Receptor-mediated Regulation of the Nonselective Cation Channels TRPC4 and TRPC5*

Mammalian transient receptor potential channels (TRPCs) form a family of Ca2+-permeable cation channels currently consisting of seven members, TRPC1–TRPC7. These channels have been proposed to be molecular correlates for capacitative Ca2+ entry channels. There are only a few studies on the regulation and properties of the subfamily consisting of TRPC4 and TRPC5, and there are contradictory reports concerning the possible role of intracellular Ca2+ store depletion in channel activation. We therefore investigated the regulatory and biophysical properties of murine TRPC4 and TRPC5 (mTRPC4/5) heterologously expressed in human embryonic kidney cells. Activation of Gq/11-coupled receptors or receptor tyrosine kinases induced Mn2+ entry in fura-2-loaded mTRPC4/5-expressing cells. Accordingly, in whole-cell recordings, stimulation of Gq/11-coupled receptors evoked large, nonselective cation currents, an effect mimicked by infusion of guanosine 5′-3-O-(thio)triphosphate (GTPγS). However, depletion of intracellular Ca2+ stores failed to activate mTRPC4/5. In inside-out patches, single channels with conductances of 42 and 66 picosiemens at −60 mV for mTRPC4 and mTRPC5, respectively, were stimulated by GTPγS in a membrane-confined manner. Thus, mTRPC4 and mTRPC5 form nonselective cation channels that integrate signaling pathways from G-protein-coupled receptors and receptor tyrosine kinases independently of store depletion. Furthermore, the biophysical properties of mTRPC4/5 are inconsistent with those ofI CRAC, the most extensively characterized store-operated current.

Mammalian homologues of the Drosophila cation channel TRP form a novel gene family within the superfamily of cation channels with six transmembrane segments (1). A first hint as to how mammalian TRPCs 1 could be regulated came from the Drosophila visual signaling cascade. In Drosophila rhab-domeres, illumination is followed by G-protein-mediated activation of phospholipase C (PLC) and results in the stimulation of a light-induced current that is generated by TRP and TRPL (TRP-like) cation channels (2)(3)(4). The light-induced current may integrate several functions, including (i) depolarization of the membrane and generation of a receptor potential, (ii) augmentation of Ca 2ϩ -dependent signaling processes, and (iii) replenishment of internal calcium stores. Thus, mammalian homologues may serve similar functions.
In mammalian cells, activation of PLCs by extracellular signaling molecules leads to the production of inositol 1,4,5trisphosphate (InsP 3 ) and diacylglycerol and couples to intracellular signaling cascades by increasing the cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ). These changes in [Ca 2ϩ ] i result from InsP 3 -mediated Ca 2ϩ release from intracellular stores and/or Ca 2ϩ entry from the extracellular space. Ca 2ϩ entry may be regulated by different mechanisms. In many cell types, depletion of intracellular Ca 2ϩ stores following Ca 2ϩ release or inhibition of Ca 2ϩ uptake leads to Ca 2ϩ entry from the extracellular space, a process often called capacitative calcium entry that is mediated by store-operated channels (5,6). The best characterized current through store-operated channels is the calcium release-activated Ca 2ϩ current (I CRAC ), but other, less Ca 2ϩ -selective channels have also been described (7). Other Ca 2ϩ entry mechanisms downstream of receptor activation are mediated by second messengers, but independently of store depletion.
There is good agreement that TRPCs are activated downstream of G-protein-coupled receptors, which induce PLC-mediated phosphoinositide breakdown. However, the downstream signaling pathways that finally activate TRPCs remain highly controversial. For nearly all of the functionally expressed TRPCs, there is at least one report proposing a store-operated mechanism of activation (1, 8 -13). On the other hand, there is growing evidence for the involvement of store-independent pathways in the regulation of TRPC3 (14 -17), TRPC5 (18), TRPC6 (17,19), and TRPC7 (20). For TRPL, TRPC3, and TRPC6, direct activators have been identified that stimulate the channels in a membrane-confined manner. Polyunsaturated fatty acids were shown to gate Drosophila TRPL (21) and diacylglycerols to activate TRPC3, TRPC6, and TRPC7 (17,20).
Phylogenetic analysis of TRPCs revealed two subfamilies, namely the TRPC3/6/7 subfamily and the TRPC4/5 subfamily. From the close structural relationship of TRPC4 and TRPC5, similar regulatory and biophysical properties might be expected. Indeed, both bovine TRPC4 and rabbit TRPC5 have been reported to form store-operated, relatively Ca 2ϩ -selective channels with an inwardly rectifying current-voltage relation (9,10). Despite relatively large whole-cell currents in cells expressing TRPC4, single-channel openings could not be resolved in cell-attached patches, suggesting that the singlechannel conductance might be low (9). Similarly, rat TRPC4 has been reported to mediate store-operated Ca 2ϩ entry when expressed in Xenopus oocytes (22). In contrast, however, mouse TRPC5 has been convincingly shown to be activated following receptor stimulation, but not by store depletion (18). Thus, the available data on TRPC4/5 cannot be integrated into a consistent model of signaling pathways leading to activation of these channels.
To study the activation mechanism and biophysical properties of TRPC4 and TRPC5, we cloned murine TRPC4 and TRPC5 (mTRPC4/5) and functionally expressed them in human embryonic kidney (HEK) cells. We provide evidence that mTRPC4 and mTRPC5 are nonselective cation channels regulated independently of the filling state of intracellular Ca 2ϩ stores. Signaling pathways of G-protein-coupled receptors and receptor tyrosine kinases converge to stimulate mTRPC4/5, presumably downstream of phospholipases C. The regulatory and biophysical properties indicate that mTRPC4 and mTRPC5 form a novel subfamily of receptor-stimulated, nonselective cation channels predominantly expressed in neuronal tissues.

EXPERIMENTAL PROCEDURES
Molecular Biology-TRPC4 and TRPC5 were isolated from mouse brain total RNA by specifically primed reverse transcription (Super-Script II, Life Technologies, Inc.) and polymerase chain reaction (Expand-HF, Roche Molecular Biochemicals, Mannheim, Germany). The primers for polymerase chain reaction were 5Ј-AGCATGGCTCAGTTC-TATTACAAA (mTRPC4 sense), 5Ј-CAACGGTGAAAGCAAAAGCAG (mTRPC4 reverse), 5Ј-ACCATGGCCCAGCTGTACTACAAG (mTRPC5 sense), and 5Ј-GATGACGGGGATTTGACTTAGAG (mTRPC5 reverse). The amplified cDNAs were subcloned in a eucaryotic TA-TOPO cloning system (Invitrogen, Groningen, The Netherlands). Several clones containing the open reading frames of mTRPC4 or mTRPC5 were functionally expressed by intranuclear microinjection in CHO-K1 cells. Positive clones were selected for their ability to enhance Mn 2ϩ entry in fura-2loaded cells when stimulated with 100 M ATP. By sequencing the positive clones, mTRPC5-4 was confirmed to be the wild-type mTRPC5. Two submitted sequences for mTRPC5 differ at amino acid 303. Our clones confirm the alanine (GenBank TM /EBI Data Bank accession number AJ006204) rather than a valine (AF029983). Clone mTRPC4-L4 was mTRPC4B (U50921) with one point mutation leading to the exchange C176R. Wild-type mTRPC4B was obtained by exchanging the mutated fragment for the corresponding part of clone mTRPC4-L6. Sequencing was done on a ABI Prism 377 DNA sequencer.
Enhanced green fluorescent protein (eGFP) was fused to the C terminus of mTRPC4 in a custom-made pcDNA3-EGFP vector lacking the start codon and the translation initiation sequence of eGFP. mTRPC4 and hTRPC6 were fused to eGFP by in-frame ligation of BamHI/XhoIdigested mTRPC4 or EcoRI/NcoI-digested hTRPC6 into pcDNA3-EGFP. The replacement of 21 (mTRPC4) or 9 (hTRPC6) C-terminal amino acids by eGFP did not affect the regulatory and biophysical properties of the respective channel. The stop codon of mTRPC5 was replaced by an XhoI cutting site by polymerase chain reaction-based mutagenesis. In-frame ligation of a NdeI/XhoI fragment into pcDNA3-EGFP resulted in a cDNA encoding mTRPC5 C-terminally fused to eGFP. The amino acid sequences of fusion proteins were confirmed by DNA sequencing. The functional properties of mTRPC5-eGFP were the same as those of wild-type mTRPC5.
Cell Culture and Transient Transfection-CHO-K1 cells were maintained as described previously (17). HEK 293 cells (American Type Culture Collection, Manassas VA) were maintained according to the supplier's recommendations. Cells were transiently transfected with a transfection reagent (Fugene 6, Roche Molecular Biochemicals) and subcultured on glass coverslips. The cDNA (4 g) of the respective channel or the expression vector without insert was supplemented with 40 -120 ng of pEGFP-C1 (CLONTECH, Palo Alto, CA). For experiments on excised patches, cells were seeded on poly-L-lysine-coated coverslips. The experiments were performed on HEK cells 2-3 days after transfection.
Fluorescence Imaging-Transfected HEK cells were loaded for 30 min with fura-2/AM (2-4 M; Molecular Probes, Inc., Eugene, OR) in HEPES-buffered saline (pH 7.4) containing 135 mM NaCl, 6 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 5.5 mM glucose, 10 mM HEPES, and 0.2% (w/v) bovine serum albumin. Coverslips were mounted in a monochromatorequipped (Polychrome II, TILL-Photonics, Martinsreid, Germany) inverted microscope (Axiovert 100, Carl Zeiss, Göttingen, Germany). Fluorescence was recorded with a 12-bit CCD camera (IMAGO, TILL-Photonics). The fluorescence of fura-2 and eGFP was excited at 340, 358, 380, and 480 nm and filtered through a 516-nm long-pass filter. Fura-2 was not excited at 480 nm. However, eGFP contributed to fluorescence excited at 340 -380 nm. By transfecting low amounts of reporter cDNA, eGFP-derived fluorescence was minimized. An additional correction was performed: the fluorescence of eGFP relative to the intensity at the 480-nm excitation wavelength was 9.3% at 380 nm, 4.5% at 358 nm, and 1.4% at 340 nm. After subtraction of background signals, eGFP-derived fluorescence at 340 -380 nm was estimated from the fluorescence intensities at 480 nm and subtracted from the respective data sets. R max , R min , and F 380(min) /F 380(max) were determined by equilibrating fura-2-loaded HEK cells in HEPES-buffered saline containing 10 M ionomycin and either 10 mM Ca 2ϩ or 10 mM EGTA. Intracellular Ca 2ϩ concentrations were calculated as described (23). GFP fusion proteins were visualized with an LSM510 confocal laser scanning microscope (Carl Zeiss) and a Plan-Apochromat 63ϫ/1.4 objective. eGFP was excited with the 488-nm line of an argon laser. Emitted light was filtered through a 505-nm long-pass filter, and pinhole diameters were set to yield optical sections of ϳ0.6 m.
Statistical Methods-In each imaging experiment, data of 30 -80 individual cells were sampled. After averaging the data of each single experiment, the means of five to eight independent experiments were calculated and expressed as mean Ϯ S.E.. The kinetics of fura-2 quench by Mn 2ϩ was quantified after averaging the changes in F 358 over 5-s intervals and expressed as percentages over time. Mean basal and stimulated [Ca 2ϩ ] i as well as quench of fura-2 fluorescence were compared by Student's t test of unpaired data. Significance was accepted at p Ͻ 0.05. In Mn 2ϩ quench experiments in which thapsigargin was applied, the intra-assay S.D. is indicated by vertical lines on the mean values of F 358 and [Ca 2ϩ ] i .
Electrophysiological Techniques-For whole-cell experiments, the standard extracellular solution contained 140 mM NaCl, 5 mM CsCl, 2 mM CaCl 2 , 1 mM MgCl 2 , 10 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). For Na ϩ -free solutions, Na ϩ was replaced by N-methyl-D-glucamine (NMDG ϩ ). For Ca 2ϩ -free solutions, Ca 2ϩ was replaced by 1 mM EGTA. The standard intracellular solution contained 110 mM cesium methanesulfonate, 25 mM CsCl, 2 mM MgCl 2 , 0.362 mM CaCl 2 , 1 mM EGTA, and 30 mM HEPES (pH 7.2 with CsOH) with a calculated [Ca 2ϩ ] of 100 nM. In some experiments, Ca 2ϩ -free (10 mM EGTA) solutions or solutions with a higher Ca 2ϩ buffer capacity (3.62 mM CaCl 2 and 10 mM EGTA) were used. For experiments with 1 and 10 M Ca 2ϩ , the solutions contained 0.851 mM CaCl 2 and 1 mM EGTA, and 0.098 mM CaCl 2 and 1 mM HEDTA, respectively. Whole-cell recordings were made with an EPC-7 amplifier using Pulse software (HEKA, Lambrecht, Germany). Patch pipettes, made of borosilicate glass, had resistances of 3-5 megaohms when filled with the standard intracellular solutions. Cells were held at a potential of Ϫ60 mV, and current-voltage (I-V) relations were obtained from voltage ramps from Ϫ100 to ϩ100 mV with a duration of 400 ms applied at a frequency of 0.2 Hz. Ramp data were acquired at a frequency of 4 kHz after filtering at 1 kHz. The holding current was acquired at 30 Hz. Series resistance compensation of Ն50% was used in most experiments. Fluctuation analysis was done as described (24). Briefly, the single-channel current (i) and total number of channels in the patch (N) were estimated by fitting the equation 2 ϭ iI Ϫ I 2 /N to plots of current variance ( 2 ) against mean current amplitude (I). Single-channel experiments were performed as described previously (17). Bath solutions (pH 7.4) contained 120 mM sodium isethionate, 2 mM magnesium gluconate, 0.575 mM calcium gluconate, 1 mM EGTA, 10 mM glucose, and 10 mM HEPES (solution B1); 120 mM CsCl, 0.62 mM calcium gluconate, 2 mM HEDTA, and 20 mM HEPES (solution B2); and 120 mM CsCl, 2 mM magnesium gluconate, 1.87 mM calcium gluconate, 2 mM EGTA, and 20 mM HEPES (solution B3). The pipette solution (pH 7.4) contained 120 mM CsCl, 1 mM magnesium gluconate, 1.8 mM calcium gluconate, and 10 mM HEPES (solution P1). All solutions were adjusted to 290 -310 mosmol/liter with mannitol. In experiments in which chloride in the bath solution was substituted by isethionate, the ground electrode was connected to the bath through an agar bridge. Unless otherwise indicated, single-channel data were filtered at 2.5 kHz and analyzed with pCLAMP7 software (Axon Instruments, Inc., Foster City, CA). Channel activity is expressed as NP o , the product of the number (N) of channels in the patch and the open probability (P o ) calculated for consecutive 5-s periods. For analysis of mean open time, currents were filtered at 5 kHz and sampled at 50 kHz. Openings with durations shorter than 0.1 ms were excluded from the analysis.

Subcellular Localization of mTRPC4 and mTRPC5 Fused to
Green Fluorescent Protein-To visualize the subcellular localization of the presumptive cation channels in living HEK cells, cDNA constructs encoding mTRPC4, mTRPC5, and hTRPC6 C-terminally fused to eGFP were generated. All fusion proteins were functional channels with regulatory and biophysical properties indistinguishable from the respective channels without eGFP (data not shown). The subcellular distribution of mTRPC4-eGFP and mTRPC5-eGFP was followed by confocal fluorescence microscopy and compared with that of hTRPC6-eGFP. The most prominent mTRPC4-eGFP and hTRPC6-eGFP signals were found in the plasma membrane, whereas a considerable amount of mTRPC5-eGFP was retained in a perinuclear compartment, probably the Golgi apparatus (Fig. 1E). In the plasma membrane, mTRPC4-eGFP and mTRPC5-eGFP showed a clustered appearance, thus contrasting sharply with the homogeneous membrane expression pattern of hTRPC6-eGFP ( Fig. 1, C, F, and I). In HEK cells, a similar punctate distribution pattern has been described for correctly targeted L-type calcium channels (25). To discriminate between Ca 2ϩ release from intracellular stores and Ca 2ϩ entry from the external medium, the quench of fura-2 fluorescence by extracellularly applied Mn 2ϩ was monitored at the isosbestic wavelength. Following the addition of Mn 2ϩ to the bath, a significant acceleration of the basal decrease in fura-2 fluorescence was observed only in mTRPC5expressing cells (Ϫ0.1 Ϯ 0.03%/s, n ϭ six independent experi-ments), but not in pcDNA3-transfected (Ϫ0.02 Ϯ 0.02%/s, n ϭ 5) or mTRPC4-transfected (Ϫ0.04 Ϯ 0.01%/s, n ϭ 8) cells. The addition of carbachol resulted in an immediate decrease in fura-2 fluorescence in mTRPC4-expressing (Ϫ4.6 Ϯ 0.3%/s, 308 cells, n ϭ 8) and mTRPC5-expressing (Ϫ2.5 Ϯ 0.6%/s, 278 cells, n ϭ 6) cells, but not in control cells (Ϫ0.16 Ϯ 0.08%/s, 334 cells, n ϭ 5) (Fig. 2, D-F). At a higher temporal resolution, a significant stimulation of Mn 2ϩ influx in mTRPC4-expressing cells was detectable within 50 -200 ms after the initial increase in [Ca 2ϩ ] i (data not shown). The kinetics of Mn 2ϩ influx indicated a transient increase in membrane permeability. Increases in [Ca 2ϩ ] i and Mn 2ϩ influx like those obtained in HEK cells were also observed in CHO-K1 cells expressing mTRPC4 and mTRPC5 following activation of a coexpressed histamine H 1 receptor or an endogenous P2Y purinoreceptor.
Because some TRPCs have been reported to mediate capacitative Ca 2ϩ entry, we tested whether mTRPC4 and mTRPC5 are regulated by the filling state of intracellular Ca 2ϩ stores. Passive depletion of internal Ca 2ϩ stores by thapsigargin (2.5 M) strongly reduced or abolished carbachol-induced [Ca 2ϩ ] i transients in control cells in the presence (Figs. 3 and 4A) and absence (data not shown) of extracellular Ca 2ϩ . Re-addition of Ca 2ϩ after store depletion resulted in larger increases in [Ca 2ϩ ] i in mTRPC4-expressing cells than in pcDNA3-transfected control cells (Fig. 3). However, the same cells already displayed higher recalcification signals prior to thapsigargin treatment. Mn 2ϩ entry in carbachol-stimulated, mTRPC4/5expressing cells was not mimicked by depletion of internal Ca 2ϩ stores with thapsigargin (Fig. 4B). When carbachol was applied after thapsigargin treatment, mTRPC4/5-expressing (but not pcDNA3-transfected) cells responded with a transient increase in [Ca 2ϩ ] i (Fig. 4A) and a pronounced increase in Mn 2ϩ influx (Fig. 4B). Interestingly, the thapsigargin-induced [Ca 2ϩ ] i signal was strongly reduced in mTRPC4-expressing cells and was almost abolished in mTRPC5-expressing cells (Fig. 4A).
In serum-deprived HEK cells, application of epidermal growth factor (EGF; 50 ng/ml) resulted in a transient, genistein-sensitive increase in [Ca 2ϩ ] i . The response to EGF was detectable in up to 90% of the cells. In mTRPC4-and mTRPC5expressing cells, the increase in [Ca 2ϩ ] i was accompanied by Mn 2ϩ influx. In a subpopulation of cells that did not respond to EGF with a [Ca 2ϩ ] i transient, Mn 2ϩ entry was also absent and could be induced only by the subsequent addition of carbachol (Fig. 5A). In the presence of genistein (10 M), EGF-induced (but not carbachol-induced) Mn 2ϩ influx was abolished. Pretreatment of cells with the phospholipase C inhibitor U73122 (10 M for 5 min) abolished both the EGF-and carbacholinduced stimulation of Mn 2ϩ influx in mTRPC4-expressing (Fig. 5B) and mTRPC5-expressing (data not shown) cells. The inactive analogue of U73122, U73343, prevented the activation of neither mTRPC4 (Fig. 5A) nor mTRPC5 (data not shown) by EGF or carbachol. Other blockers that did not affect the stimulation of mTRPC4/5 by carbachol include the sphingosine kinase inhibitor dihydrosphingosine (30 M), the serine/threonine kinase inhibitors staurosporine (10 M) and bisindolylmaleimide I (1 M), the tyrosine kinase inhibitor genistein (10 M), the phosphatase inhibitor sodium orthovanadate (1 mM), the phosphatidylinositol 3-kinase inhibitor wortmannin (10 M), the cyclooxygenase and phospholipase A 2 inhibitor indomethacin (up to 300 M), and the diacylglycerol kinase inhibitor RHC-80267 (50 M). The membrane-permeant protein kinase C activators phorbol 12-myristate 13-acetate (1-10 M), phorbol didecanoate (1-10 M), oleoylacetyl-sn-glycerol (up to 300 M), dioctanoylglycerol (up to 100 M), and l-monooleoyl-rac-glycerol (300 M) did not activate mTRPC4/5. Because polyunsaturated fatty acids activate Drosophila TRPL (20), we tested the effects of arachidonic, linoleic, linolenic, and oleic acids on mTRPC4/5. At concentrations up to 300 M, none of these compounds stimulated Mn 2ϩ entry through mTRPC4/5 or prevented the channels from being activated by carbachol. A minor (Ͻ2-fold) and delayed acceleration of Mn 2ϩ entry into pcDNA3-transfected or mTRPC4/5-expressing cells was promoted by arachidonic acid and may reflect another non-capacitative Ca 2ϩ entry mechanism recently described for HEK cells (26).
The activation of either endogenous muscarinic receptors or coexpressed histamine H 1 receptors by the respective addition of carbachol or histamine (100 M) to the extracellular solution resulted in the rapid, transient activation of an inward current at a holding potential of Ϫ60 mV in mTRPC4-or mTRPC5expressing cells (Fig. 6, A and B). In some pcDNA3-transfected control cells, a small inward current (Ϫ3.8 Ϯ 1.5 pA/pF, n ϭ 7; C m ϭ 10.8 Ϯ 0.6 pF) was activated by carbachol. However, this did not resemble the currents of mTRPC4 and mTRPC5. In cells expressing mTRPC4 or mTRPC5, the current amplitudes were highly variable, but reached peak values of several nanoamperes in many experiments (Fig. 6E). At Ϫ60 mV, the mean peak inward current densities in response to carbachol (100 M) were Ϫ232.0 Ϯ 75.3 pA/pF (n ϭ 13; C m ϭ 13.8 Ϯ 0.4 pF) and Ϫ228.1 Ϯ 57.1 pA/pF (n ϭ 10; C m ϭ 13.7 Ϯ 0.9 pF) for mTRPC4-and mTRPC5-expressing cells, respectively. Exchange of the extracellular solution for a Na ϩ -free (NMDG ϩ ) or Na ϩ /Ca 2ϩ -free solution (NMDG ϩ and EGTA) almost completely abolished the inward current. The I-V relations of the agonist-induced currents had a characteristic doubly rectifying form and displayed reversal potentials close to 0 mV (Fig. 6, C  and D). Currents with identical properties were measured in CHO-K1 cells expressing mTRPC4/5 after activation of coexpressed histamine receptors.
Effects of Store Depletion Protocols and Inositol Phosphates-To test for a role of intracellular store depletion in the activation of mTRPC4/5, protocols were used that have previously been shown to activate store-operated channels (27). Infusion of InsP 3 (10 -100 M) in a Ca 2ϩ -free 10 mM EGTA pipette solution resulted in no increase in holding current in cells expressing mTRPC4 (n ϭ 11) and a slight, slow increase in current at Ϫ60 mV (approximately a doubling over 5 min) in most mTRPC5-transfected cells showing spontaneous activity (n ϭ 7). Similar results were obtained with InsP 3 in solutions containing 100 nM free Ca 2ϩ buffered with 1 (n ϭ 3 and 7 for mTRPC4 and mTRPC5, respectively) or 10 (n ϭ 7 for TRPC4/5) mM EGTA (Fig. 7A). In Jurkat T lymphocytes, infusion of the latter solution fully activated I CRAC within 30 -60 s after patch rupture (data not shown). Slow increases in holding current in mTRPC5-expressing cells showing spontaneous activity were also observed using the same pipette solutions without InsP 3 . After 5 min of InsP 3 infusion, the effect of agonist application on whole-cell currents was tested. No response to carbachol or histamine was observed in six cells expressing mTRPC4 using a Ca 2ϩ -free intracellular solution with 10 mM EGTA. With the same solution in mTRPC5-expressing cells, three out of seven cells responded to agonist. In a solution with 10 mM EGTA and 100 nM free Ca 2ϩ , three out of seven cells expressing mTRPC4 and four out of seven cells expressing mTRPC5 responded. With 1 mM EGTA and 100 nM free Ca 2ϩ , three out of three cells expressing mTRPC4 and six out of seven cells expressing mTRPC5 responded to agonist application. The addition of thapsigargin (1 M) to the extracellular solution during wholecell recording did not result in an activation of mTRPC4 (n ϭ 4) or mTRPC5 (n ϭ 5) with an intracellular solution containing 10 mM EGTA and no Ca 2ϩ (Fig. 7B). In cells showing spontaneous mTRPC5 activity, thapsigargin application frequently caused a slight decrease in the current at Ϫ60 mV (Fig. 7B). Channel activation by carbachol was unaffected by infusion of the InsP 3 receptor antagonist heparin (5 mg/ml) for 5 min prior to carbachol application (n ϭ 5 for both channels). Since InsP 4 has been reported to activate cation channels in endothelial cells (28), we investigated the effects of InsP 4 (10 M) at [Ca 2ϩ ] ϭ 100 nM and 10 M. At 100 nM Ca 2ϩ , no mTRPC4/5 currents were observed during InsP 4 infusion into cells that subsequently responded to carbachol (n ϭ 4 for both channels). Infusion of solutions with a higher [Ca 2ϩ ] (1 or 10 M) without InsP 4 produced a small, slow, transient stimulation of mTRPC4/5 immediately following break-in in many cells. In cells that later responded to carbachol, the responses to carbachol were 5-94-fold larger than those to Ca 2ϩ (n ϭ 7 for both channels). In two cells with 10 M Ca 2ϩ , however, larger, rapid responses, like those to carbachol or GTP␥S (see below), were observed. Similar results to those obtained with 1 or 10 M Ca 2ϩ alone were obtained after infusion of InsP 4 in 10 M Ca 2ϩ (n ϭ 5 for both channels).
Effects of GTP␥S Infusion on Whole-cell Currents-To determine whether activation of G-proteins can stimulate mTRPC4/5, GTP␥S (500 M) was added to the intracellular solution. With various delays following GTP␥S infusion, a slow initial phase of current increase was often followed by a larger transient current whose time course resembled that seen after receptor stimulation (Fig. 8, A and B). During the initial phase of activation, single-channel events with an amplitude of around Ϫ2 pA could be resolved in a large number of cells. The maximum inward current densities at Ϫ60 mV observed during GTP␥S infusion were Ϫ108.6 Ϯ 15.5 pA/pF (n ϭ 38; C m ϭ 13.9 Ϯ 0.5 pF) for mTRPC4 and Ϫ63.4 Ϯ 7.8 pA/pF (n ϭ 38; C m ϭ 14.1 Ϯ 0.9 pF) for mTRPC5. The I-V relations for mTRPC4 and mTRPC5 close to maximum GTP␥S stimulation (Fig. 8, C and D) were indistinguishable from those obtained during receptor stimulation of the channel currents (Fig. 6, C and D). For mTRPC4, the current decayed rapidly and completely, whereas for mTRPC5, decay was incomplete, and a very low level of activity was maintained. Like receptor-activated currents, GTP␥S-activated inward currents were abolished in Na ϩ -free or Na ϩ /Ca 2ϩ -free bathing solutions. Current activation by GTP␥S was associated with a clear increase in current noise. Using fluctuation analysis, the estimated values of singlechannel currents at Ϫ60 mV were Ϫ1.6 Ϯ 0.1 pA for mTRPC4 and Ϫ1.8 Ϯ 0.2 pA for mTRPC5 (n ϭ 4 for both channels).
Because currents following GTP␥S stimulation decayed less rapidly than agonist-induced currents, we determined the ion selectivity under these conditions. Reversal potentials were estimated from current ramps during the decaying phase of the current. The extracellular solution was first changed from the standard solution to a Na ϩ -containing, Ca 2ϩ -free solution and then to a Ca 2ϩ -containing (10 mM), Na ϩ -free (replaced by NMDG ϩ ) solution. The mean reversal potentials were Ϫ1.5 Ϯ 1.5 mV in Na ϩ alone and Ϫ39.7 Ϯ 3.3 mV in 10 Ca 2ϩ -containing, Na ϩ -free solution (n ϭ 4) for mTRPC4 and Ϫ3.3 Ϯ 4.1 and Ϫ29.2 Ϯ 5.3 mV (n ϭ 5) in the respective solutions for mTRPC5 (Fig. 8, E and F). From the shifts of the reversal potentials, the P Ca /P Na values calculated using the Goldman equation for divalent and monovalent cations (29) were 1.05 for mTRPC4 and 1.79 for mTRPC5.
The activation of mTRPC4/5 by GTP␥S was dependent on extracellular Ca 2ϩ . No clear activation of mTRPC4 was observed during GTP␥S infusion in a Ca 2ϩ -free extracellular medium. The addition of Ca 2ϩ to the extracellular solution after 5 min of GTP␥S infusion resulted in a small increase in current with an I-V shape typical for mTRPC4. In contrast, infusion of GTP␥S into mTRPC5-expressing cells resulted in a clear, but small activation of current in the absence of extracellular Ca 2ϩ . Upon re-addition of Ca 2ϩ after 5 min of GTP␥S infusion, a large increase in current was observed. Interestingly, a similar result was reported for mTRPC5 following agonist activation (18). Raising the extracellular Ca 2ϩ concentration from 2 to 10 mM (by replacing Na ϩ ) close to the peak of current activation also potentiated the current response in mTRPC4/5-expressing cells.
One characteristic biophysical feature of I CRAC is a specific block by low micromolar concentrations of La 3ϩ . A surprising finding was that 100 M La 3ϩ did not inhibit, but, like 10 mM Ca 2ϩ , even potentiated GTP␥S-induced currents through mTRPC4/5 without changing the reversal potential. The addition of La 3ϩ to the extracellular solution after the peak of inward current led to large, rapid, and reversible current increases. Analysis of voltage ramps indicated that the current at both positive and negative membrane potentials was increased by La 3ϩ . Likewise, carbachol-induced Mn 2ϩ entry through mTRPC4 and mTRPC5 in fura-2-loaded HEK cells was not impaired by La 3ϩ at concentrations up to 300 M.
Activation and Properties of mTRPC4/5 in Membrane Patches-Since fluctuation analysis suggested that unitary currents through mTRPC4/5 should be resolvable, we looked for their presence in membrane patches. In cell-attached patches from HEK cells cotransfected with the histamine H 1 receptor and mTRPC4, little channel activity was observed prior to agonist application, whereas some activity was observed in patches from mTRPC5-expressing cells (Fig. 9A). The addition of histamine (80 M) to the extracellular solution resulted in transient 3-50-fold increases in channel activity in six out of eight patches from mTRPC4-expressing cells (Fig. 9A). For mTRPC5, seven out of nine patches responded to histamine with a 10 -200-fold increase in activity (Fig. 9A). No currents were observed in control cells transfected with pcDNA3 and the histamine H 1 receptor.
The properties of the unitary events were studied in detail in inside-out membrane patches. Following patch excision from mTRPC4-and mTRPC5-expressing cells, single-channel events with a low frequency of opening were observed at a patch potential of Ϫ60 mV. The addition of GTP␥S (10 M) to the internal membrane surface resulted in large increases in NP o (Fig. 9B) in 23 out of 29 patches for mTRPC4 (5-300-fold) and in 28 out of 32 patches for mTRPC5 (10 -1000-fold). The stimulatory effect of GTP␥S, although strongest in the first minutes after application, was maintained for the length of the recording (Fig. 9B). In all 14 patches isolated from pcDNA3-transfected cells, GTP␥S failed to activate single-channel events. Raising the [Ca 2ϩ ] from 100 nM to 10 M at the internal face of the membrane had a stimulatory effect in the absence of GTP␥S (up to 50-fold; n ϭ 5 for each channel). Under symmetrical buffer conditions (solution B3), the single-channel I-V relations for mTRPC4 and mTRPC5 closely resembled those for whole-cell currents, showing a doubly rectifying shape and a reversal potential close to 0 mV (Fig. 9C). The single-channel chord conductances at Ϫ60 mV were 41 Ϯ 1 (n ϭ 10) and 63 Ϯ 1 (n ϭ 7) picosiemens for mTRPC4 and mTRPC5, respectively. In an attempt to explain the doubly rectifying I-V relation, we tested the possibility that Mg 2ϩ blocks the channel. In symmetrical Mg 2ϩ -free solutions (solution B2), inward and outward currents through mTRPC4 and mTRPC5 channels were increased, and the I-V relation displayed slight outward rectification (Fig. 9D). In the absence of Mg 2ϩ , the chord conductances at Ϫ60 mV were 55 Ϯ 2 (n ϭ 9) and 88 Ϯ 2 (n ϭ

DISCUSSION
Based on structural homology, TRPC4 and TRPC5 form one branch of the phylogenetic tree of mammalian TRP homologues (1). We could demonstrate that mTRPC4 and mTRPC5 are not only structurally related, but also display similarities in their mechanism of regulation and their biophysical properties.
The activity of mTRPC4/5 is dependent on the activation of phospholipases C and on the presence of intracellular Ca 2ϩ , yet is independent of the filling state of internal Ca 2ϩ stores. Although the activation of mTRPC4 and mTRPC5 by GTP␥S in excised inside-out patches implicates activated G-proteins in channel activation, further evidence suggests that subsequent PLC activation is a necessary step. Carbachol-induced channel activation is prevented by the phospholipase C inhibitor U73122, and channels are activated following EGF receptor stimulation. The signaling pathways downstream of EGF receptor tyrosine kinase include phosphoinositide breakdown via PLC␥ independent of heterotrimeric G-proteins. We conclude that mTRPC4 and mTRPC5 are dually activated via PLC␤ or PLC␥. Since both signaling cascades converge at the level of phosphoinositide breakdown, it is likely that activation of mTRPC4/5 depends on this process. However, the steps leading to channel activation following PLC activation remain unclear. Consistent with a PLC-dependent (30, 31), but InsP 3 receptorindependent (32) activation of light-induced current in the Drosophila rhabdomere, our results indicate that InsP 3 is not involved in the pathways leading to activation of mTRPC4/5. None of the protocols used to deplete Ca 2ϩ stores resulted in increased Mn 2ϩ entry or activated cation currents. The sphingosine kinase inhibitor DL-threo-dihydrosphingosine was shown to block I CRAC in rat basophilic leukemia cells (33). Involvement of second messenger formation by sphingosine kinase was ruled out for mTRPC4/5 by the inability of DL-threodihydrosphingosine to prevent channel activation.
The lack of involvement of store depletion in the activation of TRPC4/5 is in conflict with a number of reports. Studies on bovine and rat TRPC4 and rabbit TRPC5 describe an activation by depletion of intracellular Ca 2ϩ stores with thapsigargin or InsP 3 infusion (9,10,22,34). However, receptor stimulation was not tested in these studies. In contrast, our data on the regulatory properties of both mTRPC4 and mTRPC5 showed activation following stimulation of G-protein-coupled receptors, but not after store depletion. In addition, InsP 3 infusion was without effect in cells expressing mTRPC4 or mTRPC5. Thus, channel gating by conformational coupling to InsP 3 receptors as suggested for TRPC3 (11) is unlikely for mTRPC4/5. Okada et al. (18) demonstrated that, after passively depleting the internal Ca 2ϩ stores with thapsigargin, recalcification did not increase Ca 2ϩ influx in mTRPC5-expressing cells compared with untransfected control cells. We confirmed the store-independent activation of mTRPC5 with the exception that mTRPC5-overexpressing cells displayed a higher basal Ca 2ϩ influx than control cells. A potentiation of [Ca 2ϩ ] i transients after re-addition of external Ca 2ϩ to thapsigargin-treated cells does not necessarily support a store-dependent mechanism of activation. It may also reflect basal activity of a Ca 2ϩ -permeable cation channel.
The properties of bovine TRPC4 have been reported to resemble those of store-operated channels like I CRAC (9,35). These include regulation by store depletion, a high selectivity for Ca 2ϩ , and an inwardly rectifying I-V relation. The inability to resolve single-channel events, despite relatively large wholecell currents, was taken as an indication that the single-channel conductance might be low (9), like that of I CRAC (35). In contrast, the present study shows that mTRPC4 and mTRPC5 form cation channels with similar permeabilities for Na ϩ , Cs ϩ , and Ca 2ϩ and respective single-channel conductances of 41 and 63 picosiemens in the presence of Mg 2ϩ . The I-V relations for both whole-cell and single-channel currents have a characteristic doubly rectifying shape and reversal potentials close to 0 mV. The conductance and the I-V relation shape of singlechannel currents depended on the extra-and intracellular Mg 2ϩ concentrations. In the absence of Mg 2ϩ on both sides of the membrane, double rectification was lost, and both channels showed outward rectification and higher chord conductances. Unitary currents through channels mediating I CRAC were observed only in the absence of extracellular divalent cations (36), whereas mTRPC4 and mTRPC5 were highly conductive also in the presence of divalent cations. Moreover, I CRAC is strongly inhibited by La 3ϩ at low micromolar concentrations, whereas currents through mTRPC4/5 were potentiated by La 3ϩ at concentrations up to 1 mM. Thus, the regulatory and biophysical hallmarks of mTRPC4/5 are clearly different from those of I CRAC .
The reasons for the marked differences in regulatory and biophysical properties between our data and those obtained with bovine TRPC4 (9) and rabbit TRPC5 (10) remain unclear. As clones were obtained from different species, there are variations in the primary sequences that might explain the obvious differences in functional properties. Most strikingly, sequence alignments of murine, bovine, and rat TRPC4 reveal a gap in the primary sequence of the rat orthologue (GenBank TM /EBI Data Bank accession number AB008889) that comprises the entire predicted second transmembrane segment. It remains to be clarified whether the deletion of this segment affects correct folding and the functional integrity of the protein. In addition, multiple frameshift mutations in the submitted sequence of rat TRPC4 as compared with murine and bovine TRPC4 make the results of functional characterization highly questionable. Nevertheless, rat TRPC4 was functionally characterized as a storeoperated channel by expression in Xenopus oocytes (22).
The TRPC3/6/7 subfamily of the TRPCs has been reported to be activated subsequent to PLC stimulation, either as a result of InsP 3 -mediated signaling cascades (11,12) or by the lipid second messenger diacylglycerol (17,20). In contrast to TRPC3 and TRPC6, no stimulation by diacylglycerols was evident for mTRPC4/5 (17), suggesting a different regulatory mechanism. Besides the differential regulation by diacylglycerols, TRPC3/6 and TRPC4/5 may be discriminated by biophysical differences. Openings of hTRPC3/6 were short, making open levels difficult to resolve. In contrast, longer open events were evident with mTRPC4/5. Furthermore, mTRPC4 and mTRPC5 were less selective for Ca 2ϩ over Na ϩ than hTRPC3/6. Both mTRPC4 and mTRPC5 are expressed in distinct areas of the brain (10,18,37). Although nonselective cation channels have been described in neurons of the regions where mTRPC4 and mTRPC5 are localized, their biophysical characterization is insufficient to allow a direct comparison with mTRPC4/5. Surprisingly, a current in native tissues with the clearest similarities, both in its regulation and functional properties, is one present in ileal smooth muscle (38). This channel is activated by acetylcholine through a G-protein and is also activated by GTP␥S infusion. It is nonselective, dependent on Ca 2ϩ for its activation, and potentiated by La 3ϩ (39). It remains to be seen whether this or other currents that have been described in native tissues are formed by TRPC4 or TRPC5. Considering that the TRPC4/5 and TRPC3/6/7 subfamilies are regulated independently of the filling state of intracellular Ca 2ϩ stores, the molecular correlates of capacitative calcium entry and I CRAC are still obscure. Thus, the conclusion that any of the mammalian TRPCs are primarily regulated by store depletion has to be critically revised.