Transient Receptor Potential-Like Channels Are ... - Semantic Scholar

Copyright © 2005 by the Genetics Society of America DOI: 10.1534/genetics.104.035139

Transient Receptor Potential-Like Channels Are Essential for Calcium Signaling and Fluid Transport in a Drosophila Epithelium Matthew R. MacPherson,*,1 Valerie P. Pollock,*,1 Laura Kean,* Tony D. Southall,* Maria E. Giannakou,* Kate E. Broderick,* Julian A. T. Dow,* Roger C. Hardie† and Shireen A. Davies*,2 *IBLS Division of Molecular Genetics, University of Glasgow, Glasgow G11 6NU, United Kingdom and † Department of Anatomy, University of Cambridge, Cambridge CB2 3DY, United Kingdom Manuscript received August 18, 2004 Accepted for publication December 9, 2004 ABSTRACT Calcium signaling is an important mediator of neuropeptide-stimulated fluid transport by Drosophila Malpighian (renal) tubules. We demonstrate the first epithelial role, in vivo, for members of the TRP family of calcium channels. RT-PCR revealed expression of trp, trpl, and trp␥ in tubules. Use of antipeptide polyclonal antibodies for TRP, TRPL, and TRP␥ showed expression of all three channels in type 1 (principal) cells in the tubule main segment. Neuropeptide (CAP2b)-stimulated fluid transport rates were significantly reduced in tubules from the trpl 302 mutant and the trpl;trp double mutant, trpl 302;trp 343. However, a trp null, trp 343, had no impact on stimulated fluid transport. Measurement of cytosolic calcium concentrations ([Ca2⫹]i) in tubule principal cells using an aequorin transgene in trp and trpl mutants showed a reduction in calcium responses in trpl 302. Western blotting of tubule preparations from trp and trpl mutants revealed a correlation between TRPL levels and CAP2b-stimulated fluid transport and calcium signaling. Rescue of trpl 302 with a trpl transgene under heat-shock control resulted in a stimulated fluid transport phenotype that was indistinguishable from wild-type tubules. Furthermore, restoration of normal stimulated rates of fluid transport by rescue of trpl 302 was not compromised by introduction of the trp null, trp 343. Thus, in an epithelial context, TRPL is sufficient for wild-type responses. Finally, a scaffolding component of the TRPL/TRP-signaling complex, INAD, is not expressed in tubules, suggesting that inaD is not essential for TRPL/TRP function in Drosophila tubules.

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ALCIUM signaling plays a crucial role in physiological processes (Berridge 1997). While calcium influx is particularly important to calcium-signaling mechanisms in excitable cells, both calcium release from intracellular stores and calcium influx are important in nonexcitable, secretory cells (Shuttleworth 1997; Petersen et al. 1999). Calcium entry in these cells is typically mediated by phospholipase C (PLC)-dependent mechanisms, which include the major route of store-operated calcium influx. Consequently, the identity of plasma membrane channels (“store-operated” channels) involved in this process has been extensively researched. Such storeoperated channels have remained elusive, but potential candidates have included the family of transient receptor potential (TRP) channels. The trp gene was first identified in Drosophila photoreceptors (Montell et al. 1985) and subsequently found to encode a calciumpermeable channel (Hardie and Minke 1992). Two other genes encoding proteins with homology to the

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These authors contributed equally to this work. Corresponding author: Division of Molecular Genetics, Institute of Biomedical and Life Sciences, Anderson Complex, University of Glasgow, 56 Dumbarton Rd., Glasgow G11 6NU, United Kingdom. E-mail: [email protected] 2

Genetics 169: 1541–1552 (March 2005)

TRP protein, trpl (trp-like; Phillips et al. 1992) and trp␥ (trp-gamma; Xu et al. 2000), have since also been identified in Drosophila, with trpl being involved in phototransduction. Around 20 mammalian TRP proteins have been identified (Clapham et al. 2001), falling into at least three subfamilies. Those most closely related to the Drosophila TRPs are all believed to be activated downstream of PLC and may include the elusive store-operated calcium channels activated by depletion of internal calcium stores by inositol 1,4,5 trisphosphate (InsP3 ; Clapham et al. 2001). In Drosophila photoreceptors, TRP represents a highly calcium-selective cation channel (PCa:PNa ⬎ 100), while trpl encodes a nonselective cation channel with moderate calcium permeability (PCa:PNa, 4:1). The light-sensitive current is completely abolished in trpl:trp double mutants lacking both TRP and transient receptor potential-like (TRPL; Niemeyer et al. 1996; Reuss et al. 1997). The newly identified third member of this family, TRP␥, may form heteromultimers with TRPL (Xu et al. 2000). Both TRP and TRPL are activated downstream of PLC but the precise mechanism of activation of any of these channels, or indeed their vertebrate counterparts, remains controversial. Neither TRP nor TRPL appears to require

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InsP3 or the InsP3 receptor for activation (Acharya et al. 1997; Hardie and Raghu 1998; Raghu et al. 2000a), raising the possibility that diacylglycerol, its downstream metabolites (polyunsaturated fatty acids), or reduction in phosphatidylinositol 4,5, bisphosphate levels may be involved (Chyb et al. 1999; Raghu et al. 2000b; Hardie and Raghu 2001). In Drosophila photoreceptors, response to light is dependent on the close interaction of TRP and TRP-related channels with other signaling proteins (rhodopsin, phospholipase C, protein kinase C, and calmodulin) mediated through the scaffolding protein INAD (inactivation no-afterpotential D; Shieh et al. 1997; Adamski et al. 1998; van Huizen et al. 1998). Recent work has shown that INAD is required for correct localization of TRP-containing supramolecular complexes in the eye (Chevesich et al. 1997; Xu et al. 1998; Li and Montell 2000). As much of the focus of in vivo studies of TRP and TRPL function have been in photoreceptors, the role of TRP and TRPL-like channels in nonvisual systems is poorly understood. The Drosophila Malpighian tubule is a tractable genetic model for fluid-secreting epithelia in which cell-specific signaling events can be linked to physiological function (Dow and Davies 2003). Using GAL4-directed aequorin transgene expression to specific tubule cell subtypes, it has been possible to show that stimulation of fluid transport by neuropeptides of the capa family (capa-1, capa-2, and cardioacceleratory peptide 2b, CAP2b) occurs as a result of a rise in cytosolic calcium concentrations ([Ca2⫹]i) (Rosay et al. 1997; Kean et al. 2002). Capa-induced calcium signaling and fluid transport is reduced in severe alleles of IP3R, suggesting that release of calcium from intracellular stores occurs upon capa peptide stimulation (Pollock et al. 2003). However, a major role of extracellular calcium is also implicated in capa action: CAP2b-elicited calcium and secretion responses are sensitive to reductions in extracellular calcium (Rosay et al. 1997) and to L-type/ cyclic-nucleotide gated calcium channel blockers (MacPherson et al. 2001; Broderick et al. 2003). To further define the contribution of plasma membrane calcium channels to calcium-signaling events and fluid transport in vivo, we have investigated the role of the TRP channel family in this epithelial context. We show that TRP, TRPL, and TRP␥ are expressed in tubule principal cells. In contrast to the visual system, functional TRPL is required for calcium signaling and epithelial fluid transport; INAD, an essential component of the TRP complex in eye, is not required for epithelial function.

MATERIALS AND METHODS Drosophila stocks: Drosophila were maintained on a 12 hr light:12 hr dark cycle on standard cornmeal-yeast-agar medium at 25⬚. Wild-type flies used were Oregon-R. trp alleles used in this study were trp hypomorph trp 301 (Pak 1979; Reuss

et al. 1997) and trp null trp 343 (Scott et al. 1997). The trp 343 line was a kind gift of W. Pak, Purdue University. trpl alleles used were trpl 302 (Niemeyer et al. 1996) and the double mutant trpl 302;trp 343 (Scott et al. 1997). To rescue trpl 302, a transgenic line containing the trpl transgene under heat-shock control, cntrpl[hstrpl]; trp34/TM6B (Niemeyer et al. 1996), a kind gift of C. S. Zuker, University of California at San Diego, was used to generate cntrpl[hstrpl];TM2/TM6B flies (maintained at 18⬚ to minimize “leaky” expression), which were heat-shocked at 37⬚ before use. This line was also used to generate cntrpl[hstrpl];trp 343 for this study. To produce flies in which tubule calcium measurements could be made using the calcium reporter aequorin (Rosay et al. 1997), it was necessary to place trp and trpl mutations in an aequorin background under control of a hsGAL4 promoter (Broderick et al. 2003), as shown in Figure A1 in the appendix. This was achieved with all lines with the exception of the trpl 302; trp 343 double mutant and the rescue lines. Maintenance of mutant phenotypes in the aequorin background was assessed by photoreceptor responses to light stimuli, as well as by tubule fluid transport assays in the presence of CAP2b. All alleles generated in the aequorin background retained the mutant phenotypes in the eye as previously documented, as well as in tubules (results not shown). Verification of aequorin expression was achieved at the final stages of the crossing procedure by measuring total light output in dissected, intact tubules after lysis in Triton/CaCl2 as described below. For tubule dissections, flies were cooled on ice and then decapitated prior to isolation of whole tubules. Materials: Coelenterazine was purchased from Molecular Probes (Eugene, OR) and dissolved in ethanol before use. Schneider’s medium (GIBCO) was obtained from Invitrogen (San Diego). The neuropeptide CAP2b (ELYAFPRV-amide) was synthesized by Research Genetics, now Invitrogen. Fluorescein-labeled anti-rabbit secondary antibody was obtained from Vector Labs (Burlingame, CA). All other chemicals were obtained from Sigma (St. Louis). Polyclonal rabbit anti-TRP, -TRPL, and -TRP␥ antibodies were synthesized by Genosphere Biotechnologies (Paris) to the following peptides: TRP, KALG SRLDYDLMMAEE; TRPL, ENSGMDVSSANKKER; and TRP␥, PAAEAGVQHNPAQLV. Reverse-transcriptase PCR: Twenty tubules were dissected, and poly(A)⫹ RNA was extracted (Dynal mRNA direct kit) and reverse transcribed with Superscript Plus (GIBCO BRL, Gaithersburg, MD) as described previously (Dow et al. 1994b). One microliter of the reverse transcription reaction, corresponding to cDNAs derived from one tubule (ⵑ160 cells), was used as a template for polymerase chain reaction containing the relevant gene-specific primer pairs based on the following published sequences: trp, GenBank M21306 (Montell and Rubin 1989); trpl, GenBank M88185 (Phillips et al. 1992); trp␥, GenBank AJ277967 (Xu et al. 2000); and inaD, GenBank U15803 (Shieh and Niemeyer 1995). Primers were designed to bracket introns, and PCR reactions were carried out on genomic DNA and cDNA prepared from Drosophila heads in all experiments. RT-PCR for trp: Forward primers to region 4361–4382 (5⬘AGAATACTTTCGCCTCCGATCC-3⬘) and backward primers to region 4900–4921 (5⬘-CCTGGTTTCTTGTCATCCGTTG-3⬘) were expected to generate a product of 467 bp using cDNA templates. RT-PCR for trpl : Forward primers to region 2991–3014 (5⬘GCTACTCAACCAAATCAGTGCTGAG-3⬘) and backward primers to region 3470–3490 (5⬘-TGGCAATGGAGCTAATGTCGG3) were expected to generate a product of 500 bp using cDNA templates. RT-PCR for trp␥: Forward primers to region 3213–3234 (5⬘AGTCGGAAACGTGAGCAAAATG-3⬘) and backward primers

TRPL Function in Epithelial Transport to region 3734–3757 (5⬘-TGGAGTTCACTGACGTATTGGATG3⬘) were expected to generate a product of 545 bp using cDNA templates. Cycle conditions were as follows: 94⬚ (1 min), 30 cycles of 94⬚ (30 sec), 57⬚ (30 sec), 72⬚ (2 min), and 72⬚ (5 min), with annealing temperature for trp at 52⬚. RT-PCR for inaD: Forward primers to region 1094–1115 (5⬘CGTCAAGCCCATCAAAAAGTTC-3⬘) and backward primers to region 1610–1590 (5⬘-CGTGACATGGTTGTTCTTGCC-3⬘) were expected to generate a product of 517 bp using cDNA templates. PCR was carried out under several different cycle conditions as follows: 94⬚ (1 min), 30 cycles of 94⬚ (30 sec), 45⬚–55⬚ (30 sec), 72⬚ (2 min), and 72⬚ (5 min) for up to 40 cycles. The PCR products obtained were cloned using PCRII.1 vector (Invitrogen). Cloned plasmids were purified (QIAGEN, Chatsworth, CA) and sequenced to confirm their identity. Immunocytochemistry: Localization of TRP, TRPL, and TRP␥ in intact tubules using rabbit polyclonal antibodies was performed with the Zenon direct labeling kit, using an amended protocol based on one previously described for tubules (MacPherson et al. 2001). Briefly, intact tubules were placed on poly-l-lysine-treated slides in phosphate buffered saline (PBS) and fixed in 4% (v/v) paraformaldehyde for 30 min. Samples were then washed extensively for 3 ⫻ 15 min in PBS before permeabilisation with 0.3% (v/v) Triton X-100 in PBS for 30 min. Tubules were then incubated overnight in PBS/0.5% (w/v) Sigma cold fraction V bovine serum a lbumin/0.2% (v/v) T riton X-100 (PAT). Five microliters of Zenon rabbit IgG labeleling reagent (Molecular Probes) was added to 20 ␮l each of anti-TRP, -TRPL and -TRP␥ antibody solution; these mixtures were diluted in PAT in a 1:100 ratio and incubated for 5 min at room temperature. The mixtures were applied to appropriate tubule samples (as described in Figure 2 legend) and incubated for 1 hr in the dark. The samples were washed for 3 ⫻ 15 min before mounting in VectaShield mounting medium (Vector Labs). Whole-mount tubules were examined under fluorescence with a Leica microscope and a Zeiss Axiocam system. Transport (fluid secretion) assays: Malpighian tubules were isolated into 10-␮l drops of 1:1 mixture of Schneider’s medium and Drosophila saline (in millimolar per liter: NaCl, 117.5; KCl, 20; CaCl2, 2; MgCl2, 8.5; NaHCO3, 10.2; NaH2PO4, 4.3; HEPES, 15; glucose 20) under liquid paraffin, and fluid secretion rates measured as detailed (Dow et al. 1994a) under the different conditions described in the text. For maximum stimulation, 10⫺7 m CAP2b was added as solution in assay medium at 30 min. Measurements of intracellular calcium concentration [Ca2ⴙ]i : For each assay, 20–40 tubules from 4- to 14-day-old adults were dissected in Schneider’s medium 4 hr after heat shock (37⬚ for 30 min for 3 days). Tubules were pooled in 160 ␮l of the same buffer containing the apoaequorin cofactor, coelenterazine (2.5 ␮m final concentration); reconstitution of aequorin occurred upon incubation in the dark for 4–6 hr (Rosay et al. 1997). Bioluminescence recordings were made using a luminometer (LB9507, Berthold Wallac); recordings were made every 0.1 sec for each tube. Each tube of 20 tubules was used for a single data point: after recording [Ca2⫹]i levels, tissues were disrupted in 350 ␮l lysis solution [1% (v/v) Triton X-100/100 mm CaCl2], causing discharge of the remaining aequorin and allowing estimation of the total amount of aequorin in the sample. Calibration of the aequorin system and calculation of calcium concentrations were performed as previously described (Rosay et al. 1997). Mock injections with Schneider’s medium were applied to all samples prior to treatment with neuropeptide. [Ca2⫹]i values for each data set were calculated as described in the figure legends.

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Western blot analysis: Western blots using anti-TRP antibody (1:500 dilution), anti-TRPL antibody (1:500 dilution), and anti-TRP␥ antibody (1:500 dilution) were performed according to standard protocols of the BioRad Mini-Protean blotting system. Protein samples were prepared from tubules from each line (described under Drosophila stocks) homogenized in ice-cold SMART buffer (Xu et al. 1997) of 0.2% dodecyl-maltoside, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, 500 mm NaCl, 5 mm EDTA, 5 mm EGTA, 5 mm MgCl2, 2 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 0.1 mm PMSF, 10 mm NAPPi, 50 mm NaF, and 1 mm GTP (pH 7.5) and centrifuged at 12,000 ⫻ g for 5 min to remove debris. Thirty micrograms of protein was run on 4–15% precast gels (BioRad, Richmond, CA). Immunolabeling was visualized using Amersham ECL secondary antibody at 1:8000 dilution. Uniformity of loading was assessed on blots by Ponceau S staining. Band densities were quantified by ImageJ (http://rsb.info.nih. gov/ij/docs/menus/analyze.html) using the gel analysis macro of the ImageJ package after background correction. Statistics: Data are presented as mean ⫾ SEM. Where appropriate, the significance of differences between data points was analyzed using Student’s t-test for unpaired samples with P ⬍ 0.05 as the critical level.

RESULTS

trp, trpl, and trp␥ are expressed in tubules: Results from RT-PCR using head and tubule cDNA template (Figure 1) demonstrate expression of trp, trpl, and trp␥ in tubules. Expression of these calcium channels is not confined to Drosophila photoreceptors, suggesting that TRP, TRPL, and TRP␥ must also have an epithelial role. Expression of TRP, TRPL, and TRP␥ in tubule principal cells: Immunolocalization of TRP, TRPL, and TRP␥ in intact tubules demonstrates that these calcium channels are expressed in the principal cells of the tubule main segment (Figure 2, A, C, and E). The insets in Figure 2, A, C, and E, show magnified regions of tubules, which clearly indicate that stellate cells are not stained. Controls were performed using a tubule preparation with no primary antibody (Figure 2F) and also using available documented null alleles of trp and trpl, trp343, and trpl 302. Figure 2B shows that the trp null does not express TRP (compare Figure 2B with 2A); similarly, Figure 2D shows lack of anti-TRPL staining in trpl 302 tubules (compare Figure 2D with 2C). Given the lack of signal in the null mutants, the immunolocalization of TRP and TRPL in wild-type tubules is unlikely to be nonspecific. Comparison of TRP␥ staining with a control preparation that lacked primary antibody (no TRP␥ mutant was available) suggests that the fluorescence observed in Figure 2E was due to specific staining of TRP␥. Interestingly, while TRPL was clearly expressed only in the main segment, TRP and TRP␥ seem to be expressed in the transitional segment also. The tubule transitional segment is associated with calcium transport (Dube et al. 2000b); localization of TRP and TRP␥ to this region may suggest a role for these channels in transepithelial calcium transport.

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Figure 1.—Drosophila melanogaster Malpighian tubules express trp, trpl, and trp␥. RT-PCR using D. melanogaster genomic template (G) and head (H) and tubule (T) cDNA template with primers directed against trp (A), trpl (B), and trp␥ (C). Top arrows denote PCR products obtained with genomic DNA; bottom arrows denote PCR products obtained with cDNA. L, 1-kb DNA ladder (Invitrogen). Identity of products was confirmed by sequencing.

However, only the main segment of the tubule is fluid secreting (O’Donnell and Maddrell 1995); given the identical localization of all three channels in this region, it is likely that the TRP family channels are directly involved with calcium-signaling mechanisms that regulate fluid transport. CAP2b-stimulated fluid transport is sensitive to mutations in trpl: Given the localization of TRP and TRPrelated channels in the fluid-transporting region of the tubule, the impact of mutations in genes encoding these channels on fluid transport was assessed using available mutants. Figure 3 shows that CAP2b-stimulated fluid transport rates are significantly reduced in tubules from trpl 302 and trpl 302; trp 343. Fluid transport was also significantly reduced in another double mutant, trpl 302; trp CM (Reuss et al. 1997; data not shown). Significantly, while trp 301 is a severe hypomorph in photoreceptors, maximum rates of stimulated fluid transport are not statistically different from Oregon-R (Figure 3A:b). Furthermore, the trp null, trp 343 (Figure 3A:c), shows near-normal fluid secretion rates, which suggests that the significant reduction of fluid transport in the double mutant, trpl 302;trp 343, is due to the lack of TRPL. In all lines, basal secretion rates were unaffected compared to control. Therefore, while TRPL has a major role in stimulated fluid transport, maintenance of resting fluid transport rates is dependent on alternative signaling mechanisms. These data show that the impact of trp mutations on tubule secretion differs from that in photoreceptor function, which suggests that the requirement for functional TRP may be tissue specific. The intracellular [Ca2ⴙ]i rise induced by CAP2b is significantly altered by a mutation in trpl: As fluid transport in the tubule is stimulated by increases of intracellular calcium, resting and hormone-stimulated cytosolic calcium levels were measured in intact tubules from trp

and trpl alleles. The use of hsGAL4-directed aequorin transgene expression allows the measurement of [Ca2⫹]i in both principal and stellate cells in all regions of the tubule. However, specificity of the response is provided by a neuropeptide ligand: capa peptides, including CAP2, have been shown to raise [Ca2⫹]i levels only in principal cells of the main, fluid-secreting segment of the tubule (Rosay et al. 1997; Kean et al. 2002). In all mutants tested, basal [Ca2⫹]i levels were similar to control tubules (Figure 4A). In wild-type tubules, the CAP2b response, representing calcium influx (Rosay et al. 1997), is biphasic and composed of a primary, rapid peak followed by a slow secondary rise as shown in Figure 4A. This calcium signature is also observed using other neuropeptides of the capa family (Kean et al. 2002). We demonstrate that a mutation in trpl results in a distinct [Ca2⫹]i signature, which correlates with the corresponding transport phenotype. In the trp null, trp 343, the CAP2b-induced [Ca2⫹]i response is unchanged compared to wild type. However, the trp hypomorph, trp 301, shows a potentiated primary peak (Figure 4, A:b and B). These calcium signals correlate well with physiological function: maximum rates of CAP2b-induced fluid transport in these mutants are not significantly different from wild type (Figure 3), and increased [Ca2⫹]i in trp 301 may result in the altered kinetics (biphasic response) of stimulated fluid transport in this line. By contrast, trpl 302 tubules (Figure 4, A:d and B) show a significantly attenuated [Ca2⫹]i response. This strongly suggests that TRPL contributes to calcium-signaling mechanisms induced by CAP2b in the principal cell, thus resulting in the transport phenotype observed (Figure 3). Epithelial phenotypes of trp and trpl alleles are dependent on TRPL: Western analysis of tubule preparations

TRPL Function in Epithelial Transport

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Figure 2.—Immunocytochemical localization of TRP, TRPL, and TRP␥ in intact tubules. Fluorescent staining of intact tubules using anti-TRP, anti-TRPL, and anti-TRP␥ antibodies observed at ⫻50 magnification. Results shown are representative of many separate experiments. Tubule diameter can be taken as 35 ␮m in A–F. (A) Staining with anti-TRP antibody, wild-type tubules. (B) Staining with antiTRP antibody, trp 343 tubules. (C) Staining with anti-TRPL antibody, wild-type tubules. (D) Staining with anti-TRPL antibody, trpl 302 tubules. (E) Staining with anti-TRP␥ antibody, wild-type tubules. (F) Control with wild-type tubule, with no primary antibody. Segments of the tubule are delineated according to (Sozen et al. 1997): m, main segment; l, lower tubule; t, transitional segment; i, initial segment; u, ureter. Insets for A, C, and E show unstained stellate cells in tubules.

from trp and trpl alleles was performed to determine TRP, TRPL, and TRP␥ levels and to see, for example, if there were any effects of trp alleles on TRPL or TRP␥ expression (Figure 5). TRP: Results in Figure 5A show that TRP levels are significantly reduced in the trp hypomorph, trp 301. Also, TRP is virtually absent in the trp null tubules. In trpl 302 tubules, TRP levels are not significantly different from wild type, which suggests that the severe tubule phenotype observed in this line is not a result of lack of TRP protein. Conversely, in trp 301 and the trp null, trp 343, in which TRP levels are reduced, a near-normal tubule phenotype is observed (Figures 3 and 4). Thus, stimulated fluid transport and calcium responses by the tubule do not correspond to TRP levels in the different mutants. TRPL: Tubule TRPL levels are significantly reduced in the trp hypomorph, trp 301 (Figure 5B). Interestingly, in this allele, the kinetics of the fluid transport response differs from wild type (Figure 3, A:b) and a hypersensitive calcium response to CAP2b is observed (Figure 4, A:b and B). By contrast, the trp null, trp 343, exhibits tubule TRPL levels that are near wild-type levels (at ⵑ75%). In the trpl 302, TRPL levels are very low; a faint band

was detectable in tubule preparations (see also Figure 6B), in contrast to the previously reported complete null in trpl 302 heads (Niemeyer et al. 1996). Analysis of photoreceptor responses of aeq; trpl 302; hsGAL4 flies generated from trpl 302 shows that these behave as previously documented for the trpl 302 null (data not shown). This suggests that our experiments have been conducted on the strain correctly identified as trpl 302, in spite of our findings of low-level TRPL protein expression in trpl 302. Severely reduced levels of TRPL result in a direct correlation with both reduced fluid transport (Figure 3B) and calcium signaling (Figure 4B). Fluid transport is severely compromised in trpl 302 and trpl 302;trp 343, while calcium signaling is markedly reduced in trpl 302. Furthermore, where transport/calcium signaling is unaffected (e.g., trp 343), TRPL levels are similar to wild type (see figure legend for pooled data, Figure 5B). Therefore, on the basis of the TRPL levels observed in trpl 302, it is likely that the secretion phenotype of the trpl 302;trp 343 double mutant (Figure 3, A:e and B) is due to the lack of TRPL. TRP␥: Levels of TRP␥ in the tubule are not compromised in any of the trp or trpl alleles tested (Figure 5C). However, the lack of a TRP␥ mutant made it impossible

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Figure 3.—CAP2b-stimulated fluid secretion is attenuated in trpl mutants. (A) Basal rates of fluid secretion were measured for 30 min before stimulation with CAP2b (10⫺7 m; arrows). Stimulated fluid secretion rates were then measured for another 30 min. a–e show typical fluid secretion rates in tubules from each line. Results are expressed as mean fluid secretion rate (nl/min) ⫾SEM. (N ⫽ 7– 10). (a) Oregon-R; (b) trp 301; (c) trp 343; (d) trpl 302; and (e) trpl 302; trp 343. Basal fluid secretion rates are unaffected in all lines. (B) To aid comparisons among secretion rates of different lines, data shown in B are expressed as the percentage change of CAP2b-stimulated fluid secretion rate compared to basal [(maximal CAP2b-stimulated secretion rate-average basal rate over 30 min/average basal rate) ⫻ 100%] for each line, as denoted. Data are expressed as mean ⫾SEM (N ⫽ 8). Statistically significant inhibition of CAP2b-stimulated fluid secretion as compared to wild-type tubules (trpl 302, trpl 302; trp 343) is denoted by an asterisk. *P ⬍ 0.05 determined with Student’s t-test (unpaired samples).

to investigate a direct role of this channel in epithelial function. Taken together, the data here suggest that in the tubule TRPL channels may constitute major regulators of CAP2b-induced calcium signaling and transport.

A trpl transgene rescues the fluid transport phenotype in trpl 302: The data shown in the previous figures suggest that tubules require TRPL for function. Figure 6 shows that rescue of trpl 302 with a heat-shock trpl transgene construct rescues the secretion phenotype associated


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Figure 4.—CAP2b-induced [Ca2⫹]i rises in tubule principal cells in trp and trpl mutants. (A) Typical traces of changes in [Ca2⫹]i (in nanomolars) in tubule principal cells stimulated by 10⫺7 m CAP2b (arrows) in the following lines: (a) aeq; hsGAL4 (wild-type control); (b) aeq;hs GAL4;trp 301(trp 301); (c) aeq;hsGAL4;trp 343 (trp 343); and (d) aeq;trpl 302;hsGAL4 (trpl 302). Each sample contains 20 intact tubules. No change in the resting intracellular calcium concentration is seen in any of the mutants. (B) Pooled results of changes in tubule [Ca2⫹]i in trp and trpl mutants in response to 10⫺7 m CAP2b. Results are expressed in mean nanomolars [Ca2⫹]i ⫾SEM (N ⫽ 8) for background (open bars), CAP2b-stimulated primary peaks (solid bars), and CAP2bstimulated secondary peaks (shaded bars) for the lines described in A. The measure of secondary peak is taken as the average [Ca2⫹]i over 4 min poststimulation with CAP2b. CAP2b-stimulated primary peaks, which are significantly different from aeq; hsGAL4 tubules, are denoted by an asterisk, and statistically significant differences in secondary peaks compared to wild-type are denoted by two asterisks, where P ⬍ 0.05 determined with the Student’s t-test (unpaired samples).

with trpl 302 (Figure 6A). CAP2b-stimulated fluid transport in heat-shocked cntrplP{hstrpl};TM2/TM6 tubules is indistinguishable from that observed in Oregon-R tubules. We also demonstrate that introduction of the trp null, trp 343, into the trpl rescue line, cntrplP{hstrpl};trp 343, is not detrimental to the secretion phenotype compared to either Oregon-R or rescued trpl 302 tubules (Figure 6A). This confirms observations shown in Figure 3, where secretion in trp 343 null tubules is not significantly different from that of Oregon-R tubules. Importantly, the data shown in Figure 6A also suggest that, in the absence of TRP, functional TRPL modulates tubule function. Finally, although TRP levels are similar between Oregon-R and trpl 302 tubules (Figure 5), expression of the

trpl transgene in trpl 302 tubules is associated with restoration of TRPL levels to those of wild-type tubules (Figure 6B), and consequently, restoration of stimulated fluid transport levels. inaD is not expressed in tubules: INAD is an essential scaffolding component of the TRP-signaling complex in photoreceptors (Li and Montell 2000). Given that TRP-TRPL complexes may be operational in the tubule, we investigated expression of established components of the TRP-signaling complex (Montell 1998) in the tubule. RT-PCR using primers directed against published sequences for other TRP-complex-associated signaling proteins, inaC (Schaeffer et al. 1989) and norpA (Bloomquist et al. 1988), were performed. Sequencing

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M. R. MacPherson et al. Figure 5.—Western analysis of trp and trpl mutants. Tubule preparations from trp and trpl lines were subjected to Western analysis using anti-TRP, anti-TRPL, and anti-TRP␥ antibody. Data are from representative single blots probed with antibody; multiple experiments were carried out. Linearity of exposures was established (data not shown), band density quantified with ImageJ software, and data expressed as arbitrary gray units, ⫾SEM (N ⫽ 4–10) in graphs for each blot. An asterisk indicates data that are significantly different from wild-type tubules, where P ⬍ 0.05. (A) Levels of TRP (band size of ⵑ140 kD) in tubules (left); (right) graph showing pooled results from band intensity analysis. For consistency, lanes have been reoriented in the order shown: Oregon-R, trp 301, trp 343, and trpl 302. TRP is undetectable in the double mutant trpl 302; trp 343 (data not shown). (B) Levels of TRPL (band size of ⵑ130 kD) in tubules (left); (right) graph showing pooled results from band intensity analysis. For consistency, lanes have been reoriented in the order shown: Oregon-R, trp 301, trp 343, and trpl 302. t-Test analysis of data points for trp 343 vs. those for Oregon-R showed that these data were not significantly different (P ⫽ 0.15 for trp 343). TRPL levels in trpl 302; trp 343 are similar to those for trpl 302 (data not shown). (C) Levels of TRP␥ in tubules (band size of ⵑ130 kD; left); (right) graph showing pooled results from band intensity analysis. Samples were run in the order shown: Oregon-R, trp 301, trp 343, and trpl 302.

of PCR products verifies that both these genes are expressed in tubules (data not shown). Furthermore, norpA has recently been shown to play a role in epithelial transport (Pollock et al. 2003), confirming a role outside phototransduction. Tubules also express protein kinase C and phospholipase C genes not associated with photoreceptors, namely, pkc53e (Rosenthal et al. 1987) and plc21 (Shortridge et al. 1991; data not shown). Therefore, while tubules are expected to express those genes not associated with excitable cells, some genes previously associated only with phototransduction or CNS function are also expressed in these transporting epithelia. Surprisingly, then, data from RT-PCR experiments for inaD using a head/tubule cDNA template (Figure 7) demonstrate that this gene is not expressed in the tubule, although strong expression is observed in the head, as expected. Furthermore, PCR was carried out under varying conditions: (1) utilizing two sets of primers made to different regions of the gene and (2) performing PCR under different cycle conditions at annealing temperatures over a large range (40⬚–55⬚) and over a different total number of cycles (40 maximum). The tubule template quality was also verified using primers to a different gene. Under these conditions, no cDNA

product was ever observed using the tubule template, although the correct product was always obtained in the head. These results are supported by expression data obtained by microarray analysis of the adult tubule (Wang et al. 2004), where inaD is shown to be depleted in the tubule compared to the rest of the fly (inaD in tubules, 9.1 ⫾ 3; inaD in the rest of the fly, 63.0 ⫾ 5.5; data expressed as Affymetrix signal ⫾SEM; N ⫽ 5 separate biological replicates). The lack of inaD expression therefore suggests that although many of the components of the TRP-signaling complex may have widespread expression, INAD is tissue specific. DISCUSSION

Until recently, organotypic roles for TRP, TRPL, and TRP␥ channels have been demonstrated only in photoreceptor function in Drosophila, with an additional role for TRP in olfactory adaptation (Stortkuhl et al. 1999). However, given the wide tissue expression of vertebrate TRP channels (Harteneck et al. 2000), it seemed possible that these channels also contribute to diverse physiological processes.


Figure 6.—Rescue of trpl 302 by trpl transgene restores wildtype fluid transport. (A) Fluid transport assays were carried out on tubules from Oregon-R (black), cntrpl[hstrpl];TM2/ TM6B (non-heatshocked control, orange), hs cntrpl[hstrpl]; TM2/TM6B (heat-shocked, red), hs cntrpl[hstrpl];trp343 (blue), and trpl 302 (green). Note that “cntrpl ” flies are equivalent to trpl 302. Tubules were stimulated with CAP2b at 30 min (arrow). Data are expressed as mean secretion rate (nanoliters/minute) ⫾SEM (N ⫽ 8–10). Existence of chromosome 3 balancers TM2 and TM6 is not detrimental to the secretion phenotype (Pollock et al. 2003; data shown here); compare secretion rates of hs cntrpl[hstrpl];TM2/TM6B with those of Oregon-R. To rule out effects of rearing the cntrpl[hstrpl];TM2/ TM6B and any derivative stocks at 18⬚, fluid transport assays were carried out on Oregon-R flies reared at 18⬚ and 25⬚. No significant differences were observed (data not shown). (B) Tubule preparations from Oregon-R (lane i), heat-shocked cntrpl[hstrpl];TM2/TM6B (two separate preparations run in lanes ii and iii), and trpl 302 (lane iv) lines were subjected to Western analysis using anti-TRPL antibody as in Figure 5. Data shown are of representative single blots probed with antibody; several experiments were carried out. Bands of ⵑ130 kD in tubules were detected in Oregon-R, cntrpl[hstrpl];TM2/TM6B (heat-shocked), and very faintly in trpl 302 (see also Figure 5). Similar levels of TRP expression were observed in all lines (data not shown; see Figure 5, Oregon-R and trpl 302).

Expression of trp, trpl, and trp␥ in tubules and localization of these gene products to principal cells containing the electrogenic vacuolar H⫹-ATPase (Dow 1999) suggest a role for these calcium channels in fluid transport (Figures 1 and 2). Interestingly, while expression of TRPL is confined to principal cells in the main, fluidsecreting segment, TRP and TRP␥ are expressed in principal cells in the transitional segment. The initial and transitional segments of anterior tubules have been shown to be the major calcium-transporting region (Dube et al. 2000b), with the initial segment storing up to 30% of total body calcium (Dube et al. 2000a). As such, TRP and TRP␥ may have roles in transepithelial calcium transport. Using available trp and trpl mutants, we have investigated direct roles for TRP and TRPL channels in neuropeptide-induced calcium signaling and fluid transport.

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Figure 7.—inaD is not expressed in tubules. RT-PCR using D. melanogaster genomic template (G) and head (H) and tubule (T) cDNA template with primers directed against inaD. Top arrow denotes PCR products obtained with genomic DNA; bottom arrow denotes PCR products obtained with cDNA. L, 1-kb DNA ladder (Invitrogen). A PCR product of expected size is observed only using head template cDNA; no product is obtained with tubule cDNA under a range of PCR conditions as described in the text (see results).

Evidence for a role of TRPL in tubule function is provided by the observations that both CAP2b-stimulated fluid transport and calcium responses are severely reduced by a trpl mutation while two protein null alleles of trp (trp 301 and trp 343), which result in severe eye phenotypes, had little or no effect on tubule function. Comparison of data obtained for fluid transport (Figure 3B) and calcium signaling (Figure 4B) with Western analysis of TRPL (Figure 5B) shows that fluid secretion and calcium signaling accurately mirror the level of TRPL expression across all the alleles tested. Interestingly, while the trpl 302 mutation, a result of an amber stop codon, is documented to be a complete null at least in the head (Niemeyer et al. 1996), very low residual TRPL levels are detectable in trpl 302 homozygote tubules (Figures 5B and 6B). Stop codon readthrough occurs at rates of 10⫺4 in Drosophila (Sato et al. 2003) and can occur at much higher rates for specific genes, including hdc in tracheal epithelial cells (Steneberg et al. 1998; Steneberg and Samakovlis 2001) and Kelch in eggchamber ring canals (Robinson and Cooley 1997). It may be that a single stop codon without a favorable 3⬘ context in the trpl gene is sufficient to confer a functional, but not a protein, null phenotype in tubules; indeed, UAGG, the tetranucleotide in the trpl 302 mutation, is known to be a particularly poor stop signal in eukaryotes (Brown et al. 1990). In the light of these findings, trpl 302 can be considered a hypomorph at least in the context of the tubule. The importance of TRPL in tubule function is con-

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firmed by rescue of the trpl 302 with a trpl transgene, which restores stimulated fluid transport levels to those of wild type. Introduction of the trp null (trp 343) into the trpl rescue line does not impact on the rescued transport phenotype by these tubules. Previous work has shown that loss of either the primary or the secondary calcium signal in tubules is associated with reduction of fluid transport. Mutations in IP3R (Pollock et al. 2003), pharmacological blockade of L-type/cyclic nucleotide gated channels (MacPherson et al. 2001; Broderick et al. 2003), and reduction of external calcium (Rosay et al. 1997) is sufficient to reduce or abolish calcium signaling, with associated loss in fluid transport. Therefore, it is likely that rescue of trpl 302 results in restoration of the wild-type calcium signal and fluid transport. We suggest that possible TRP-TRPL heteromultimers (Xu et al. 1997) may mediate fluid secretion in wildtype tubules, but that in the absence of TRP protein (as in trp 301, trp 343 ), TRPL homomultimers (or TRPL-TRP␥) can also support tubule function. TRP and TRPL have been shown to act as separate channels in wild-type light-activated currents (Reuss et al. 1997), suggesting the existence of homomultimers. A role for functional TRPL-TRPL complexes is further supported by work in trp 301 photoreceptors, which suggested that TRPL-TRPL homomultimers can account for the measured current (Hardie et al. 1997). Given the estimated 10-fold increase in TRP compared to TRPL channels in photoreceptors, with TRP forming the major conductance component via probable TRP-TRP homomultimers, it follows that increased severity of the trp phenotype compared to that of trpl will be observed (Leung et al. 2000; Montell 2001). This may explain the very different trp mutant phenotypes observed between photoreceptors and tubules. TRPL has also been proposed to stabilize TRP in a heteromultimeric complex (Leung et al. 2000). This could explain why the absence of TRPL, in spite of wildtype levels of TRP (in trpl 302), results in a severe epithelial phenotype. In summary, data presented here suggest that in tubules TRPL plays a singular role in modulating calcium signaling and fluid transport. It is also possible that a third channel, encoded by trp␥, contributes to the tubule phenotype. As TRP␥ has been shown to interact with TRPL (Xu et al. 2000), and TRP␥ is expressed at wild-type levels in all trp and trpl alleles tested (Figure 5C), it is possible that TRPL-TRP␥ heteromultimers constitute a major channel in tubules. These functional channel complexes may also substitute for TRP-TRPL channels in the absence of TRP (trp 301, trp 343 ). The TRP-signaling complex is localized and assembled by the scaffolding protein, INAD, in photoreceptors (Li and Montell 2000). If TRP is also organized into signaling complexes in tubules, the scaffolding proteins involved in complex assembly must differ from those of photoreceptors in that inaD is not detectably expressed. Recent work has demonstrated that TRPL is

not a component of the INAD complex (Paulsen et al. 2000). If TRPL, as opposed to TRP, is indeed the major regulator of calcium signaling and fluid transport in tubules, INAD may not be required in this tissue. Which, then, are possible candidates for TRP complex scaffolding proteins in tubule principal cells? Drosophila is known to contain at least 86 PDZ (postsynaptic density protein-95, discs large, ZO-1)-containing proteins, the majority of which are encoded by novel genes (SMART database, EMBL). Known PDZ-containing proteins in vertebrate renal epithelia include the sodium/hydrogen exchanger regulatory factor, NHERF. Intriguingly, NHERF has been shown to assemble TRP4 and phospholipase C (Tang et al. 2000). We have verified expression of the Drosophila NHERF gene sip1 (Spradling et al. 1999) in tubules; however, reporter gene expression of a P-element insertion in NHERF (Sip106373) shows expression of this gene only in tubule type II (stellate) cells (data not shown). As such, Drosophila NHERF is unlikely to be a scaffolding partner for TRP and TRP-related proteins in tubules, given that these proteins are located only in principal cells. Several genes encoding scaffolding proteins have been implicated in epithelial development in Drosophila, for example, arc, Bazooka, and scribbled. Bazooka has been shown to colocalize apically with atypical protein kinase C (Wodarz et al. 2000). Both Baz and aPKC are expressed in adult tubules (data not shown); as PKC function is important in signaling associated with TRP function, Baz may encode a potential candidate for TRP complex scaffolding proteins in tubules. We have demonstrated a novel role for trp, trpl, and trp␥ in transporting epithelia and provided the first demonstration of an organotypic role for members of the Drosophila TRP family outside the eye. The availability of the complete Drosophila genome, together with the molecular genetic tools in tubule work, allow a comprehensive dissection of the role of calcium channels in transporting epithelia. As such, this may allow inferences to be drawn regarding calcium homeostasis and calcium signaling in vertebrate renal function. We are very grateful to W. L. Pak, Purdue University, for the kind gift of trp 343, and C. S. Zuker and R. Hardy, University of California at San Diego, for trpl rescue lines and for helpful advice. In the early stages of this work, we received aliquots of anti-TRPL and anti-TRP antibodies as kind gifts from C. Montell, Johns Hopkins Medical School, and C. S. Zuker. We are very grateful for these reagents. This work was supported by the United Kingdom Biotechnology and Biological Sciences Research Council in the form of grants (S.D., J.A.T.D., R.C.H.), committee studentships (M.R.M., T.D.S.), and a David Phillips Fellowship (S.D.); the Wellcome Trust ( J.A.T.D., R.C.H.); and the UK Medical Research Council (R.C.H.)

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APPENDIX

Figure A1.—Summary of the crossing scheme to generate flies homozygous for each of the heatshock GAL4 drivers on chromosome 2 (hsGAL4), the trp 301 allele on chromosome 3, and the UASapoaequorin reporter line on the X chromosome (UAS::aeq). Y indicates the Y chromosome. Other markers (homozygous lethal) used are CyO, the Curly of Oster balancer chromosome, which prevents recombination on chromosome 2 and carries a curly-wing dominant visible marker; TM6, a balancer chromosome for chromosome 3, carrying a dominant “tubby” phenotype; Bl, Bristle, a dominant mutation on chromosome 2, carrying a dominant stubbly bristle phenotype; TM2, a balancer chromosome for chromosome 3, carrying a dominant pigmented ebony phenotype. For trpl on chromosome 2, a crossing scheme involving the heat-shock GAL4 driver on chromosome 3 and the UAS-apoaequorin reporter line on the X chromosome (UAS::aeq) was used (Broderick et al. 2003).