from 2 to 4 experiments using cells prepared through 2-3 transfections.

Measurement of Mn²⁺ Influx-- Mn²⁺ entry was measured through monitoring the decline of fluorescence of fura-2 induced by binding of Mn²⁺. Fluorescence quenching was studied using the fura-2 isosbestic excitation wavelength at 360 nm and recording emitted fluorescence at 510 nm. The system used was the same as in the [Ca²⁺]_i measurement. The fluorescence intensity relative to the initial values were obtained on a pixel by pixel basis. MnCl₂ and ATP dissolved in water were diluted to their final concentrations in nominally Ca²⁺-free HBS from which KH₂PO₄ was omitted.

Electrophysiology-- For electrophysiological measurements, coverslips with cells were placed in a perfusion chamber. Cells prepared in this manner had membrane capacitance of 8-26 picofarads (n = 13). Patch electrodes having a resistance of 4-6 megohms (when filled with the pipette solution described below) were fabricated from 1.5-mm Pyrex capillaries, using an automatic multiple-stage micropipette puller (P-97, Sutter Instruments, USA), and heat-polished in a microforge bearing a thin indium-platinum heating wire (Narishige, Tokyo, Japan). Voltage generation and current signal acquisition were implemented through a high-impedance low-noise patch clamp amplifier (EPC-7, List Electronics, Darmstadt, Germany). Ramp currents were sampled at 2 kHz after low pass filtering at 1 kHz and analyzed with pCLAMP 6.02 software (Axon Instruments, Foster City, CA) for Fig. 8C. For long term recordings such as illustrated in Fig. 8A, current signals were stored on a computer hard disc (filtered at 50 Hz), using an A/D, D/A converter, MacLab/4 (AD Instruments, New South Wales, Australia). The details for the nystatin-perforated recording have been described previously (44). The pipette solution contained (in mM) 140 CsCl, 2 MgCl₂, 10 HEPES, 20 glucose, adjusted to pH 7.2 with Tris base. For the conventional whole-cell recording, the pipette solution contained (in mM) 140 CsCl, 2 MgCl₂, 10 BAPTA, and 0, 3, or 5 CaCl₂, 2 ATP (free acid), 0.1 GTP (lithium salt), 10 HEPES, adjusted to pH 7.2 with Tris base. The Ca²⁺-free external solution contained (in mM) 140 NaCl, 0.5 EGTA, 10 HEPES, 20 glucose, adjusted to pH 7.4 with Tris base. For the nominally Ca²⁺-free external solution, EGTA was simply omitted from this solution. For 2 mM Ca²⁺-containing external solution, 2 mM CaCl₂ was added to the nominally Ca²⁺-free solution. For the cesium external solution, Na⁺ in the Ca²⁺-free solution was simply replaced by 140 mM Cs⁺. The 10 mM divalent cation-containing external solution contained (in mM) either of 10 CaCl₂, BaCl₂, or MnCl₂, 126 N-methyl-D-glucamine-Cl (NMDG-Cl), 20 glucose, 10 HEPES, adjusted to pH 7.4 with NMDG.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Primary Structure of TRP7-- Fig. 1A shows the amino acid sequence of the mouse TRP7 deduced from the cDNA sequence in comparison with that of mouse TRP3 (41) which is structurally the most similar to TRP7 among the TRP homologues (see below). TRP7 is composed of 862 amino acid residues. Deletions of nucleotides encoding the amino acid sequences 322-376 and 261-376 in the clones p7 and p7/a11, respectively (see above under "Experimental Procedures"), are presumably resulting from alternative RNA splicing. A 382-base pair deletion in the clone p7 causes a frameshift that changes the residue 322 from phenylalanine to glutamate and terminates translation of TRP7 there. A hydropathy profile of TRP7 reveals eight hydrophobic segments and the hydrophilic N and C termini (Fig. 1B), similar to those of other TRP subtypes (14, 16, 19-25, 41). Sufficient length of hydrophobic regions to span the membrane, together with the lack of a hydrophobic N-terminal sequence indicative of the signal sequence, suggests that TRP7 is a membrane protein with a core of transmembrane segments and the flanking N- and C-terminal regions disposed on the cytoplasmic side. Deleted amino acid sequences in the spliced variants include the first hydrophobic region (H1) and the adjacent N-terminal portion. Fig. 1C depicts a phylogenetic tree of the TRP family constructed by the neighbor-joining method (45), based on the sequence alignment carried out by the ClustalW program (46). The vanilloid receptor (rVR1) that forms a non-selective cation channel activated by capsaicin is structurally related to members of the TRP family (47). The amino acid sequence of mouse TRP7 has identities/similarities of 34:56, 81:89, 40:60, 40:60, 75:84, 32:53, 33:53, and 13:34% with mTRP1 (48), mTRP3 (41), mTRP4 (41), mTRP5 (25), mTRP6 (24), dTRP (14), dTRPL (16), and rVR1 (47), respectively.

View larger version (53K): [in this window] [in a new window]   Fig. 1.   Primary structure of mouse TRP7. A, the amino acid sequence (in single letter code) of the mouse TRP7 deduced from the cDNA sequence is aligned with that of mouse TRP3 (41) by the ClustalW program (46). Pairs of identical residues at one position are enclosed with solid lines and pairs of conservative residues at one position with broken lines. The hydrophobic regions H1-H8 are shown. The bracketing arrows indicate where deletion occurs through alternative RNA splicing in TRP7. B, the Kyte-Doolittle hydrophobicity profile of TRP7 was generated with the window size of 10 amino acids (72). C, the phylogenetic tree for the TRP family was generated by the neighbor-joining method (45), based on the sequence alignment carried out by the ClustalW program (46). Members of the TRP family are as follows: dTRP (14), dTRPL (16), mTRP1 (48), mTRP3 (41), mTRP4 (41), mTRP5 (25), mTRP6 (24), ceTRP (73), and rVR1 (47).

Tissue Distribution of TRP7 RNA-- RNA preparations from different mouse tissues were subjected to Northern blot analysis using TRP7 cDNA as a specific probe. Hybridizable RNA signals of ~4.0 and >5.0 kb were detected at the highest level in the heart, lung, and eye and at lower levels in the brain, spleen, and testis (Fig. 2A). To determine which cell types express TRP7 RNA in the central nervous system, para-sagittal sections of 8-week-old B6 mouse brains were subjected to in situ hybridization histochemistry using cRNA probes specific for TRP7 (Fig. 2, B and C). Cerebellar Purkinje cells were the most prominent site of TRP7 RNA expression in the brain; intense TRP7 hybridization signals were concentrated along the Purkinje cell layer (Fig. 2, B and C). TRP7 signals were also found intensely in the mitral layer of olfactory bulb and hippocampal neurons and diffusely in other regions including the cerebellar nuclei, pons, and cerebral cortex.

View larger version (48K): [in this window] [in a new window]   Fig. 2.   Tissue distribution of TRP7 RNA. A, autoradiogram of blot hybridization analysis with the TRP7 cDNA probe of RNA from various tissues. The positions of ribosomal RNAs (18 S and 28 S) are shown on the left. B, in situ hybridization analysis of a B6 mouse brain sagittal section using the TRP7 cRNA probe. C, the magnified image of the cerebellar lobe in B.

Functional Characterization of TRP7, Fluorescence Measurements-- HEK293 cells serve as an excellent heterologous expression system to study functional properties of recombinant TRP channels, since stimulation of an endogenous P_2y2 purinoceptor induces intracellular phosphoinositide signaling but generates low endogenous activity of store-operated CCE channels (25) (see also Fig. 5). TRP7 cDNA, which is the longest form of the cloned TRP7 variants, together with a marker protein CD8 cDNA was transiently expressed in HEK cells, and intracellular Ca²⁺ concentration in TRP7-transfected cells was monitored by using fura-2 as an indicator and compared with that in vector-transfected control cells and cells transfected with TRP3, which is structurally the closest to TRP7. In the presence of 2 mM extracellular Ca²⁺, application of 100 µM ATP to vector-transfected, CD8-positive control cells induced a rapid rise in [Ca²⁺]_i that peaked within 20 s and gradually decreased to the resting level within 300 s (Fig. 3A, open circles). This transient rise in [Ca²⁺]_i was presumed to be due mainly to release from the intracellular Ca²⁺ store, because omission of extracellular Ca²⁺ did not significantly affect the peak level (Fig. 3B, open circles). The decay phase was accelerated by omission of extracellular Ca²⁺. When 100 µM ATP was applied to TRP7-transfected, CD8-positive cells in the presence of extracellular Ca²⁺, the [Ca²⁺]_i rise greatly increased and showed a sustained phase after the initial transient phase (Fig. 3A, filled circles). Under the constant stimulation with ATP, [Ca²⁺]_i levels in TRP7-transfected cells did not return completely to the resting level even 30 min after the start of ATP stimulation ([Ca²⁺]_i = 19 ± 4 nM, n = 65). ATP-induced [Ca²⁺]_i increases were enhanced and prolonged also by TRP3 expression (Fig. 3A, crosses), but [Ca²⁺]_i levels in TRP3-transfected cells were almost the resting level after 30 min of ATP stimulation ([Ca²⁺]_i = 8 ± 4 nM, n = 32). In TRP7- and TRP3-expressing cells, Ca²⁺ influx across the plasma membrane was likely to be a major cause of the [Ca²⁺]_i rise, because the ATP-induced [Ca²⁺]_i increase was much smaller in amplitude and much more transient in the absence of extracellular Ca²⁺ (Fig. 3, B and D, filled circles and crosses) than in the presence of extracellular Ca²⁺ at ATP concentration above 0.1 µM (Fig. 3, A and C, filled circles and crosses). Interestingly, [Ca²⁺]_i increase due to ATP-induced Ca²⁺ release from the internal Ca²⁺ store in TRP7-transfected cells was smaller than in control and TRP3-transfected cells (Fig. 3, B and D).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   [Ca2+]i rises due to basal and ATP-induced Ca2+ influx and Ca2+ release from internal stores in TRP7-, TRP3-, and vector-transfected HEK cells. Cytosolic Ca2+ was measured in fura-2-loaded TRP7- (filled circles), TRP3- (crosses), and vector-transfected (open circles) HEK293 cells. A, the cells were treated with 100 µM ATP in the presence of 2 mM extracellular Ca2+. B, the perfusion solution was first changed to Ca2+-free HBS containing 0.5 mM EGTA, and 100 µM ATP was applied to the cells in the absence of extracellular Ca2+. Three min after the application of ATP, 2 mM Ca2+ was further added to the extracellular solution. The duration of exposure to Ca2+-containing HBS, Ca2+-free HBS, and ATP is indicated by the filled, open, and hatched bars, respectively, above the graphs. C and D, dose-response relationships for maximum ATP-induced [Ca2+]i rises ([Ca2+]i) in individual cells in the presence of 2 mM extracellular Ca2+ (C) and in the absence of extracellular Ca2+ (D). E, ATP concentration dependence of maximum [Ca2+]i rises ([Ca2+]i) induced by the addition of 2 mM extracellular Ca2+ 3 min after the addition of ATP in individual cells. Data points are the means ± S.E. [Ca2+]i (A and B) or the means ± S.E. [Ca2+]i (C-E) in 20-65 cells. F, fluorescence quenching due to Mn2+ influx was observed in fura-2-loaded TRP7-, TRP3-, and vector-transfected cells. Mn2+ (1 mM) was added to nominally Ca2+-free HBS without phosphate. The duration of exposure to 1 mM Mn2+ is indicated by the filled bar above the graph. The results are expressed as the mean percent fluorescence intensity of the initial value in 28-53 cells.

The ATP-induced Ca²⁺ influx in TRP7- and TRP3-expressing cells was observed separately from Ca²⁺ release using the protocol as in Fig. 3B, where ATP was first applied in the absence of extracellular Ca²⁺, and 2 mM Ca²⁺ was then added to the extracellular solution after [Ca²⁺]_i returned to the level before ATP application. Addition of Ca²⁺ to the extracellular solution only slightly raised [Ca²⁺]_i above the resting level in control cells, whereas in TRP7- and TRP3-transfected cells, it elicited the large and sustained [Ca²⁺]_i rises. This sustained Ca²⁺ influx contrasts with the transient nature of TRP5-dependent Ca²⁺ influx (25). Importantly, ATP concentration required to induce the extracellular Ca²⁺-dependent [Ca²⁺]_i rise in TRP7-expressing cells was significantly lower than that needed to trigger Ca²⁺ release from the intracellular Ca²⁺ store; 1 µM ATP induced almost the maximal level of TRP7-dependent Ca²⁺ influx but did not evoke detectable Ca²⁺ release from the store (Fig. 3, D and E, filled circles). This indicates that activation of TRP7 by receptor stimulation is not coupled with Ca²⁺ release from the intracellular Ca²⁺ store or consequent depletion of the Ca²⁺ store. TRP3-dependent Ca²⁺ influx required relatively higher ATP concentrations for activation and was only half-activated by 1 µM ATP (Fig. 3E, crosses).

We have also examined the cells transfected with other alternatively spliced forms of TRP7, TRP7, or TRP7, which lack the first hydrophobic region (H1) and the adjacent N-terminal region. However, basal and ATP receptor stimulation-induced Ca²⁺ influx in these transfectants were not significantly different from vector-transfected cells, although Ca²⁺ release from the intracellular Ca²⁺ store seemed to be a little reduced by expression of TRP7 and TRP7 in the manner similar to that in TRP7-transfected cells (data not shown).

TRP7 showed a significant basal activity without any stimulation of G protein-coupled receptor by agonists such as ATP. In the presence of extracellular Ca²⁺, the resting [Ca²⁺]_i level in TRP7-expressing cells was higher than in control and TRP3-expressing cells (Fig. 3, A and B) by 15-30 nM. After removal of Ca²⁺ from the external solution, [Ca²⁺]_i in TRP7-expressing cells was reduced to the level similar to the resting [Ca²⁺]_i level in control cells (Fig. 3B). Re-addition of 2 mM extracellular Ca²⁺ raised [Ca²⁺]_i back to the previous level before the Ca²⁺ removal (data not shown). This high basal activity is an important characteristic that distinguishes TRP7 from other mammalian TRP homologues. TRP3 was also reported to have a basal activity (34), whereas this activity of TRP3 was considerably low compared with that of TRP7. Basal activities of TRP channels were clearly observed by monitoring Mn²⁺ influx. In the presence of 1 mM Mn²⁺ in the nominally Ca²⁺-free external solution, basal Mn²⁺ influx was observed in TRP7-transfected cells at levels higher than in TRP3-transfected cells (Fig. 3F). Small basal Mn²⁺ influx in TRP3-expressing cells did not derive from low expression of TRP3, since TRP7 and TRP3 elicited Mn²⁺ influx to a similar extent, when they were maximally activated by 100 µM ATP (data not shown).

Lanthanides La³⁺ and Gd³⁺ and the imidazole derivative SK&F96365 have been previously reported to block Ca²⁺-permeable channels including the TRP homologues (25). We tested these agents on the TRP7-dependent Ca²⁺ influx. In Fig. 4, 100 µM La³⁺ or Gd³⁺ (Fig. 4, A and B, filled circles) or 25 µM SK&F96365 (Fig. 4 C, filled circles) were added to the extracellular solution 1 min prior to the addition of extracellular Ca²⁺. 25 µM SK&F96365 and 100 µM La³⁺ significantly suppressed the [Ca²⁺]_i increase due to Ca²⁺ influx, whereas the effect of 100 µM Gd³⁺ on Ca²⁺ influx was not significant (Fig. 4D). These effects of the agents on TRP7 are similar to the effects on TRP5 reported previously (25).

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Pharmacological properties of ATP-induced Ca2+ influx in TRP7-transfected HEK cells. Cytosolic Ca2+ was measured in fura-2-loaded TRP7-transfected cells. The perfusion solution was changed to Ca2+-free HBS, and cells were stimulated with 100 µM ATP. Then 2 mM extracellular Ca2+ was added to the solution in the presence (filled circles) or absence (open circles) of 100 µM La3+ (A), 100 µM Gd3+ (B), or 25 µM SK&F96365 (C). The duration of exposure to these agents is indicated by the solid lines, and the duration of exposure to Ca2+-containing HBS, Ca2+-free HBS, and ATP is indicated by the filled, open, and hatched bars, respectively, above the graphs. D, effects of 100 µM LaCl3, 100 µM GdCl3, and 25 µM SK&F96365 on the amplitude of maximum [Ca2+]i rises ([Ca2+]i) induced by the addition of 2 mM extracellular Ca2+ 3 min after the addition of ATP in individual TRP7-transfected cells. For the experiments using lanthanides and their control experiments, KH2PO4 was omitted from HBS. Data points and columns are the means ± S.E. [Ca2+]i or the means ± S.E. [Ca2+]i in 17-46 cells. Bonferroni's t test following analysis of variance was employed to determine the statistical significance of differences. *, p < 0.05, compared with the control.

To examine whether TRP7 induces Ca²⁺ influx via depletion of intracellular Ca²⁺ stores, we used thapsigargin (TG), the inhibitor of sarcoplasmic and endoplasmic reticulum Ca²⁺-ATPases (49). Cells were treated with 2 µM TG in the absence of extracellular Ca²⁺ for 10 min, and the extracellular solution was subsequently changed to the 2 mM Ca²⁺-containing solution. [Ca²⁺]_i increases induced by the addition of extracellular Ca²⁺ were quite similar in TRP7-, TRP3-, and vector-transfected cells, indicating that CCE is not potentiated by expression of TRP7 or TRP3 (Fig. 5, AC). The results suggest that TRP7 and TRP3 are unresponsive to TG treatment. However, it is possible that TRP7 and TRP3 are capable of producing CCE, but activation triggers generated by TG determine the maximum limit of CCE induced in HEK cells. Furthermore, robust endogenous CCE may have masked TRP3 and TRP7 activities in the experiments. To examine these possibilities, we monitored the store depletion-dependent activities of TRP7 and TRP3 in the presence of 10 µM Gd³⁺, which effectively blocks endogenous Ca²⁺ influx in HEK cells (34) but does not significantly affect basal and ATP-induced Ca²⁺ influx mediated by TRP7 and TRP3 (data not shown). In TRP7-expressing cells, amplitude of [Ca²⁺]_i increase ([Ca²⁺]_i = 51 ± 5 nM, n = 22) induced by the addition of extracellular Ca²⁺ after TG treatment was larger than that in control cells ([Ca²⁺]_i = 19 ± 5 nM, n = 40) (Fig. 5, D and F). However, taking into account that basal Ca²⁺ influx elevated [Ca²⁺]_i in the TRP7-expressing cells (31 ± 6 nM, n = 27), TG-induced Ca²⁺ influx in TRP7-expressing cells was not significantly different from that in control cells. Gd³⁺-resistant, TG-induced Ca²⁺ influx was not potentiated by TRP3 expression (Fig. 5, E and F), which is different from the data obtained using human TRP3 (34). We also tested whether ATP is capable of activating additional Ca²⁺ influx in the TRP7- or TRP3-expressing cells where TG-induced Ca²⁺ influx is already activated.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   Thapsigargin-induced [Ca2+]i transients and ATP-induced [Ca2+]i changes after store depletion in the presence and absence of Gd3+ in TRP7-, TRP3-, and vector-transfected HEK cells. Cytosolic Ca2+ was measured in fura-2-loaded TRP7- (A and D), TRP3- (B and E), and vector-transfected (C and F) cells. A-C, the perfusion solution was changed to Ca2+-free HBS containing 0.5 mM EGTA, and 2 µM thapsigargin (TG) was applied to the cells in the absence of extracellular Ca2+, which was followed by the addition of 2 mM extracellular Ca2+ and by the addition of 100 µM ATP. D-F, 10 µM Gd3+ was added to the solution 1 min before the addition of extracellular Ca2+. The duration of exposure to Ca2+-containing HBS, Ca2+-free HBS, ATP, and thapsigargin is indicated by the filled, open, hatched, and shaded bars, respectively, above the graphs. Data points are the means ± S.E. [Ca2+]i in 22-47 cells.

When TG-induced [Ca²⁺]_i transients in the presence of extracellular Ca²⁺ decayed to almost stationary levels, external application of 100 µM ATP induced small [Ca²⁺]_i increases in TRP7-expressing cells but induced slight decreases in control cells and, to lesser extent, in TRP3-expressing cells (Fig. 5, A-C). By contrast, in the presence of Gd³⁺, [Ca²⁺]_i transients elicited by ATP application after TG treatment became prominent in TRP7- and TRP3-expressing cells compared with those measured in the absence of Gd³⁺ but not in the vector-transfected control cells (Fig. 5, D-F). Thus, the results obtained using TG suggest that TRP7 is activated in response to G_q-coupled stimulation through mechanisms independent of store depletion.

Interestingly, [Ca²⁺]_i transients induced by TG treatment in the absence of extracellular Ca²⁺ in TRP7-expressing cells were significantly smaller than those in control and TRP3-expressing cells (Fig. 5). This observation suggests that suppression of ATP-induced Ca²⁺ release from the store by TRP7 expression described above (Fig. 3, B and D) derived from the reduced Ca²⁺ content or size of internal stores.

Hofmann et al. (36) reported that human TRP3 and TRP6 are activated by diacylglycerols in a membrane delimited fashion, whereas human TRP1, mouse TRP4, and mouse TRP5 are unresponsive to the lipid mediators. We tested the effects of membrane-permeable diacylglycerol analogues 1,2-dioctanoyl-sn-glycerol (DOG) and 1-oleoyl-2-acetyl-sn-glycerol (OAG) on Mn²⁺ influx mediated by TRP7 (Fig. 6, A and B). Usage of Mn²⁺ enables measurement of divalent cation influx even when Ca²⁺ release from the internal stores coincides. When 100 µM DOG was added to the extracellular solution containing 100 µM Mn²⁺, Mn²⁺ influx was activated in 15 out of 21 TRP7-transfected, CD8-positive cells within 15 s after DOG application (Fig. 6A). Only 1 out of these 15 DOG-responsive cells and no DOG-unresponsive cells showed additional Mn²⁺ influx induced by subsequent 100 µM ATP application. No vector-transfected, CD8-positive control cells responded to DOG up to 130 s (Fig. 6A). However, enhancement of Mn²⁺ influx was observed after 135-170 s of DOG perfusion in 2 out of 16 control cells which showed no response to subsequent application of 100 µM ATP. When 100 µM OAG was applied, Mn²⁺ influx was activated in 17 out of 20 TRP7-transfected cells within 20 s after OAG application (Fig. 6B). Twelve out of 17 cells that immediately responded to OAG showed second enhancement of Mn²⁺ influx after 65-190 s of OAG perfusion. One out of 3 cells that did not respond immediately to OAG showed Mn²⁺ influx enhanced after 90 s of OAG incubation. Subsequent application of 100 µM ATP did not enhance Mn²⁺ influx in any TRP7-transfected cells. No control cells responded to OAG within 1 min, but 15 out of 18 cells showed Mn²⁺ influx 65-150 s after OAG application (Fig. 6B). No control cells responded to 100 µM ATP applied subsequently. The diacylglycerol lipase inhibitor RHC80267 was also tested for its effect on TRP7-mediated Mn²⁺ influx. Treatment of cells with the agent was expected to inhibit metabolization of diacylglycerols and to increase passively diacylglycerol content. Slight enhancement of Mn²⁺ influx was induced in TRP7-transfected cells by perfusion with 50 µM RHC80267-containing solution. In contrast to the experiment above using DOG and OAG, subsequent application of 1 µM ATP induced further Mn²⁺ influx in TRP7-expressing cells (Fig. 6C).

View larger version (28K): [in this window] [in a new window]   Fig. 6.   Effect of diacylglycerol analogues and a diacylglycerol lipase inhibitor on Mn2+ influx in TRP7- and vector-transfected cells, and effect of PKC inhibitor and PKC activator on Mn2+ influx in TRP7-transfected cells. Fluorescence quenching due to Mn2+ influx was observed in fura-2-loaded TRP7- (thick line in A-C, thick and thin line in D and E) and vector-transfected cells (thin line in A-C). A-C, Mn2+ (0.1 mM) was added to nominally Ca2+-free HBS without phosphate, and then one of the membrane-permeable diacylglycerol analogues, DOG (A) and OAG (B) at 100 µM, or a diacylglycerol lipase inhibitor, RHC80267 at 50 µM (C), was added to the solution. Finally, 100 µM ATP was added to the solution. D, thick line, TRP7-transfected cells were treated with a PKC inhibitor BIM I (1 µM), and 0.1 mM Mn2+ and then 100 µM OAG were added to the external solution. E, thick line, TRP7-transfected cells were treated with a PKC activator PMA (1 µM) in the presence of 0.1 mM extracellular Mn2+, and subsequently 100 µM OAG was added to the external solution. D and E, the results of the control experiments without the PKC inhibitor and activator are shown as thin lines. The duration of exposure to Mn2+, one of diacylglycerol analogues and RHC80267, and ATP is indicated by the filled, shaded, and hatched bars, respectively, above the graphs. The duration of exposure to BIM I or PMA is indicated by the solid lines. The results are expressed as the mean percent fluorescence intensity of the initial value in 16-29 cells.

Induction of Mn²⁺ influx by diacylglycerols in TRP7-expressing cells does not involve activation of protein kinase C (PKC), since treatment with a PKC inhibitor bisindolylmaleimide I (BIM I, 1 µM) (50) rather enhanced OAG-induced Mn²⁺ influx (Fig. 6D). Furthermore, a PKC activator phorbol 12-myristate 13-acetate (PMA, 1 µM), which did not induce Mn²⁺ influx by itself, abolished OAG-induced Mn²⁺ influx (Fig. 6E). Similar effects of BIM I and PMA were observed also on DOG- and ATP-induced Mn²⁺ influx in TRP7-expressing cells (data not shown).

Arachidonic acid, a polyunsaturated fatty acid, which is synthesized from diacylglycerol by diacylglycerol lipase and activates Drosophila TRP and TRPL directly (35), slowly accelerated Mn²⁺ influx in TRP7-transfected cells, but a similar acceleration of Mn²⁺ influx by arachidonic acid was observed also in control cells (data not shown).

We also tested effects of U-73122, the inhibitor of PLC (51), since PLC is directly responsible for the production of diacylglycerol by stimulation of G_q-coupled P_2Y2 ATP receptor. As expected, IP₃-dependent Ca²⁺ release induced by 3 µM ATP was almost completely suppressed by U-73122 (1 µM). However, the level of Ca²⁺ rises due to Ca²⁺ influx in TRP7-transfected cells was only slightly reduced by U-73122, whereas the Ca²⁺ rises were significantly slowed by the inhibitor (Fig. 7A, filled circles). U-73343 (1 µM), an analogue of U-73122 that acts as a very weak inhibitor of PLC (52), showed little effect on Ca²⁺ release from the stores or on Ca²⁺ influx induced by 3 µM ATP (Fig. 7A, open squares).

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Effect of PLC inhibitor and calmodulin antagonist on ATP-induced Ca2+ influx in TRP7-transfected cells. Cytosolic Ca2+ was measured in fura-2-loaded TRP7-transfected cells. A, TRP7-transfected cells were treated with 1 µM U-73122, a PLC inhibitor (filled circle), or its analogue, 1 µM U-73343, which acts as a very weak inhibitor of PLC (open square). B, TRP7-transfected cells were treated with a calmodulin antagonist W-13 at 100 (filled circles) or 30 µM (open squares). The perfusion solution was changed to Ca2+-free HBS containing 0.5 mM EGTA, and ATP (3 µM (A) or 100 µM (B)) was applied to the cells in the absence of extracellular Ca2+. Finally, 2 mM Ca2+ was further added to the extracellular solution. Both in A and B, the results of the control experiments without the PLC inhibitors and calmodulin antagonist are shown by open circles. The duration of exposure to Ca2+-containing HBS, Ca2+-free HBS, and ATP is indicated by the filled, open, and hatched bars, respectively, above the graphs. The duration of exposure to U-73122, U-73343, or W-13 is indicated by the solid lines above the graphs. Data points are the means ± S.E. [Ca2+]i in 22-54 cells.

The significance of intracellular Ca²⁺ in activation of TRP7 was suggested through electrophysiological recordings where the effects of intrapipette Ca²⁺ concentration on TRP7 activity were tested (see below in Fig. 9) and by the previous report on TRP3 (33). We therefore examined involvement of Ca²⁺-calmodulin by using its antagonist N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W-13) (53). TRP7-mediated Ca²⁺ influx was dose-dependently inhibited by W-13 (Fig. 7B). Interestingly, Ca²⁺ release was increased similarly by 30 and 100 µM W-13.

Functional Characterization of TRP7, Electrophysiological Measurements-- To investigate directly the functional characteristics of TRP7, ionic currents in TRP7-transfected cells were examined in comparison with those in control cells at a holding potential of 60 mV using the nystatin-perforated patch recording (Fig. 8A). The nystatin-perforation method allowed us to observe the development of significant spontaneous inward currents in 17 out of 17 TRP7-transfected cells in the 140 mM Na⁺-containing, nominally Ca²⁺-free extracellular solution, as the series resistance fell on progression of membrane perforation by nystatin (Fig. 8B, lower trace; 251 ± 44 pA; range: 58-756 pA). These inward currents were markedly inhibited when the extracellular solution was changed to 2 mM Ca²⁺-containing solution (data not shown) and increased by a subsequent change to EGTA-containing, Ca²⁺-free solution (Fig. 8A, lower trace). Stimulation by 100 µM ATP gradually increased the magnitude of inward currents for about 1 min, which was then followed by a gradual decline, in all the TRP7-transfected cells examined. The degree of current increase by ATP varied depending on the cells tested. As exemplified in Fig. 8A, the cells having relatively small spontaneous inward currents (<200 pA in the Ca²⁺-free solution; n = 6) tended to exhibit a large increase in current amplitude (2.6-11.2-fold) in response to ATP. In contrast, ATP appeared to less potently stimulate the cells which initially exhibited large spontaneous inward currents (>200 pA; n = 4; 1.3-4.0-fold increase by 100 µM ATP). Addition of 2 mM Ca²⁺ to the external solution caused a rapid reduction of the currents enhanced by ATP and subsequently a gradual increase that continued until the termination of ATP stimulation (Fig. 8A, lower trace). In the presence of 2 mM Ca²⁺, further addition of 100 µM Gd³⁺ only slightly inhibited the currents induced by ATP (82 ± 5% that in the presence of 2 mM Ca²⁺). This is consistent with our finding in [Ca²⁺]_i measurement that ATP-induced Ca²⁺ entry is not appreciably affected by the addition of 100 µM Gd³⁺ in TRP7-transfected cells (see Fig. 4).

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Electrophysiological characterization of the TRP7 channel by nystatin-perforated patch clamp recording. A, shown are time courses of ionic currents recorded from a vector- (upper trace) and a TRP7-transfected (lower trace) cell at a holding potential of 60 mV and under 2-s voltage ramps from 120 to 80 mV. The perfusion solution was first changed to the Ca2+-free solution, and 100 µM ATP was applied to the cells in the absence of extracellular Ca2+. During ATP stimulation, the external solution was changed back to the 2 mM Ca2+-containing solution. Finally, ATP was washed out with the 2 mM Ca2+-containing solution. The duration of exposure to the 2 mM Ca2+-containing solution, the Ca2+-free solution, and ATP is indicated by the filled, open, and hatched bars, respectively, above each trace. B, time courses of conductance increase in the process of nystatin perforation after the formation of gigaohm seals on a vector- (upper trace) and a TRP7-transfected (lower trace) cell. C, current-voltage relationships of the TRP7 channel. The difference current trace 8-(5, 6) was yielded by subtracting the average of the currents evoked by the voltage ramps numbered 5 and 6 in A from the current numbered 8. The difference current traces 9-(1-4), 11-(1-4), and 13-(1-4) were yielded by subtracting the average of the currents numbered 1-4 from the currents 9, 11, and 13, respectively. D, time courses of ionic currents recorded from TRP7-transfected cells at a holding potential of 60 mV. In the nominally Ca2+-free solution, 1 µM calphostin C, a PKC inhibitor (upper trace), or 250 nM phorbol 12,13-dibutyrate, a PKC activator (lower trace), was applied to the cell. In the upper trace, 100 µM OAG, a diacylglycerol analogue was further added to the external solution. The duration of exposure to calphostin C or phorbol 12,13-dibutyrate was indicated by the solid line above each trace, and the duration of exposure to OAG by the hatched bar above the upper trace.

The amplitude of spontaneous inward current in the Ca²⁺-free, Na⁺-containing solution was reduced to 48 ± 3% (n = 6), 13 ± 2% (n = 4), 27 ± 1% (n = 3), and 18 ± 1% (n = 3) of the control level at 60 mV, by extracellularly applied 1.2 mM Mg²⁺, 2.0 mM Ca²⁺, 25 µM SK&F96365, and 300 µM amiloride, respectively. Similar extents of inhibition by Ca²⁺ (2 mM) and SK&F96365 (25 µM) were observed for ATP-induced inward current (12 ± 4%, n = 5, and 39 ± 4% of control, n = 2, respectively). The blocking effect of SK&F96365 was more significant at positive than at negative membrane potentials (data not shown). In control cells, neither spontaneous or ATP-induced inward currents were detectable (3 out of 3 cells) (Fig. 8, A and B, upper trace).

Fig. 8C demonstrates the current-voltage relationships examined using rising voltage ramps from 120 to 80 mV for 2 s in the same TRP7-transfected cell as shown in Fig. 8A. The averages of spontaneous inward currents obtained by voltage ramps in 2 mM Ca²⁺-containing (1-4 in Fig. 8A) and Ca²⁺-free solution (5 and 6 in Fig. 8A) were subtracted from the currents after ATP application in the corresponding solutions (9, 11, and 13, and 7 and 8 in Fig. 8A, respectively). The current-voltage relationships of difference currents evaluated in this way were almost linear, showing a slight flattening approximately between 0 and 20 mV and outward rectification at more positive potentials. The reversal potential of difference currents did not significantly shift as the ATP-induced inward current increased in 2 mM Ca²⁺-containing solution (2.0 to 2.8 mV; compare 9-(1-4), 11-(1-4), and 13-(1-4)).

The effects of the lipid mediators observed in Mn²⁺ influx measurements were reproducible in the electrophysiological current recordings. Responsiveness of TRP7 currents to OAG treatment (Fig. 8D, upper trace) and ATP receptor stimulation (data not shown) was not impaired by a PKC inhibitor calphostin C (1 µM) (54). In addition, a PKC activator phorbol 12,13-dibutyrate (250 nM) significantly reduced spontaneous TRP7 inward currents (Fig. 8D, lower trace).

Divalent cations, Ca²⁺, Ba²⁺, and Mn²⁺ permeate the TRP7 channels responsible for the spontaneous and ATP-enhanced inward currents. When the external solution was changed to that containing one of these divalent cations (10 mM) and NMDG⁺ (substituted for the rest of cations), a small but measurable magnitude of inward currents was observed at 30 mV under both unstimulated (1.7 ± 0.5 pA in 10 mM Ca²⁺, n = 7; 4.3 ± 1.3 pA in 10 mM Ba²⁺, n = 6; 1.0 ± 0.3 pA in 10 mM Mn²⁺, n = 5) and stimulated conditions with ATP (13.3 ± 2.7 pA in 10 mM Ca²⁺, n = 4; 16.0 ± 4.9 pA in 10 mM Ba²⁺, n = 3; 5.0 ± 2.0 pA in 10 mM Mn²⁺, n = 2), in TRP7-expressing cells.

In order to determine the cationic selectivity of spontaneous and ATP-enhanced inward currents, we next calculated their reversal potentials (E_rev) from the current-voltage relationships measured in the Ca²⁺-free, Cs⁺-containing solution, the Ca²⁺-free, Na⁺-containing solution, and the solution containing 10 mM Ca²⁺ or Ba²⁺. Relative permeability ratios were calculated as follows using Equations 1 and 2 derived from the Goldman-Hodgkin-Katz equations for the biionic conditions.

(Eq. 1)

and

(Eq. 2)

where E_rev is a change in the reversal potential, (E_rev(Na⁺ or X²⁺)  E_rev(Cs⁺)), and X²⁺ is either Ca²⁺ or Ba²⁺; F is the Faraday constant; R is the gas constant, and T is the absolute temperature. The reversal potentials of spontaneous currents were 1.2 ± 0.4 (n = 9), 1.9 ± 0.5 (n = 10), 23.8 ± 2.0 (n = 7), and 11.1 ± 0.7 (n = 10) mV, and those of ATP-enhanced currents were 0.67 ± 0.2 (n = 4), 4.1 ± 0.8 (n = 2), 2.3 ± 0.7 (n = 4), and 5.0 ± 0.6 mV (n = 3), in the Cs⁺-, Na⁺-, 10 mM Ca²⁺-, and 10 mM Ba²⁺-containing solutions, respectively. The calculated relative permeabilities (P_Cs:P_Na:P_Ca:P_Ba) were 1:1.0:1.9:3.5 for the spontaneous current and 1:1.1:5.9:5.0 for the ATP-enhanced current.

Previous reports have identified intracellular Ca² as an important positive regulator inactivation of TRP3 (33) and TRP5 (25). To investigate the dependence of TRP7 channel activity on [Ca²⁺]_i, pipette solutions with various free Ca²⁺ concentration were used in the whole-cell mode of patch clamp recording. After development of spontaneous inward currents in nystatin-perforated patch recordings, patched membranes were broken in to establish the whole-cell configuration. After break-in of membranes, as the pipette solution containing 10 mM BAPTA (calculated free Ca²⁺ concentration was <1 nM (55)) was intracellularly perfused, constitutively activated inward currents in TRP7-expressing cells at a holding potential of 60 mV were suppressed time-dependently (Fig. 9, A, upper trace, and B). Similar but weaker suppression of TRP7 currents was observed when the pipette solution containing 10 mM BAPTA and 3 mM Ca²⁺ (54 nM calculated free Ca²⁺ concentration) was used (Fig. 9B). In contrast, intracellular perfusion of the pipette solution containing 10 mM BAPTA and 5 mM Ca²⁺ (125 nM calculated free Ca²⁺ concentration) potentiated TRP7-mediated inward currents (Fig. 9A, lower trace and 9B). The current-voltage relationships of these potentiated currents were similar to those of ATP-enhanced TRP7 currents in Fig. 8 (data not shown). We also used Ca²⁺ ionophore ionomycin to increase [Ca²⁺]_i. However, application of 100 µM ionomycin in the presence of 2 mM extracellular Ca²⁺ did not augment but rather slightly suppressed inward currents in TRP7-expressing cells (data not shown).

View larger version (37K): [in this window] [in a new window]   Fig. 9.   Effect of [Ca2+]i on constitutive and ATP-induced, TRP7-mediated current characterized by whole-cell patch clamp recording. A, time courses of ionic currents recorded from TRP7-transfected cells at a holding potential of 60 mV. The external solution was the nominally Ca2+-free solution supplemented with 1.2 mM Mg2+. After the chemical perforation by nystatin was completed and an inward cationic current reached a steady level, a brief negative pressure was given into the pipette to establish the whole-cell configuration by monitoring capacitative surges elicited by short small depolarizing pulses (see e.g. vertical deflections in A marked by arrowheads). The pipette solution contained 10 mM BAPTA (upper trace) or 10 mM BAPTA/5 mM Ca2+ (lower trace). B, currents that reached steady levels after intracellular perfusion with the pipette solution containing 10 mM BAPTA, 10 mM BAPTA/3 mM Ca2+, or 10 mM BAPTA/5 mM Ca2+, normalized by currents measured in the nystatin-perforated configuration. Data columns are the means ± S.E. normalized current in 4 or 5 cells. C, time courses of ionic currents induced by 100 µM ATP recorded from TRP7-transfected cells perfused intracellularly with the pipette solution containing 10 mM BAPTA (upper trace) or 10 mM BAPTA/5 mM Ca2+ (lower trace). The extracellular solution was the nominally Ca2+-free solution. D, ATP-induced currents measured by whole-cell patch clamp recordings using the pipette solution containing 10 mM BAPTA, 10 m