articles

OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity

Rainer Strotmann*, Christian Harteneck*, Karin Nunnenmacher*, Gnter Schultz* and Tim D. Plant*
*Institut fr Pharmakologie, Universittsklinikum Benjamin Franklin, Freie Universitt Berlin, Thielallee 6773, 14195 Berlin, Germany email:tplant@zedat.fu-berlin.de

Ca2+-permeable channels that are involved in the responses of mammalian cells to changes in extracellular osmolarity have not been characterized at the molecular level. Here we identify a new TRP (transient receptor potential)-like channel protein, OTRPC4, that is expressed at high levels in the kidney, liver and heart. OTRPC4 forms Ca2+-permeable, nonselective cation channels that exhibit spontaneous activity in isotonic media and are rapidly activated by decreases in, and are inhibited by increases in, extracellular osmolarity. Changes in osmolarity of as little as 10% result in significant changes in intracellular Ca2+ concentration. We propose that OTRPC4 is a candidate for a molecular sensor that confers osmosensitivity on mammalian cells.

SM-9, a putative TRP-like channel protein from Caenorhabditis elegans, has been shown to have a key function in osmosensitivity in the nematode. Behavioural experiments have shown that OSM-9 is involved in odorant perception and the avoidance response of C. elegans to solutions of high osmolarity1. Furthermore, it is also involved in sensory adaptation in neurons that require cyclic nucleotide-gated channels for olfactory transduction1. However, nothing is yet known of the biophysical properties of this channel, nor of its regulation, and a mammalian homologue of this protein with similar functions has not yet been identified. OSM-9 belongs to the increasingly diverse family of Ca2+-permeable cation channels that is formed by the homologues of the Drosophila TRP cation-channel protein2. All of these channels have similar predicted transmembrane topologies, with six-segment transmembrane domains; these topologies are also shared with some K+ channels, cyclic nucleotide-gated channels, hyperpolarization-activated cyclic nucleotide-gated channels and voltage-gated Na+ and Ca2+ channels. On the basis of sequence homology, TRP channels (TRPCs) can be divided into three subfamilies3. The classical subfamily, which includes the Drosophila homologues TRP and TRPL (transient receptor potential-like protein4), is involved in Ca2+ entry after activation of receptors coupling to phospholipases C. To date, seven mammalian isoforms (TRPC17, refs 514) have been described in this channel subfamily. Another subfamily of TRPCs is structurally similar to OSM-9. We have named it the OTRPC subfamily3, after its first member1. Some members of this family are activated by physical and chemical stimuli. Mammalian TRPC homologues that belong to the OTRPC family include the vanilloid receptor (VR1, refs15, 16), the vanilloid receptor-like protein (VRL-1, also called GRC)17,18, and the putative epithelial Ca2+ channels (ECaC)19, and calcium transport protein 1 (CaT1, ref. 20). VR1 is a Ca2+-permeable, nonselective cation channel that was cloned from rat dorsal root ganglia15. It is sensitive to heat and to the pain-inducing vanilloid capsaicin, and is thought to have a function in sensory transduction in nociceptors. The recently-cloned relative of VR1, VRL-1, has been shown to be activated by high levels of noxious heat and may be involved in high-threshold nociception17. Its more widespread expression pattern may be an indication of other functions and of a further mechanism of regulation17, as recently indicated by the regulation of its translocation by insulin-like growth factor-1 (ref. 18). Other isoforms were cloned from rabbit kidney (ECaC)19 and rat duodenum

(CaT1, ref. 20), and are thought to function as Ca2+-influx channels in 1,25-dihydroxyvitamin-D3-responsive epithelia19,20. Here we identify OTRPC4, a new mammalian TRP-like channel that is expressed in the kidney, heart and liver and that is structurally related to the members of the OTRPC subfamily. OTRPC4 is responsive to changes in extracellular osmolarity in a physiologically relevant range, and its functional properties indicate that it may be involved in cellular osmoreception.

Results
Cloning and structure of OTRPC4. To identify new members of the TRP channel family, we searched the expressed-sequence tag (EST) database with complementary DNA sequences encoding channels of the OTRPC subfamily. The murine ESTs AA139413 and W53556 were detected by their homology to the putative pore region and the amino-terminal sequence of the mammalian TRP isoforms VR1 and VRL-1 (Fig. 1a). Screening of a genomic library using the expressed-sequence tag (EST) cDNAs as probes showed that both sequences are localized on the same gene. A combination of reverse transcription and the polymerase chain reaction (RTPCR) across the remaining gap indicated that the sequences belong to the same transcript. We then selected RNA from mouse kidney, in which expression is high (see below), to complete the 5 terminus of the clone by rapid amplification of cloned ends (RACE). We cloned a sequence of 3,270 base pairs (bp), with an open reading frame of 2,616 bp. We then subcloned the coding sequence into a mammalian expression vector in frame with enhanced green fluorescent protein (eGFP), which resulted in an expressed protein with eGFP fused to the carboxy terminus of OTRPC4. As deduced from the open reading frame, OTRPC4 is composed of 871 amino-acid residues. The hydropathy profile predicts 6 transmembrane segments, with a pore-forming loop between segments 5 and 6. Sequence analysis revealed three ankyrin-like domains in the N-terminal sequence. The structure of OTRPC4 is thus similar to other TRPC family members. However, the lack of a proline-rich motif in the C-terminal sequence, and the significant degree of homology of the transmembrane regions with those of VR1 and VRL-1, indicate a closer relationship to the OTRPC subfamily. Sequence alignment within this group (Fig. 1a, c) showed respective overall amino-acid similarities of 41.5%, 39.1% and 22.9% to VR1, VRL-1 and EcaC, respectively. The region that
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VR1 VRL-1 ECaC OTRPC4 V V I I M M M M I I I I E Q Q Q K K K K M V M I I I I L L L F F R R G K D D D D L L L L C L M F R R R R F F F F M L C L F L W L V V L V Y Y M Y L L A L V V V L F F V F L L I M F F L I G G G G F F F Y S A A A T V S S A A A A V L F L V V V T S T L L L I S E R D E G A K R L N S N N P P S K C L A T P P N M E M E D K S N V T N C P S H D H T I E K V T D C T F Q R E Q S G Q T N S P E C A T D T V P V G N P Q N T C E L Y K E G P P E E A G P F C

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VR1 VRL-1 ECaC OTRPC4 N A S R S P D D Y Y Y S N R P E S S T T I A L L L D F F Y A S S A T S F C S T L L L F L E E E D L L L L F F F F K K K F F L L T T T T I I I I G G I G M M D M G G G G D E P D L L A L E A N E F F Y M T Q S L E E V S N Q D S Y L L A D R P K F F F Y K R M P A G V V V V F V Y F I L C I I L I L L L T L L L Y L L L A V A A A T Y Y F Y V V A I I L I I L L I L T T A T Y Y T F I V L V L L L L L L M L L L L L N N N N M M L M L L F L I I I I A A A A L L M L M M M M G S G G

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VR1 VRL-1 ECaC OTRPC4 E E D E T T T T V V H V N N W G K H R Q I V V V A A A S Q D Q K E N E E S S R S K W D K N S E H I I L I W W W W K K R K L L A L Q Q Q Q R K V W A A V A I I A T T S T T I V T I L L V L D E M D T M L I E E E E K N R S G S F Y F L W P K W V C C F M R L R R R K K R K A K K A F H M F R R P R S E R S G G F G K R L E L L W M L L P V Q K R T

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mTRPC4 mTRPC5 mTRPC1 mTRPC7 mTRPC3 mTRPC6 mTRPC2 VR1 VRL-1 OTRPC4 ECaC OSM-9 40 30 20 10 0

Figure 1 Close relatives of OTRPC4 and tissue distribution of the OTRPC4 transcript. a, Protein sequence alignment of a region including the fifth and sixth transmembrane domains, and the adjacent cytosolic region of the OSM-9-like mammalian TRP channel (OTRPC) subfamily members. Sequences that are predicted to form transmembrane segments (TM5 and TM6) and the putative pore-forming loop are indicated. Conserved amino-acid residues are highlighted. b, Autoradiograph of northern-hybridization analysis with an OTRPC4 cDNA probe of RNA from different mouse tissues. The positions and sizes (in kb) of DNA markers are shown on the left. A 3.3-kb fragment was detected in heart, liver, kidney and testis; a further

2.3-kb fragment was localized in liver and kidney. c, Phylogenetic tree of TRP-like channels. Two distinct subgroups can be identified by their phylogenetic relationships. The upper set shows murine members of the classical TRP-related subfamily that have been intensively investigated. To date, seven isotypes have been found in mammals. The lower set shows a subgroup within the TRP family that has structural similarity to the C. elegans TRP-channel OSM-9. The recently-cloned VR1, VRL-1 and EcaC, as well as OTRPC4, fall within this group. Scale represents evolutionary distance calculated by Clustal analysis.

includes transmembrane segments 5 and 6, the putative pore region, and the adjacent cytosolic section exhibit sequence similarities of 46.2%, 42.7% and 24.5%, respectively. Northern hybridization of a mouse tissue panel with the EST cDNA sequences showed signals at a transcript length of 3.3 kilobases (kb) in liver, kidney, heart and testis, with a further fragment of 2.3 kb in liver and kidney (Fig. 1b). To localize OTRPC4 expression within the kidney, we carried out an in situ hybridization analysis. Autoradiographs showed the presence of high levels of OTRPC4 transcripts in the inner cortex and a punctate distribution in the outer cortex (Fig. 2a). Controls using a sense probe showed no specific hybridization (Fig. 2b). Higher-resolution microscopy of emulsion-dipped slides indicated a high density of grains in the epithelium of the distal convoluted tubule (Fig. 2c). Increased basal cytosolic Ca2+ levels in OTRPC4-expressing HEK293 cells. We studied the functional properties of OTRPC4 after heterologous expression of the protein in HEK293 cells. As shown by RT-PCR, using specific primers for the human OTRPC4 sequence, HEK293 cells do not express an endogenous OTRPC4 isoform. In contrast, a strong signal was observed in the human kidney (data not shown). Confocal imaging of HEK293 cells transiently transfected with the OTRPC4GFP fusion protein revealed strong fluorescence at the plasma membrane, but also a stronger signal in the endoplasmic reticulum (data not shown) than for other C-terminally GFP-tagged TRPCs21. After transient transfection with OTRPC4GFP, we measured
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the cytosolic Ca2+ concentration [Ca2+]i of HEK293 cells using the Ca2+-sensitive dye fura-2. Figure 3 shows that at an extracellular Ca2+ concentration of 1 mM, the basal fluorescence ratio F340/F380 in OTRPC4-expressing cells (50 cells in 3 independent experiments) was significantly increased (P < 0.01) relative to controls (63 cells in 3 independent experiments), indicating a significantly higher resting [Ca2+]i in these cells. To determine whether the increased basal [Ca2+]i resulted from Ca2+ influx through a divalent-cation-permeable pathway, we carried out fluorescence-quench experiments using Mn2+, while simultaneously measuring [Ca2+]i in fura-2-loaded cells. Addition of 200 mM MnCl2 to the extracellular solution led to a rapid reduction in fluorescence in transfected cells, indicating Mn2+ influx, whereas no reduction in fluorescence intensity was observed in control cells. Concomitant [Ca2+]i measurements, however, showed a reduction in F340/F380 during the application of MnCl2, indicating that Mn2+ may partially inhibit the Ca2+-entry pathway. Furthermore, removal of extracellular Ca2+ from cells showing spontaneous activity rapidly reduced the F340/F380 to a value close the resting fluorescence ratio observed in control cells. OTRPC4 mediates [Ca2+]i responses to changes in osmolarity. We searched for stimuli that may activate OTRPC4. Knowing that OSM-9 is involved in responses to changes in extracellular osmolarity in C. elegans, we tested the effects of hypo- and hypertonic extracellular media on OTRPC4-expressing cells. Upon reduction of extracellular osmolarity, large, reversible increases in [Ca2+]i were
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Figure 2 Localization of OTRPC4 in the kidney. In situ hybridization of OTRPC4 antisense (a) and sense (b) cRNA probes to sections of the whole mouse kidney. c, Bright-field micrograph of a section of the cortex of a mouse kidney showing the localization of OTRPC4 mRNA. Note the presence of a glomerulus (upper left) and the high density of silver grains in the distal convoluted tubule (lighter areas).

observed in OTRPC4-transfected cells, but not in control cells (Fig. 3b). However, simultaneous measurements of cell volume (see, for example, Fig. 3a) showed uniform volume increases in both groups. Similar responses were observed as a result of removal of mannitol or with dilution of the standard salt solution. Conversely, increasing the osmolarity led to a reduction in [Ca2+]i (Fig. 4, inset). Increases in [Ca2+]i in response to hypotonic stimulation were also observed in other cell types (CHO-K1 and rat basophilic leukaemia (RBL) cells) transiently transfected with OTRPC4GFP. In contrast to OTRPC4, all other members of this channel subfamily (VR1, VRL-1 and ECaC) showed no response to application of hypotonic solutions (data not shown). Following a variable delay after exchange of the extracellular medium, [Ca2+]i responses in individual cells to changes in osmolarity were rapid, reaching peak values within 30 s (Fig. 3, inset). Although the rising phase of the response in individual cells was steep, the variable delay before the onset of the Ca2+ response led to an averaged signal with a slower rise time (Fig. 3). After reaching a maximum, [Ca2+]i decreased slowly, reaching half-maximal values after ~15 min. The Ca2+ signal quickly returned to basal levels after switching to an isoosmotic superfusion medium. We investigated the sensitivity of the response by testing the effects of changes in osmolarity from 300 to 330, 270, 250, 230 and 200 mosmol l1. Significant Ca 2+ changes were already observed in response to reductions or increases in osmolarity of 30 mosmol l1 (P < 0.0001, n = 65 in 3 independent experiments). The dependence of [Ca2+]i on extracellular osmolarity is shown in Fig. 4. The increases in [Ca2+]i in response to hypotonic solutions (200 mosmol l1) in HEK293 cells expressing OTRPC4 had amplitudes similar to, or slightly larger than, those observed in response to activation of the endogenous muscarinic receptor.
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Figure 3 Ca2+ increase in OTRPC4-transfected HEK293 cells in response to a reduction in extracellular osmolarity. a, Measurements of relative cell volumes of OTRPC4GFP-expressing and control HEK293 cells loaded with fura-2. The relative cell volume was calculated from the fura-2 fluorescence at the isosbestic point (see text). Cells were initially kept in isotonic solution (300 mosmol l1) containing 100 mM mannitol and 1 mM CaCl2. The upper horizontal bar indicates a reduction of the osmolarity of the superfusion medium to 200 mosmol l1 by omitting mannitol. b, Fluorescence ratio F340/F380 in the same cells. At the time marked by the bar, extracellular Ca2+ was replaced with 1 mM EGTA. Traces are means obtained from 17 cells (OTRPC4) and 21 cells (control), in the same experiment. The shaded area represents s.e.m. Non-transfected cells on the same coverslip, identified by absence of GFP fluorescence, were used as controls. Fluorescence images were recorded at 25 magnification. Inset, traces from single cells in the same experiment, showing individual differences in the latency of response after hypotonic challenge.

Increases in [Ca2+]i can result from entry of Ca2+ from the extracellular space or release of Ca2+ from intracellular stores. To discriminate between these mechanisms in OTRPC4-expressing cells, we replaced Ca2+ in the superfusion medium with EGTA during responses to hypotonic solutions (Fig. 3). Removal of extracellular Ca2+ led to a reduction in [Ca2+]i to concentrations just above the basal level within 5 min. Re-addition of Ca2+ restored [Ca2+]i to a level slightly below that before removal of Ca2+. We investigated further the possibility that Ca2+ released from intracellular stores contributes to the observed signal by depletion of the stores before hypotonic stimulation. After treatment with thapsigargin (5 M), an inhibitor of the Ca2+-ATPase in the endoplasmic reticulum22, the Ca2+ response to hypotonic challenge had a similar amplitude and time course to that observed in OTRPC4-expressing cells without thapsigargin treatment. We tested the effect of lanthanum (La3+), which blocks many Ca2+-permeable cation channels, including members of the TRPC family10,13,18, and some volume-regulating mechanisms (reviewed in ref. 23), on OTRPC4. LaCl3 (100 mM) reduced the [Ca2+]i response to hypotonic stimulation by 50% relative to OTRPC4-transfected cells not treated with LaCl3. Members of the classical TRP channel family are activated by diverse signals secondary to phospholipase C activation514. To test for activation mechanisms of this type in the response of OTRPC4, we used the endogenous muscarinic receptor of HEK293 cells to stimulate the Gq-coupled signal cascade. Application of 100 mM
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Figure 4 Osmolarity-dependence of [Ca2+]i in fura-2-loaded HEK293 cells expressing OTRPC4. Dependence of the mean peak fluorescence ratio on the extracellular osmolarity. Responses to steps in osmolarity from 300 mosmol l1 to the indicated values were measured in at least 60 cells in 3 independent experiments. Error bars represent s.e.m. The shaded range indicates the normal extracellular osmolarity. Fluorescence images were recorded at 16 magnification. Inset, representative single cell traces of the fluorescence ratio after application of solutions with osmolarities of (from bottom to top) 330, 270, 250, 230 and 200 mosmol l1.

carbachol to OTRPC4-transfected HEK293 cells evoked a Ca2+ response with a similar amplitude to that observed in non-transfected control cells. Because of the large degree of homology of the transmembrane region of OTRPC4 to those of VR1 and VRL-1, we tested stimuli that have been found to activate these channels for their effect on OTRPC4. Capsaicin (10 mM) and resiniferatoxin (10 mM), which activate VR1 (ref. 15), had no effect on OTRPC4. Elevation of the temperature of the superfusion solution up to 65 C, which has been reported to activate both VR1 and VRL-1 (refs 15, 17), also had no effect on [Ca2+]i in OTRPC4-expressing cells. Capsazepine (10 mM), a competitive inhibitor of VR1 (refs 15, 16), but not of VRL-1 (ref. 17), had no effect on OTRPC4. In contrast, ruthenium red (10 mM), which inhibits currents through VR1 (refs 15, 16) and VRL-1 (ref. 17), markedly reduced both the elevated basal [Ca2+]i and the hypotonicity-induced increase in [Ca2+]i in OTRPC-4expressing cells. Electrophysiological characterization of OTRPC4. In accordance with the increased basal [Ca2+]i , spontaneous currents were observed in whole-cell recordings from cells expressing OTRPC4. Because of the rapid run-down of the current in normal wholecell recordings, we carried out all the experiments presented here using the perforated-patch technique. In standard extracellular solutions, the current voltage (IV) relationship of the spontaneous current, measured from voltage ramps, had an outwardlyrectifying form and a reversal potential close to 0 mV (mean Erev = 1.2 0.5 mV, n = 17). Replacement of the extracellular solution with a Na+- and Ca2+-free solution strongly shifted the reversal potential to negative potentials and almost abolished the inward currents (data not shown). The mean currents at 100 mV and +100 mV, obtained from the ramp protocols, at the start of recording were 12.8 1.1 and +32.2 2.7 pA pF1 (n = 17; Cm = 9.6 0.5 pF). These values contrast with those from control cells, which exhibited only small currents (-2.6 0.7 and +3.9 0.9 pA pF1, n = 5, Cm = 13.4 1.5 pF), with an almost linear IV relationship and Erev of 16.0 2.1 mV.
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Changing the extracellular medium from the standard solution to one containing 100 mM NaCl and 100 mM mannitol (320 mosmol l1) shifted Erev to more negative potentials (11.2 1.6 mV, n = 14), as expected for a cation current upon reduction of extracellular Na+ concentration. Furthermore, both inward and outward currents were reduced (see, for example, Fig. 5c, inset) to 7.0 0.8 and 22.7 2.7 pA pF1 at 100 and +100 mV, respectively. Application of a hypotonic solution (220 mosmol l1) resulted, with a delay of a few seconds (1050 s, mean 18 s), in an increase in both inward and outward currents (Fig. 5), which reached a maximum after around 50 s (2080 s). The peak inward and outward current densities were 16.9 1.4 and 66.0 6.1 pA pF1 (n = 13). The I-V-relationship had an Erev of 5.6 0.7 mV and, like that of the spontaneous currents, exhibited outward rectification. Removal of Na+ and Ca2+ from the extracellular solution completely and reversibly abolished the inward current and reduced the outward current component (Fig. 5a). After returning to the 320 mosmol l1 solution, currents returned to a level similar to that before application of the hypotonic solution. In some control cells, similar reductions in osmolarity led to the activation of a current with properties similar to those described for volume-activated Cl currents24. Compared to the current in OTRPC4-expressing cells, this current showed a much longer delay before activation and a slower activation time course, and was unaffected by removal of Na+ and Ca2+ from the extracellular solution (data not shown). Activation of this current was largely prevented in control cells in the presence of the Cl-channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 50 mM) in the extracellular solution. The cation currents activated in response to hypotonic solutions in OTRPC4-expressing cells were unaffected by NPPB. The sensitivity of the OTRPC4-mediated cation currents to variations in osmolarity was similar to that of changes in [Ca2+]i. Thus, as indicated above, currents in most cells were reduced by an increase in osmolarity of 20 mosmol l1 (Fig. 5c, inset). Furthermore, a decrease in osmolarity from 300 to 270 mosmol l1 resulted in clear increases in current in 3 out of 5 cells (Fig. 5b). Subsequent reduction of the osmolarity from 270 to 220 mosmol l1 resulted in a clear response in all 5 cells. Interestingly, cells in which no response to 270 mosmol l1 was detectable showed only small responses to 220 mosmol l1. Currents activated by hypotonic solutions were reversibly inhibited by the trivalent cations Gd3+ and La3+. Gd3+ (100 mM) had the strongest effect, reducing the current by 70 6% (n = 5), whereas La3+, at the same concentration, had a weaker inhibitory effect (27 7%, n = 5). Ruthenium red (10 mM), an inhibitor of VR1 and VRL-1 (refs 1518), abolished currents through OTRPC4. To study the ion selectivity of the current activated by hypotonic solutions, we activated currents by removing mannitol, as described above. After the current had reached a maximum, we replaced the standard hypotonic solution (220 mosmol l1) containing both Na+ and Ca2+ with hypotonic solutions containing Na+ or 20 mM Ca2+ only. The measured Erev values were 14.5 0.8 mV (n = 5) in 100 mM Na+ and +5.7 1.4 mV (n = 5) in 20 mM Ca2+. These values gave permeability ratios of 6.3 0.5 for PCa/PNa and 0.8 0.03 for PNa/PCs. In cell-attached patches from OTRPC4-expressing cells, but not from control cells, spontaneous openings of single channels were observed with the properties illustrated in Fig. 6. With the standard extracellular solution in the pipette and a bath solution containing 140 mM KCl to zero the resting potential, currents displayed outward rectification and an Erev close to 0 mV (Fig. 6a, b). The shape of the single channel IV relationship (Fig. 6b) closely resembles that of the whole-cell currents (Fig. 5). From the current amplitudes at membrane potentials of 60 and +60 mV (Fig. 6B), the respective chord conductances were 30.4 1.5 (n = 8) and 88.2 2.8 pS (n = 7). In seven out of nine cell-attached patches that showed spontaneous OTRPC4 events, channel activity was reversibly increased (between 5- and 1,000-fold, mean value 262fold) by reducing the osmolarity of the bath solution from 300 to
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Figure 6 Single-channel properties of OTRPC4 in cell-attached patches. a, Examples of spontaneous currents recorded in cell-attached patches from OTRPC4expressing cells at the indicated membrane potentials (assuming a resting potential of 0 mV). b, Currentvoltage relationship for single-channel currents from experiments such as that described in a. Data are expressed as mean amplitude s.e.m. from 48 patches. c, Stimulation of channel activity in a cell-attached patch by application of a hypotonic solution. Plot of NPo against time during an experiment, showing stimulation of channel activity by application of a hypotonic bath solution. Insets, currents recorded at the times indicated by (1) and (2). Cell-attached recordings were obtained with the standard extracellular solution (140 Na+, 5 Cs+) in the pipette, and a bath solution containing 140 KCl to zero the membrane potential. The hypotonic stimulus (from 300 to 200 mosmol l1) applied at the time indicated by the bar was obtained by diluting the bath KCl solution.

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Figure 5 Whole-cell currents in cells expressing OTRPC4. a, Reduction in osmolarity results in activation of a cation current. Whole-cell currents recorded during voltage ramps from 100 to +100 mV in an external solution with an osmolarity of 320 mosmol l1 and after exchange of the solution to a hypotonic solution of 220 mosmol l-1 (by removing mannitol). During the application of the hypotonic solution, Na+ and Ca2+ were removed from the extracellular solution (and replaced with NMDG+). Finally, the solution was returned to 320 mosmol l1. Inset, current values at -100 and +100 mV obtained from the ramps during the course of the experiment. Bars indicate the times during which the different solutions were applied. Filled symbols indicate values obtained from the ramps shown in this figure. All values of osmolarity are in mosmol l1 b, Sensitivity of current responses to solutions of different osmolarities. Currents were recorded in solutions of 300, 270 and 220 mosmol l1. As in a, the inset shows the current values throughout the experiment. c, Inhibition of currents by Gd3+. Currents were recorded in a hypotonic solution (220 mosmol l1) before and during the application of 100 M Gd3+ to the extracellular solution, and after wash-out. Inset, current values throughout the experiment. Note the reduction in both inward and outward currents upon changing the osmolarity of the extracellular solution from 300 to 320 mosmol l1.

and control cells. However, none of these channel types was consistently seen. To test the possibility that membrane stretch is responsible for activation of OTRPC4, we tested the effects of negative and positive pressure applied to the patch pipette on channel activity in the cell-attached mode. Changes in pressure to levels that disrupted the patch or the seal had no effect on spontaneous activity of the OTRPC4 channel (10 and 6 patches for negative and positive pressure, respectively). In three patches, including two of those showing OTRPC4 activity, a channel (with an amplitude of ~2.5 pA at +60 mV) that was different to OTRPC4 was stimulated by the application of negative pressure.

Discussion
We have cloned and functionally characterized a new mammalian TRP homologue, OTRPC4, that is predominantly expressed in kidney (particularly in the distal nephron), liver and heart. OTRPC4 is activated after treatment of cells with hypotonic solutions. On the basis of its sequence, OTRPC4 belongs to the structurally and functionally distinct OTRPC subfamily of TRP homologues that are related to the C. elegans protein OSM-9 (ref. 1). In mammals, this family comprises the TRP-like channels VR1, VRL1 and ECaC (OTRPC13, respectively1520). Within the TRPC family, OTRPC4 has the closest structural homology to VR1 and VRL1, and a more distant relationship to ECaC and CaT1. Recently, another cDNA, encoding the stretch-inhibited channel (SIC), which can be aligned with the sequences of the OTRP-channel family members, has been cloned from rat kidney25. Sequence
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200 mosmol l1 (Fig. 6c). Like the increases in [Ca2+]i and activation of whole-cell currents, channel activation occurred after a variable delay. Occasionally, other channel types that responded to hypotonic stimulation were observed in patches from OTRPC4-expressing
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comparison revealed that the N terminus and the transmembrane domains, including the putative pore region, are identical to those of VR1. However, the N terminus of SIC is substantially shorter, beginning at the amino-acid residue 308 of VR1 and including only one ankyrin domain. In contrast, the cytosolic C terminus of SIC is homologous to the C terminus of OTRPC4, with a similarity of 96.9%. Until the structure of this channel is verified, it seems premature to assign it to the OTRPC family. A mechanosensory transduction channel, NOMPC, that was recently cloned from Drosophila and C. elegans26 is related to the known TRPCs in its transmembrane region, but, because of its extremely large number of N-terminal ankyrin repeats, is likely to form a new TRPC subfamily. In contrast to the members of the classical TRP channel family (TRPC17) that are activated secondary to the activation of PLCcoupled receptors513, the OTRPCs, so far with the exception of ECaC, have been proposed to be involved in the transduction of physical and chemical stimuli1517,19,20. This also holds true for OTRPC4, which is activated in response to treatment with hypotonic solutions, but not by stimulation of PLC-coupled receptors. In transiently-transfected HEK293 cells, both cation currents and [Ca2+]i are markedly increased after superfusion with hypotonic solutions. This increase in [Ca2+]i results exclusively from influx of Ca2+ from the extracellular space, with no involvement of Ca2+ release from intracellular stores. Conversely, increases in extracellular osmolarity reduce [Ca2+]i and currents.. Thus, our results indicate that in OTRPC4-expressing cells, the sensitivity of [Ca2+]i to changes in osmolarity results from modulation of the activity of a Ca2+-permeable, nonselective cation channel. By what mechanism is the channel regulated? Several studies of responses to hypotonic stimuli have indicated that increases in [Ca2+]i result from the activation of mechanosensitive, nonselective cation channels in the cell membrane, which respond to membrane stretch caused by increases in cell volume27,28. However, OTRPC4 activity was unaffected by pressure-induced stretching of the membrane. Although this finding limits the mechanisms that could be involved, it is still unclear how OTRPC4 is regulated. Channel activation could be an early step in the cellular response to changes in osmolarity or cell volume, or, as indicated by the observed delay before channel activation, could be a more distal step in a signalling cascade that couples changes in cell volume to channel gating. OTRPC4 is highly sensitive to changes in extracellular osmolarity and, in HEK293 cells, responds to superfusion with 10% hypo- or hypertonic media. After a delay, currents and responses to reductions in osmolarity reached a maximum within <1 min. The speed of response and sensitivity are consistent with those described for increases in [Ca2+]i and for cation channels activated by increases in cell volume27,29. Interestingly, the activation of OTRPC4 currents is more rapid than that of endogenous volume-regulated Cl currents in the same cells. The activation of these chloride channels, at least in endothelial cells, occurs through tyrosine kinases30 after reductions in intracellular ionic strength as a result of cellular swelling31. The observed responses of OTRPC4 indicate that the channel can respond to changes in extracellular osmolarity within the physiological range, and can signal both increases and decreases in osmotic pressure. The only other member of the OTRPC family that shows spontaneous activity is ECaC, for which a regulatory mechanism has not yet been elucidated19,20. Other channels in this subfamily that have been reported to be involved in responses to changes in osmolarity seem to respond in the opposite direction to that of OTRPC4. OSM-9 is involved in the behavioural responses of C. elegans to hypertonic solutions1, and SIC responds to cell shrinkage in hypertonic solutions and is inhibited by membrane stretching25. There is evidence that all mammalian members of the OTRPC subfamily, like the classical TRPCs, form Ca2+-permeable channels. This has been directly demonstrated for VR1 (refs 15, 16), VRL-1 (refs 17, 18) and ECaC32. The relative Ca2+ permeability (PCa/PNa) for OTRPC4 was ~6, compared with values for VR1 of 9.6 with
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capsaicin15 and 3.8 with heat16, 2.9 with heat for VRL-1 (ref. 17) and 8.9 for GRC(VRL-1) in the IGF-I response18, and 107 for ECaC32. Thus, all but ECaC, which is highly Ca2+ selective, have a slightly higher permeability for Ca2+ than for Na+. In contrast to the members of the OTRPC subfamily, SIC has been shown to be more permeable to Na+ than to Ca2+ (PCa/PNa = 0.24; ref. 25). This result is surprising, as the transmembrane region, including the pore, is identical to that of VR1. Furthermore, SIC has a much higher unitary conductance (250 pS; ref. 25) than VR1 (35 pS; ref. 15), or any other member of the TRPC family. Interestingly, the single-channel conductance of OTRPC4 is similar to that of VR-1 and shows a similar potential dependence, being higher at positive (88 and ~80 pS for OTRPC4 and VR1, respectively) than at negative membrane potentials (30 and 35 pS, respectively)15,16. The potential-dependence of whole-cell currents through OTRPC4 is similar to that of VR1 (refs 15, 16), VRL-1 (refs 17, 18) and TRPL33, and exhibits outward rectification. Unfortunately, there are no specific blockers of nonselective cation channels of the TRP family. However, the lanthanides Gd3+ and La3+ inhibit a variety of cation channels. For the OTRPC family, both responses involving GRC (VRL-1, ref. 18) and ECaC19,34 are inhibited by La3+. Furthermore, there is evidence that mechanoand osmosensitive channels found in native cells are inhibited by lanthanides27,35,36. Most studies of cell-swelling-dependent [Ca2+]i signals23 report suppression by micromolar concentrations of La3+, but differing effects of Gd3+. In this study, La3+ partially inhibited Ca2+ influx and currents through OTRPC4, whereas Gd3+ was a stronger inhibitor of currents. In response to hypotonic solutions, cells first swell, and then regulate their volumes back towards their original values by the loss of intracellular ions through volume-activated K+ and Cl channels (reviewed in ref. 37). Many studies have shown that this regulatory-volume decrease (RVD) following osmotic stress is dependent on Ca2+ (ref. 37). Release of Ca2+ from inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular stores has been shown to be involved in RVD after cell swelling secondary to the uptake of osmoticallyactive substances, whereas RVD after hypotonic cell swelling is often dependent on extracellular Ca2+ (ref. 38). RVD is prevented in the absence of extracellular Ca2+, or if intracellular Ca2+ is buffered to low levels by membrane-permeable Ca2+ chelators39,40. A Ca2+permeable channel that reacts to changes in extracellular osmolarity or to membrane stretching has been proposed to be the osmosensor37,39. However, most studies of RVD have measured changes in cell volume or [Ca2+]i, and data regarding the biophysical properties of the channel involved, which would allow comparison with OTRPC4, are lacking. Nevertheless, the properties that OTRPC4 confers on HEK cells are similar to those of Ca2+-entry mechanisms that are activated in response to cell swelling. Interestingly, OTRPC4 is expressed in a region of the kidney where cells may be exposed to hypotonic fluid, at least on their luminal surfaces. In addition to a function in the regulation of cell volume, OTRPC4-mediated influx of Ca2+ could be involved in the regulation of electrolyte or fluid transport in the distal nephron. We have identified OTRPC4 as a cation channel with an activity that is controlled by changes in the osmolarity of the extracellular medium within the physiological range. Data from northern hybridization indicate that high levels of the OTRPC4 transcript are present in tissues that are exposed to changing osmotic environments. OTRPC4 is therefore a candidate for an osmosensor channel that is involved in regulation of cellular volume.

Methods
Molecular biology.
ESTs W53556 and AA139413 were identified by database homology searches using sequences of known TRP-related channels as templates. Further screening of a genomic P1 artificial chromosome (PAC) library (RPCI-21 female (129/SvEvTA CfBr)) Mouse PAC Library (Roswell Park Cancer Institute), provided as spotted filters by the Resource Center of the German Genome Project (Berlin) showed that the EST probes identified the same PAC clones.

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To determine the tissue distributions of the transcripts, northern hybridization was carried out using a mouse multiple-tissue northern blot (Clontech). Probes of 594 bp and 2,207 bp were excised from ESTs W53556 and AA139413 and radiolabelled with [a-32P]dATP. After 1 h of prehybridization in ExpressHyb hybridization solution (Clontech), hybridization was carried out overnight at 65 C in the same solution. Blots were washed according to the manufacturers high-stringency washing protocol (3 times with 2 SSC at 65 C, twice with 0.1 SSC at 55 C for 1 h each) and autoradiographed on Kodak XAR film for 72 h. For PCR cloning, total RNA was purified from 129/SvEv mouse kidney using TRIzol LS (Life Technologies) according to the standard protocol. Reverse transcription was carried out from 1 mg of total RNA using 200 U of SuperScript II reverse transcriptase (Life Technologies) and 5 pM of primer 5-CCAGTGAGCAGAGTGACGAGGACTCGAGCTCAAGCTTTTTTTTTTTTTTTTT for poly TcDNA and gene-specific primers for the RACE protocol. The reaction was incubated at 42 C for 50 min. The complete cDNA sequence was obtained by anchored PCR using the EST W53556 sequence information. 5-RACE was carried out according to the manufacturers instructions (5-RACE System, Life Technologies). The full coding sequence was then re-amplified from poly-A-primed cDNA using the primers 5CCAAGCTTGCCACCATGGCAGATCCTGGTGATGGT and 5-CCGGATCCAGTGGGGCATCGTCCGT under the following PCR conditions: 1 min initial denaturation at 96 C; 30 cycles: 94 C for 15 s, 52 C for 30 s, 72 C for 150 s; final extension at 72 C for 7 min. The PCR reaction mixture contained 30 pM of each primer, 3 ml cDNA, 200 M of each dNTP, 1.5 mM MgCl2 and 2.6 U of polymerase-enzyme mixture (ExpandHF, Roche). The PCR fragment was subsequently digested with BamHI and HindIII and subcloned into the pEGFP-N1 GFP-expression vector (Clontech). Sequence identity with the EST sequences was confirmed by DNA sequencing. For PCR amplification of a diagnostic human OTRPC4 fragment from cDNA libraries, the following primers were used: 5-CCGGCCACCTGTCTTGCTCCTATG and 5-AGCACCGGCAAATCCCAGACACTA. The measuring buffer contained 88 mM NaCl, 5 mM KCl, 1 mM MgCl2, 5.5 mM glucose, 0.1% mM BSA, 10 mM HEPES and 1 mM CaCl2 (pH 7.4 with NaOH). In the Ca2+-free measuring buffer, CaCl2 was replaced with 1 mM EGTA. Osmolarity was adjusted by addition of appropriate amounts of mannitol.

Patch-clamp recordings.
Recordings of whole-cell currents from single cells were made with an EPC-7 amplifier using Pulse software (HEKA, Lambrecht, Germany). Borosilicate glass patch pipettes had resistances of 35 MW when filled with the standard intracellular solutions. In initial experiments, the standard whole-cell mode of the patch-clamp technique was used. Most experiments, however, used the perforated-patch technique41 with amphotericin B (250 mg ml1, Sigma) as the ionophore for current recording42. Measurements of currents were started when the series resistance approached a plateau at <30 MW. To measure channel currents, cells were held at a potential of 0 mV and ramps from 100 to +100 mV with a duration of 400 ms were applied at a frequency of 0.2 or 0.1 Hz. Ramp data were acquired at a frequency of 4 kHz after filtering at 1 kHz. Currents from cell-attached patches were recorded on DAT (Biologic Science Instruments, Claix, France). Data were subsequently filtered (12.5 kHz), and digitized (1025 kHz) and analysed using pClamp6 software (Axon Instruments, Foster City, California). The standard pipette solution contained 110 mM CH3O3SCs (caesium methane sulphonate), 25 mM CsCl, 2 mM MgCl2, 0.362 mM CaCl2, 1 mM EGTA and 30 mM HEPES (pH 7.2 with CsOH). The standard extracellular solution contained 140 mM NaCl, 5 mM CsCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose and 10 mM HEPES (pH 7.4 with NaOH). For experiments to test the effects of hypotonic solutions, cells were initially bathed in a solution containing 100 mM NaCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 mM glucose and 100 mM mannitol (pH 7.4 with NaOH); mannitol was then removed to reduce the osmolarity without affecting ion concentrations. For Na+-free solutions, Na+ was replaced with N-methyl-D-glucamine (NMDG+). For Ca2+-free solutions, Ca2+ was omitted and 0.1 or 1 mM EGTA was added. For cell-attached recordings, the standard bath solution was exchanged for a solution containing 145 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and 10 mM HEPES (pH 7.4 with KOH). To reduce the osmolarity, this solution was diluted appropriately. The osmolarity of the solutions was measured using a freezing-point-depression osmometer (Roebling, Berlin). NPPB was obtained from RBI (Deisenhofen, Germany).
RECEIVED 26 NOVEMBER 1999; REVISED 26 APRIL 2000; ACCEPTED 14 JUNE 2000; PUBLISHED 8 SEPTEMBER 2000.

Cell culture and transfection.

HEK293 cells were cultured in MEMEarle medium (Biochrom, Berlin, Germany), supplemented with 10% (v/v) fetal calf serum (Biochrom), 100 U ml1 penicillin and 100 mg ml1 streptomycin. Cells were plated onto glass coverslips 2448 h before transfection. Cells were transiently transfected with 0.8 mg DNA and 6 ml of FuGENE 6 transfection reagent (Roche) in 94 ml of OptiMEM medium (Life Technologies) per 85-mm dish. Ca2+ measurements and electrophysiological studies were carried out 2436 h after transfection.

In situ hybridization.
Paraffin sections (5 mm) from mouse kidney were mounted on silane-coated glass slides. A 2.0-kB cDNA fragment comprising 500 bp of the 3-untranslated region of OTRPC4 was subcloned in pBluescribe. Plasmid DNA was linearized by digesting the BamHI or HindIII site (on the vector) and transcribed using T3 and T7 MAXIscript RNA polymerase (Ambion, Austin, Texas) in the presence of [a-35S] UTP. Probe length was adjusted to 150 bases by alkaline treatment. Sections were partially digested by 0.5% pronase in 0.2 M HCl and acetylated with 0.25% acetic anhydride in 0.1 M triethanolamine (pH 8.0) after deparaffinization and rehydration. Hybridization was carried out for 1618 h at 50 C with antisense or sense RNA at 350,000 c.p.m. per section in hybridization buffer (50% formamide, 0.3 M NaCl, 10 mM TrisHCl pH 7.5, 10 mM NaPO4 pH 6.8, 0.02% polyvinylpyrrolidone, 0.02% Ficoll 400, 0.02% BSA, 1 mg ml-1 yeast tRNA, 5 mM EDTA, 10 mM dithiothreitol (DTT) and 10% dextran sulphate). Sections were washed twice with posthybridization solution (50% formamide, 10 mM DTT, 0.5 M NaCl, 0.1 M TrisHCl, 0.05 M EDTA and 0.1 M NaH2PO4/Na2HPO4) at 52 C for 30 min and 4 h, and then incubated in TES buffer (10 mM TrisHCl pH 7.5, 1 mM EDTA and 0.5 M NaCl) at 37 C for 15 min. After treatment with RNase A (20 mg ml1 in TES buffer) for 30 min at 37 C and further incubation in TES buffer at 37 C for 15 min, slides were washed in 2 SSC and 0.1 SSC for 30 min at 21 C. After dehydration, the slides were exposed to Kodak BIOMAX film for 6 days and to Kodak NTB-2 film solution for 4 months. After development, slides were counterstained with hematoxylene/eosine.

Measurement of intracellular Ca2+ concentration.

Ca2+ measurements in single cells were made using the fluorescent Ca2+ indicator fura-2 in combination with a monochromator-based imaging system (TILL Photonics, Martinsried, Germany) attached to an inverted microscope (Axiovert 100, Zeiss). Cells were loaded with 4 mM fura-2-AM (Molecular Probes) for 60 min at 37 C in the 300 mosmol l1 standard measuring buffer supplemented with 0.2% BSA and 0.01% Pluronic F127. Coverslips were then washed in 300 mosmol l1 measuring buffer and mounted in a perfusion chamber on the microscope stage. The effective volume of the chamber was 0.3 ml, and superfusion was carried out at room temperature at a flow rate of 12 ml min1. Complete exchange of the chamber medium was assumed after 10 s. For [Ca2+]i measurements, fluorescence was excited at 340 nm and 380 nm, and the emitted light was long-pass-filtered at 520 nm. After correction for the individual background fluorescence, the fluorescence ratio F340/F380 was calculated. For manganese-quench experiments, fura-2 fluorescence was excited at the isosbestic wavelength 358 nm and the emitted light was monitored using the same filter system as for [Ca2+]i measurements. The relative cell volume was spectroscopically determined using two-dimensional fura-2 fluorescence images at the isosbestic wavelength. The integral fluorescence intensity of an area that contained the whole cell at all times was divided by the integral fluorescence of a spot within the cell. This fluorescence ratio is reciprocal to the cytosolic fura-2 concentration and thus reflects the cell volume. In all experiments, transfected cells were identified by their GFP fluorescence at an excitation wavelength of 465 nm. Each experiment reflects the fluorescence ratios averaged over at least 20 GFP-positive cells. GFP-negative cells on the same cover slip were used as controls. Other control experiments were carried out using cells transfected with pEGFP-N1. Data are expressed as means s.e.m. To keep the ion concentrations in the superfusion solution constant while varying the osmolarity, [Na+] was reduced to 88 mM compared to standard buffers, and the medium osmolarity was adjusted to 300 mosmol l1 by supplementation with mannitol. Solutions of lower osmolarity were obtained by reducing the mannitol concentration. In the experimental setup, hypotonic challenge was achieved by rapidly changing the medium from an initial osmolarity of 300 mosmol l1 to the target osmolarity.

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ACKNOWLEDGEMENTS We thank M. Schaefer for discussions and I. Reinsch for technical assistance. This work was supported by grants from the DFG and Fonds der Chemischen Industrie. Correspondence and requests for materials should be addressed to T.D.P. The cDNA sequences encoding the mouse and the human OTRPC4 orthologues have been deposited at GenBank under accession numbers AF208026 and AF258465, respectively.

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