Molecular characterization of an epithelial Ca²⁺ channel-like gene from crayfish Procambarus clarkii

The crustacean molting cycle serves as a model for studying mechanisms of calcium (Ca²⁺) homeostasis(Wheatly, 1999; Wheatly et al., 2002) because directionality and magnitude of transepithelial Ca²⁺ flux are dramatically regulated. Crustaceans exhibit Ca²⁺ balance in intermolt, transition to net loss in premolt associated with cuticular demineralization and, following ecdysis, switch to impressive postmolt uptake to calcify the new exoskeleton. Crustaceans residing in different environments have developed appropriate strategies for Ca²⁺ conservation and acquisition. The freshwater (FW) crayfish, Procambarus clarkii,exhibits unique adaptations for evolution into Ca-deficient (<1 mmol l^–1) inland waters, including the ability to dilute the urine(Wheatly and Toop, 1989) and impressive postmolt branchial net Ca²⁺ uptake. Because the crayfish maintains circulating hemolymph Ca²⁺ levels above ambient,unidirectional Ca²⁺ influx is necessarily active.

There is an accepted model for transcellular Ca²⁺ influx in animal models. First, because intracellular (IC) Ca²⁺ concentration is maintained at micromolar levels and the interior of a cell is negatively charged, apical Ca²⁺ entry is passive, involving simple diffusion through Ca²⁺ channels or via carrier-mediated/facilitated diffusion. Meanwhile, basolateral Ca²⁺ export is active and effected by the Na⁺/Ca²⁺ exchanger (NCX) and/or a plasma membrane Ca²⁺ ATPase (PMCA). This model has been confirmed in crustacean epithelia (reviewed by Wheatly et al., 2002). Active basolateral processes have received greater attention (Flik et al., 1994; Zhuang and Ahearn, 1996; Wheatly et al., 1999) than apical mechanisms (Ahearn and Franco,1990; Ahearn and Franco,1993; Zhuang and Ahearn,1996).

Crustacean apical Ca²⁺ channels have received relatively little experimental attention, even though, from an energetic perspective, apical Ca²⁺ entry is the rate-limiting step (`gatekeeper') in epithelial Ca²⁺ uptake and, as such, the best target for regulation. Ca²⁺ channels have been classified as voltage-operated,ligand-gated, mechanosensitive, or Ca²⁺ store-operated based largely on electrophysiological and pharmacological properties. Physiological studies in crustaceans have variously suggested that apical Ca²⁺channels are inhibitable by Ba²⁺/La³⁺ and verapamil, and are membrane potential-dependent (Ahearn and Franco, 1993; Ahearn and Zhuang, 1996; Zhuang and Ahearn, 1996; Zilli et al.,2000). Comparable studies in FW fish have concluded that a mixed population of voltage-dependent (Comhaire et al., 1998) and voltage-independent Ca²⁺ channels[trout (Perry and Flik, 1988)and tilapia (Flik et al.,1993)] exist in gill and intestine. Meanwhile, the apical Ca²⁺ channels responsible for Ca²⁺ influx in epithelial tissues remained elusive for many years. Although several distinct voltage-dependent Ca²⁺ channels are present in the apical membrane of epithelial cells (Yu et al.,1992), apical administration of known Ca²⁺ channel antagonists failed to block Ca²⁺ reabsorption.

A novel non-voltage-gated epithelial Ca²⁺ channel (ECaC) was finally cloned from rabbit kidney(Hoenderop et al., 1999a) that was exclusively expressed in Ca²⁺ absorptive epithelia responsive to calciotropic hormones, namely distal parts of the nephron (connecting tubules, cortical collecting duct), small intestine and placenta. Subsequently, the human kidney (ECaC1)(Müller et al., 2000a; Müller et al., 2001) and intestine orthologues (calcium transport protein, CaT1/ECaC2)(Peng et al., 2000a; Hoenderop et al., 2000a) were identified. Subsequent studies confirmed that ECaC1 and ECaC2 are localized adjacent to each other on the same chromosome, suggesting that they are duplications of a common ancestral gene(Müller et al., 2000b). ECaCs are calcium-selective members of the vanilloid subfamily of the transient receptor potential superfamily (TRPV) of channels. Recently,standard nomenclature for this family has been recommended (TRPV5 for ECaC1/CaT2 and TRVP6 for ECaC2/CaT1)(Montell et al., 2002). Other members encode nonselective cation channels that function as heat sensors[capsaicin receptor, VR1 (TRPV1), VRL-1 (TRPV2) and TRPV3] or osmoreceptors[OTRPC4/VR-OAC/VRL-2/TRP12 (TRPV4) (Peng et al., 2001; Peng et al.,2003a; Peng et al.,2003b)].

ECaCs have recently been cloned from rainbow trout(Perry et al., 2003; Shahsavarani et al., 2006),pufferfish (Qiu and Hogstrand,2004) and zebrafish (Pan et al., 2005). Seemingly there is a single ECaC gene in fish,suggesting that gene duplication occurred after the divergence of fish and mammals (Pan et al., 2005). In two of the fish studies (Qiu and Hogstrand, 2004; Pan et al.,2005), ECaC upregulation was associated with increased Ca²⁺ uptake rate.

The aim of the present study was to characterize crustacean ECaC. We selected antennal gland (kidney) because of significant intermolt Ca²⁺ reabsorption, suggesting high ECaC abundance. Owing to loading of external FW prior to ecdysis (shedding) and the subsequent hemodilution,postfiltrational Ca²⁺ reabsorption (and concomitantly ECaC expression) is predicted to increase in pre- and postmolt stages compared with intermolt.

Crayfish Procambarus clarkii (Girard) were obtained and maintained, and molting stages were assessed as outlined in prior laboratory publications (Gao and Wheatly,2004). ECaC was cloned from postmolt antennal gland (kidney) using reverse transcription-polymerase chain reaction (RT-PCR), followed by rapid amplification of cDNA ends (RACE) strategy. Relative ECaC expression was documented in a range of postmolt tissues (using real-time PCR and quantitative RT-PCR), including cardiac (heart) muscle, axial abdominal (tail)muscle, antennal gland, gill, hepatopancreas (liver) and egg. These techniques were also used to quantify relative expression of ECaC in antennal gland during different molting stages. ECaC expression was subsequently localized in transverse sections of antennal gland using in situhybridization.

After dissection, tissues were frozen immediately in liquid N₂and stored at –80°C. Total RNA was isolated using the Trizol reagent(Invitrogen). Briefly, 0.2 g of tissue was finely ground in liquid N₂ and lysed by adding 1.0 ml of Trizol reagent. The lysates were allowed to incubate at room temperature (RT) for 5 min. Then, 1.0 ml chloroform was added, followed by vigorous vortexing for 15 s. Samples were then incubated for 5 min at RT and centrifuged for 15 min at 13 400 g. Following removal of the aqueous phase and addition of 1.5 ml of isopropanol, samples were placed at –80°C overnight and then centrifuged for 15 min at 13 400 g. The RNA pellets were washed with 1.5 ml 75% ethanol, sedimented for 5 min at 7500 gand air-dried for 10 min before being dissolved in diethyl pyrocarbonate(DEPC)-treated water and stored at –80°C. RNA was quantified by RNA 6000 Nano assay in the Agilent 2100 Bioanalyzer (Applied Biosystems, Foster City, CA, USA). mRNA was separated from total RNA using an oligo-dT cellulose column (Stratagene).

First-strand cDNA was reverse transcribed from 400 ng of mRNA from postmolt antennal gland using the SuperScript II RNase H-reverse transcriptase(Gibco-BRL, Gaithersburg, MD, USA) with oligo(dT) 12–18 as primer. Based on four published ECaC sequences from human ECAC1 (GenBank accession number AJ271207; now known as TRPV5), rabbit ECaC (AJ133129), rainbow trout ECaC (AY256348) and pufferfish ECaC (AY232821), two degenerate primers,5′-GGVCCCTTCCATGTYATYCTTATY-3′ (sense) and 5′-AGGWACCARCGCTCCCCCAGRCC-3′ (antisense), were designed,corresponding to nucleotides 1401-2024 in rainbow trout and 1398-2031 in pufferfish. These primers targeted a fragment of approximately 626 bp located between transmembrane domain 3 and a positive protein kinase C phosphorylation site after transmembrane domain 6. PCR (total volume 50 μl) included 2μl of first-strand cDNA from postmolt antennal gland, 20 mmol l^–1 Tris HCl (pH 8.4), 50 mmol l^–1 KCl, 1.5 mmol l^–1 MgCl₂, 0.2 mmol l^–1 dNTP mix, 0.1-0.2 μmol l^–1 of each primer and 2.5 units of Taq DNA polymerase (Gibco-BRL). RT-PCR cycles were performed at 94°C for 3 min, followed by 30 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, and a final cycle of 72°C for 10 min. Negative controls in which reactions contained no template cDNA were included. RT-PCR products were analyzed by electrophoresis on a 1.0% agarose gel with 0.5 μg ml^–1 of ethidium bromide in 1× TAE buffer [40 mmol l^–1 Tris, 40 mmol l^–1 sodium acetate and 1 mmol l^–1 ethylenediamine-tetraacetic acid (EDTA), pH 7.2]. The DNA bands were visualized with ultraviolet light.

Subsequently, 3′ and 5′ RACE systems for rapid amplification of cDNA ends (Invitrogen) were used to amplify the 3′ and 5′ ends. For the 3′ RACE, a gene-specific primer,5′-GCTGCCTCGCTGCCTGTG-3′, and a nested primer,5′-GTGTGGCCTGGAGTACGGTCTGG-3′, were used. For the 5′ RACE,two gene-specific primers, 5′-CACCTCGCTGACCCTGAACACG-3′ and 5′-ACGCACAGCAGCACCACCAG-3′, and a nested primer,5′-CAGGGAGGCATAGGTGATAAGGAT-3′, were designed. The PCR conditions were the same as described above. PCR products were ligated to PCR 2.1 vector(Invitrogen) and transformed into INVF host cell (Invitrogen). Each clone was digested with appropriate restriction enzymes and subcloned for sequencing. Two independent clones were sequenced from both ends.

The cDNA clones were sequenced by automated sequencing (ABI PRISM 377, 3100 and 3700 DNA sequencers; Davis Sequencing, Davis, CA, USA). The complete sequence was assembled with DNASTAR (DNASTAR, Madison, WI, USA). Sequence homology was analyzed through the GenBank database using the BLAST algorithm(Altschul et al., 1990). Analysis of the phylogenetic relationships among all the ECaC sequences as well as with other channel proteins was undertaken by the Jotun Hein method of MEMALIGN (DNASTAR), which evaluates and scores all ancestors by pair alignment as well as concensi between progeny.

Real-time PCR was used to quantitate relative expression of ECaC mRNA in a range of epithelial and non-epithelial tissues, as well as to document relative expression in antennal gland during different molt stages (compared with intermolt). For each sample the amount of mRNA was quantified relative to 5 μg of total RNA by real-time RT-PCR. The TURBO DNA-free kit (Ambion,Austin, TX, USA) was used to eliminate genomic DNA contamination prior to RT-PCR. DNA-free total RNA from each tissue (1 μg) was reverse transcribed with random hexamers to create cDNA using the TaqMan Reverse Transcription Kit(Applied Biosystems). The resulting cDNA was employed in PCR amplifications optimized with gene-specific primers containing a fluorescent reporter molecule (SYBR Green PCR core reagents kit; Applied Biosystems).

Oligonucleotide primers for the crayfish ECaC gene[5′-GTAGCTACGCCCAGGGTCACAGG-3′ (sense) and 5′-TCGATGAGCAGGGAGATGATGTC-3′ (antisense)] (see Fig. 1 for primer location)were chosen with the Primer Express™ software (Applied Biosystems). The integrity of the cDNA from the tissues was checked by the presence of a fragment of 18s rRNA gene. The 18s rRNA primers (sense 5′-TGGTGCATGGCCGTTCTTA-3′ and antisense 5′-AATTGCTGGAGATCCGTCGAC-3′) were designed from Procambarus clarkii 18s rRNA gene (accession number AF436001). The reaction mixture(20 μl) contained 2 μl of 10× SYBR Green PCR buffer, 3 μl of 25 mmol l^–1 MgCl₂, 2 μl of dNTP mix (2.5 mmol l^–1 dATP, 2.5 mmol l^–1 dCTP, 2.5 mmol l^–1 dGTP and 5 mmol l^–1 dUTP), 0.125 μl of AmpliTaq Gold (5 U μl^–1), 0.25 μl of AmpErase UNG 91 U μl^–1), 2 μl of template cDNA and 4.08 μl of each 5μmol l^–1 primers in water.

Real-time PCR reactions were performed in a 96-well microtiter plate using the relative quantification ΔΔCt method. The threshold cycle (Ct)represents the PCR cycle at which an increase in SYBR Green fluorescence can first be detected above a baseline signal. Real-time PCR conditions were as follows: 50°C for 2 min and then 95°C for 10 min for one cycle,followed by 40 cycles of 95°C for 15 s, and then 60°C for 1 min on an ABI prism 7900HT sequence detection system (Applied Biosystems). For an 18s rRNA reaction mix, 4.08 μl of each 1 μmol l^–1 primers and 2 μl of 0.1× diluted cDNA were used. The cDNA sample was analyzed in triplicate and the fold-change relative to the control tissue (liver for differential tissue expression) or condition (intermolt for relative expression in antennal gland with molting stage) was calculated based on the relative quantification ΔΔCt method. Relative quantification (RQ)was performed by normalizing the Ct values of each sample gene with the Ct value of the endogenous control 18s rRNA gene (ΔCt), and was finally calculated using ΔCt of the control tissue/condition as calibrator.ΔΔCt corresponds to the difference between the ΔCt of the gene of interest and the ΔCt of the endogenous control 18s rRNA. Fold-change in expression was calculated as RQ=2^ΔΔCt. Several controls were performed to ensure proper PCR amplification. Negative controls consisting of no template and PCR performed on samples not subjected to reverse transcription were run on every plate. In addition, efficiency controls were performed to confirm that the target sequence amplified at the same efficiency as the endogenous control (18s rRNA) for each primer set tested.

For the quantitative RT-PCR, two primers,5′-GGCTGCCAAGGAGGGTAA-3′ (sense) and 5′-CTCTCCTGGGCCACCCT-3′ (antisense), were designed corresponding to nucleotides 868-1900 bp and targeting a fragment of 1032 bp from transmembrane domain 1 to transmembrane domain 6(Fig. 1). RT-PCR reactions (PCR total volume 50 μl) included 2 μl of first-strand cDNA, 20 mmol^–1 Tris HCl (pH 8.4), 50 mmol^–1 KCl, 1.5 mmol^–1 MgCl₂, 0.2 mmol^–1 dNTP mix, 0.1–0.2 μmol^–1 of each primer and 2.5 units of Taq DNA polymerase (Gibco-BRL). RT-PCR cycles were: 94°C, 3 min, followed by 30 cycles of 94°C for 30 s, 55°C for 1 min, 72°C for 1 min, and a final cycle of 72°C for 10 min. RT-PCR reactions contained primers to amplify a 518 bp fragment of 18s rRNA as control. PCR products from real-time and quantitative PCR (15 μl) were analyzed by electrophoresis on a 1.0%agarose gel with 0.5 μg ml^–1 of ethidium bromide in 1× TAE buffer (40 mmol l^–1 Tris, 40 mmol l^–1 sodium acetate and 1 mmol l^–1 EDTA, pH 7.2). The DNA bands were visualized with ultraviolet light.

In situ hybridization was performed as outlined previously[Wheatly et al. (Wheatly et al.,2004), as adapted for crayfish from a mammalian protocol, Key et al. (Key et al., 2001)]. Antennal glands were dissected from crayfish at different molting stages and placed in pre-chilled 4% paraformaldehyde [PFA (w/v), 0.1 mol l^–1 sodium acetate, pH 6.5–7.5] for one day. The tissue was then placed in 4% PFA/20% sucrose for 3–6 days at 4°C. After fixation, the tissues were wrapped tightly in aluminium foil, placed in a ziploc bag and stored at –80°C until processing. The tissue blocks were removed from the freezer and placed in the Cryostat (Cm3050; Leica,Nussloch, Germany) at –20°C for 30 min, before being mounted on cold specimen holders with tissue-freezing medium. Serial 20 μm transverse sections were taken, transferred on 0.2% gelatin-coated slides and stored at–80°C.

In situ hybridization was used to localize and visualize ECaC mRNA sequences by hybridizing a complementary nucleotide probe designed from the crayfish antennal gland ECaC cDNA sequence (GenBank accession number AY452713). The antisense of this probe sequence was 5′-GAACACGCACAGCAGCACCACCAGGGAGGCATAGGT-3′, and the sense of this probe sequence (used as a negative control for nonspecific hybridization) was 5′-ACCTATGCCTCCCTGGTGGTGCTGCTGTGCGTGTTC-3′. The ECaC probe was 36 bp in length and was located in the transmembrane domain 3 region (see Fig. 1 for probe location).

The probe (20 pmol l^–1) was ³⁵S labeled with a terminal deoxynucleotidyl transferase (TDT) kit (Roche Molecular Biochemicals,Indianapolis, IN, USA). The oligonucleotide probe was incubated at 37°C for 90 min as follows: 5 μl of 4 pmol μl^–1 probe, 4μl of ddH₂O, 5 μl of 5× terminal transferase buffer, 5μl of CoCl₂, 2 μl of TdT (400 U) and 4 μl of ³⁵S-dATP (1250 Ci mmol l^–1; NEN Life Sciences,Boston, MA, USA). Then, 50 μl of 0.1 mol l^–1 Tris-HCl/TEA(triethanolamine)/EDTA was added. Unincorporated radiolabel was removed in a Mini Quick Spin DNA column (Roche Molecular Biochemicals), and the probe was diluted in hybridization buffer [4× SSC, 50% formamide (v/v), 1×Denhardt, 250 μg ml^–1 yeast tRNA, 10% dextran sulfate(v/v), 10 mmol l^–1 DTT, 500 μg ml^–1boiled salmon sperm DNA] to yield approximately 0.5×10⁶ cpm 100 μl^–1, and stored at –20°C before use.

For the hybridization, the slides were pre-washed in 0.01 mol l^–1 phosphate-buffered saline (PBS; pH 7.4) for 15 min, then in 2× SSC (0.3 mol l^–1 NaCl, 0.03 mol l^–1 sodium citrate) for 30 min at RT. After washing,approximately 30 μl of dilute sterile probe hybridization solution was added to each tissue section. The slides were kept inside a humid chamber in the incubator at 37°C overnight. After hybridization, the slides were postwashed with 1× SSC at RT for 1 h, followed by three washes with 1× SSC at RT for 15 min and then with 1× SSC at 50°C for 30 min. Finally, the slides were gently rinsed with 0.01 mol l^–1PBS for 10 min at RT. After washing, the slides were placed on the slide warmer at a very low setting for 10 min, prior to being placed in the Fuji Film BAS Cassette with a BAS-IIIs imaging plate along with high and low standards to convert intensity to μCi. Fuji Films were scanned after 1–3 days of exposure in a Fuji FLA-2000 scanner (Fuji Photo Film, Tokyo,Japan) attached to a Power Macintosh 7300/200 computer with Image Gauge v3.3 software (Apple Computer, Cupertino, CA, USA). Parallel sections were examined with standard histology (staining with cresyl violet) and photographed with a KODAK EDAS 290 digital camera to correlate radioactive labeling with cellular structure of the antennal gland.

A pair of degenerate primers was successfully used in amplifying a 626 bp fragment of cDNA from crayfish postmolt antennal gland. A GenBank search confirmed that this fragment matched exclusively with ECaCs from rainbow trout(81%), pufferfish (80%), human (65%) and rabbit (66%). The deduced amino acid sequence showed 82–84% homology with rainbow trout and pufferfish and 63–71% homology with human and rabbit. Based on the 626 bp partial sequence, a 1399 bp fragment from 5′ RACE and a 662 bp from 3′RACE were successfully amplified.

The complete nucleotide sequence and deduced amino acid sequence of crayfish antennal gland ECaC (referred to as ECaC1, now TRPV5) is shown in Fig. 1. This 2687 bp nucleotide sequence consists of an open reading frame of 2169 bp, coding for 722 amino acid residues with a predicted molecular mass of 81.7 kDa. There is a 124 bp non-coding region at the 5′ terminal and a 394 bp non-coding region with a poly(A) tail at the 3′ terminal. A GenBank search using the BLAST algorithm revealed that the crayfish antennal gland ECaC matched exclusively at the mRNA level with ECaC from rainbow trout (76%), pufferfish (78%), human(62%) and rabbit (56%). The deduced amino acid sequence of crayfish antennal gland ECaC matched with published ECaCs; specifically, the percent homology was greater with fish (80–82%) and rainbow trout (80%) than mammalian species (52–60%; Fig. 2). A search in the protein database also revealed a significant but low homology(20%) to previously published ion channels, including rat capsaicin receptor(VR1, now TRPV1; VRL, now TRPV2) (Caterina et al., 1997; Caterina et al.,1999) and mouse growth factor-regulated channel (GRC, now TRPV2)(Kanzaki et al., 1999) and other transient receptor potential (TRP)-related ion channels(Birnbaumer et al., 1996).

The hydropathy profile of crayfish ECaC exhibits a putative secondary structure common to other ECaCs (Fig. 3) consisting of six transmembrane-spanning domains, a short hydrophobic stretch predicted as the pore-forming region between transmembrane domains 5 and 6, and three ankyrin repeat domains(Fig. 3). Phylogenetic analysis between crayfish ECaC and other channels(Fig. 4) suggests that crayfish ECaC belongs to the same family as ECaCs from human, rat, mouse,rabbit, rainbow trout and pufferfish; of these, the closest phylogenetic relationship is with rainbow trout and pufferfish. There is a lower phylogenetic relationship with other ion channels [transient receptor potential channel (TRPC); GRC/VRL, now TRPV2; and VR1, now TRPV1].

Real-time (Fig. 5A) and quantitative RT-PCR data (Fig. 5B) confirmed that, relative to 18s rRNA, ECaC mRNA is most abundantly expressed in postmolt epithelial tissues and was virtually undetectable in egg and muscle. Real-time PCR analysis indicated that ECaC expression in all epithelia was 1000–2000-fold greater than in non-epithelial tissues (cardiac and tail muscle; data not shown). Epithelial expression was quantified relative to hepatopancreas (lowest expression of the epithelial tissues tested); expression was 9.9-fold greater in gill, and greatest (15.3-fold greater) in the antennal gland. Quantitative RT-PCR data confirmed that, although expression of 18s rRNA was comparable in all tissues tested, amplification of the 1032 bp ECaC fragment was most abundant in gill and antennal gland (Fig. 5B). Under these experimental conditions, expression was undetectable in hepatopancreas, egg and muscle. Collectively, these data confirm that ECaC is expressed exclusively in epithelia and that expression is significantly higher in gill or antennal gland than in hepatopancreas in postmolt stage.

ECaC mRNA expression in antennal gland increased in pre- and postmolt stages compared with intermolt (Fig. 6). Real-time PCR data (Fig. 6A), using intermolt expression levels as calibrator, indicated that the expression of ECaC mRNA increased by 7.4- and 23.8-fold at premolt and postmolt stages, respectively. This was confirmed by quantitative RT-PCR(Fig. 6B), which showed significant amplification of the 1032 bp fragment in pre- and postmolt compared with virtually undetectable levels in intermolt. Meanwhile, the 518 bp 18s rRNA PCR product was expressed at constant levels.

Localization of ECaC in transverse sections of the antennal gland using in situ hybridization confirmed that ECaC mRNA expression increased in premolt with a further increase in postmolt compared with intermolt expression (Fig. 7; upper panels). Closer examination of antennal gland structure[Fig. 7; middle panels with reference to prior ultrastructural studies in Wheatly et al.(Wheatly et al., 2004)]revealed that ECaC hybridization was associated with the labyrinth and nephridial canal regions in intermolt sections, and that the increased expression during pre- and postmolt was largely restricted to these regions rather than the coelomosac or bladder. No significant binding was observed to the sense probe (Fig. 7; lower panels), confirming authenticity of the antisense probe.

This study has described the cloning and expression of an epithelial Ca²⁺ channel ECaC-like gene from the antennal gland (kidney) of the crustacean crayfish Procambarus clarkii. This is the first ECaC sequenced in an invertebrate. In addition to sharing structural features with cloned ECaCs, crayfish ECaC is expressed exclusively in epithelial tissues and abundance is proportional to Ca²⁺ influx(Hoenderop et al., 2000b).

The crayfish ECaC cDNA encodes a protein with highest identity to fish ECaCs (68–82%) and lower identity to mammalian ECaCs (52–60%). The predicted amino acid sequence of crayfish ECaC exhibits structural features common to other ECaCs, namely six transmembrane-spanning domains with a putative pore-forming region between transmembrane domains 5 and 6, three ankyrin repeats, and phosphorylation sites. (The crayfish ECaC cDNA sequence has been accepted to the GenBank database under the accession number AY452713.) Mammalian researchers(Hoenderop et al., 2000b)speculated that four monomers of ECaC constitute a functional tetrameric ion channel. ECaC contains conserved potential regulatory sites, including putative phosphorylation sites for protein kinase C, cAMP-dependent protein kinase and cGMP-dependent protein kinase. They also contain structural domains such as N-linked glycosylation sites and ankyrin repeats, which interact with the cytoskeleton to assemble and stabilize proteins in the plasma membrane (Müller et al.,2000a); the latter can also bind to diverse proteins associated with Ca²⁺ homeostasis, such as inositol-(1,4,5)-trisphosphate(IP₃) and ryanodine receptors. Hoenderop et al. has suggested that protein kinase C directly phosphorylates the channel to regulate activity(Hoenderop et al., 2002a; Hoenderop et al., 2002b).

ECaC does not possess the residues that confer sensitivity to depolarization in voltage-gated Ca²⁺ channels. Overall, the primary structure bears little resemblance to either voltage-gated or ligand-operated Ca²⁺ channels. Detailed analysis(Hoenderop et al., 2000a; Hoenderop et al., 2000b) of the pore-forming region and the region flanking transmembrane segment 6 showed a low but significant homology with capsaicin receptors and the TRP channels.

Phylogenetic analysis (Fig. 4) indicated that the crayfish ECaC is a new member of the ECaC superfamily that includes the fish ECaCs and the mammalian ECaC/CaT genes(Hoenderop et al., 1999a; Müller et al., 2000a). ECaCs from crayfish, pufferfish and trout clustered together, forming a distinct group from mammalian ECaCs, suggesting that invertebrate, fish and mammalian genes are not orthologous. Gene mapping of the two mammalian ECaC isoforms, ECaC1 (TRPV5) and ECaC2/CaT1 (TRPV6)(Müller et al., 2000b),showed that they are localized adjacent to each other on the same chromosome,suggesting gene duplication that occurred after the divergence of fish and mammals. Both phylogeny and chromosome mapping results indicated that there was only one gene encoding ECaC in zebrafish(Pan et al., 2005) and pufferfish (Qiu and Hogstrand,2004). Collectively, these studies suggest that ECaCs from crayfish, fish and mammals evolved from a common ancestral gene.

There is evolutionary distance (<30% homology) between crayfish ECaC and other ion channels, including capsaicin receptor (a non-selective cation channel that functions as a transducer of painful thermal stimuli; VR1, now TRPV1; and VRL, now TRPV2) (Caterina et al., 1997; Caterina et al.,1999), GRC (now TRPV2)(Kanzaki et al., 1999) and the canonical TRPC (proposed to mediate the entry of extracellular Ca²⁺into cells in response to depletion of IC Ca²⁺ stores)(Birnbaumer et al., 1996) or olfactory channels (OSM9) (Colbert et al.,1997).

Mammalian ECaC and CaT1 (TRPV6) have been expressed in Xenopus laevis oocytes (Peng et al.,2003a; Peng et al.,2003b) in order to characterize the physiological properties. That study has shown that ECaC mediates passive apical entry down the electrochemical gradient, that it is constitutively active and not voltage- or ligand-gated, that it is selective for Ca²⁺, has a K_m of between 0.2 and 0.66 mmol l^–1 and that it has a feedback-inhibition mechanism to prevent toxic accumulation of free Ca²⁺ in the cell. Ca²⁺ influx is not coupled to Na⁺, Cl^– or H⁺ gradients, although activity is linked to pH. Like most electrogenic processes, hyperpolarizing potential favors Ca²⁺ influx. Functional expression of pufferfish ECaC in Madin-Darby canine kidney (MDCK) cells confirmed that in addition to a role in Ca²⁺ uptake, pufferfish ECaC might serve as a pathway for zinc and iron acquisition (Qiu and Hogstrand, 2003).

The present study of postmolt tissues clearly showed that crayfish ECaC was expressed virtually exclusively in epithelial tissues implicated in Ca²⁺ transport (antennal gland, gill and hepatopancreas), and that expression levels reflected a relative role in Ca²⁺ absorption in postmolt stage. The antennal gland exhibited the highest expression,consistent with perceived high rates of postfiltrational renal Ca²⁺reabsorption (Wheatly, 1999)that are hypothesized to increase in postmolt. The next highest expression was in gill, long acknowledged as a primary route for postmolt Ca²⁺entry for mineralization. Lower expression levels in hepatopancreas suggest that Ca²⁺ entry via digestive epithelium may be less important in the immediate postmolt than postfiltrational reabsorption or branchial uptake (Zanotto and Wheatly,2002). This is in agreement with the observation that crustaceans typically refrain from feeding around ecdysis and only resume once mouthparts and other appendages are adequately hardened several days postmolt. There is reason to predict that expression levels will be higher in intermolt in this particular epithelium. Lack of expression in eggs suggests that the gene may be developmentally regulated.

In rainbow trout (Perry et al.,2003), ECaC was only identified through quantitative RT-PCR in gills, with a faint band in heart. It was undetectable in kidney, intestine,white muscle and blood. A recent study(Shahsavarani et al., 2006)reported that the rainbow trout ECaC was not restricted to mitochondria-rich cells of gills but was also expressed in pavement cells. In the FW zebrafish(Pan et al., 2005), ECaC was ubiquitously expressed in all tissues examined (brain, heart, gills,intestine, liver and kidney); however, expression was highest in the gills and kidney. In the marine pufferfish (Qiu and Hogstrand, 2004) expression was abundant in gill; ECaC was not found in the kidney, consistent with the finding that marine teleost fish do not postfiltrationally reabsorb Ca²⁺(Hickman and Trump, 1969). Expression of the ECaC transcript in pufferfish intestine was low, confirming that, as in crayfish, the intestine of teleost fish is less important than the gill in Ca²⁺ absorption (Flik and Verbost, 1993). As in invertebrates, pharmacological evidence has suggested that other types of Ca²⁺ channel may mediate brush-border membrane Ca²⁺ uptake in fish enterocytes(Larsson et al., 2002).

Mammalian ECaC is typically associated with 1,25 dihydroxyvitamin D[1,25(OH)₂D₃]-responsive epithelia that facilitate Ca²⁺ absorption (Hoenderop et al., 1999a; Hoenderop et al.,2000a; Hoenderop et al.,2000b; Peng et al.,1999; Zhuang et al.,2002). In rabbit kidney(Hoenderop et al., 1999a),ECaC mRNA and protein were expressed primarily in the distal part of the nephron, the region associated with Ca²⁺ regulation. ECaC protein was immunolocalized at the apical domain of the connecting tubule(Peng et al., 2000b). Importantly, ECaC was colocalized with calbindin-D_28K (the Ca²⁺-binding protein that facilitates cytosolic diffusion of Ca²⁺ from apical influx to basolateral efflux sites), NCX and PMCA(Peng et al., 2000b).

In rabbit, ECaC was also identified in placenta and in the proximal small intestine (duodenum, jejunum); however, it was not detected in the ileum,colon, lung, muscle, liver or brain. In intestine, ECaC was present in a thin layer along the apical membrane of the duodenal villus tip, whereas a complete colocalization was observed between ECaC, calbindin-D_9K and PMCA,but not NCX (Peng et al.,2000b). In humans it was also detected in other epithelial tissues, such as testis, prostate and placenta(Müller et al., 2000a),and in non-epithelial tissues, such as brain and pancreas. This raises the interesting question of the role ECaC plays in non-epithelial tissues. Mammalian researchers have suggested that it serves to control Ca²⁺entry and regulate IC Ca²⁺ concentration, and may be involved in cell proliferation, differentiation and signal transduction(Putney, 2001; Zhuang et al., 2002). In endocrine cells it may regulate cytosolic Ca levels in order to modulate depolarization-stimulated insulin release.

In human, CaT1 (now TRPV6) (Peng et al., 2000a; Weber et al.,2001) is abundant in the proximal small intestine (primarily duodenum), the site of Ca²⁺ absorption. Strong signals were also detected in placenta and exocrine tissues (salivary gland, prostate and pancreas) where it probably mediates reuptake of Ca²⁺ following its release by secretory vesicles. Although kidney and intestine both engage in Ca²⁺ absorption, there are differences in the vitamin D-regulated calbindins involved (D_9K versus D_28K, respectively), and so it is not surprising that different ECaC proteins are involved(Peng et al., 1999).

The present study demonstrated increased expression of crayfish ECaC mRNA in antennal gland in the premolt and postmolt phases of the molting cycle as compared with baseline intermolt levels(Fig. 6); furthermore, it localized these increases to the labyrinth and nephridial canal regions of antennal gland slices, areas long associated with ion reabsorption. This suggests that the ECaC abundance at a transporting epithelium is proportional to the magnitude of unidirectional Ca²⁺ influx. In intermolt the crayfish reabsorbed 97% of Ca²⁺ filtered at the antennal gland(renal unidirectional influx of 70 μequiv kg^–1h^–1) (Wheatly and Toop,1989), which exceeded unidirectional influx at the gill(Wheatly, 1999). In the late premolt phase, external FW was loaded into the extracellular fluid, causing a hemodilution of 40% as reflected in transitory (48 h) reductions in circulating Na⁺ and Cl^–(Wheatly, 1996). Hemolymph Ca²⁺, meanwhile, remained remarkably constant, suggesting that renal Ca²⁺ reabsorption increased in pre- and postmolt. During this time, activities of ion transport enzymes (carbonic anhydrase, PMCA) increased in the antennal gland (Wheatly,1997), indirectly confirming that tubular ion reabsorption increased. Measurement of the chemical composition of the urinary filtrate during pre- and postmolt has presented some technical difficulty because urinary cannulation has only been achievable in intermolt stage.

Increased expression of ECaC during pre- and postmolt is consistent with our original hypothesis that abundance is correlated with Ca²⁺influx rates. Other studies in our laboratory have used similar techniques to document upregulation (8–18-fold increases) of the primary basolateral Ca²⁺ efflux mechanisms [PMCA(Gao and Wheatly, 2004; Wheatly et al., 2004); NCX(Stiner et al., 2004)] during pre- and postmolt. This would suggest that genes controlling apical Ca²⁺ entry (ECaC) and basolateral exit from cells (PMCA, NCX) are closely regulated during periods of elevated transcellular Ca²⁺flux. However, the point of entry is logically the gatekeeper and, as such, a prime target for endocrine control of Ca²⁺ influx.

In situ hybridization revealed that the ECaC upregulation in crayfish antennal gland during pre- and postmolt was localized in the periphery of the transverse sections. When viewed under higher magnification and correlated with prior ultrastructural studies(Wheatly et al., 2004), these regions corresponded to the labyrinth and nephridial canal(Maluf, 1939); hybridization with the coelomosac (site of ultrafiltration) and bladder (urine storage) was less intense. The labyrinth epithelium is composed of cuboidal to columnar cells possessing a brush border, basal invaginations of the plasma membrane,and extensive surface blebbing (Peterson and Loizzi, 1974a; Peterson and Loizzi, 1974b; Fuller et al., 1989). The presence of microvilli, the abundance of mitochondria in the proximity of these microvilli, and the occurrence of endocytotic vesicles along the apical cell membrane collectively suggest an energy-requiring reabsorptive function of this region of the antennal gland. In FW crayfish active ion reabsorption is also strongly associated with the nephridial canal, a region that is missing in marine species that produce isosmotic urine. The histology of the nephridial tubule epithelium resembles that of other cell types pumping ions against a concentration gradient (cells lack a microvillous border but display intense basal invaginations of the plasmalemma associated with numerous mitochondria). A prior study in our laboratory (Wheatly et al.,2004) has shown that the increased PMCA expression associated with elevated unidirectional Ca²⁺ influx (postmolt compared with intermolt) at the antennal gland is similarly localized predominantly in the nephridial canal and labyrinth. Collectively, these studies confirm that apical and basolateral mechanisms effecting transcellular Ca²⁺influx in crayfish kidney are coordinated spatially as well as temporally. Similarly, in mammalian studies ECaC has been localized in apical membranes of distal convoluted tubule 2 and connecting tubules of the human kidney cortex,as well as brush border of duodenal and jejunal villi(Müller et al., 2001);furthermore, in these tissues ECaC has been colocalized with basolateral proteins involved in active transcellular Ca²⁺ transport (NCX,PMCA).

Studies in other species have confirmed that ECaC expression is associated with epithelial Ca²⁺ flux. In zebrafish(Pan et al., 2005), ECaC expression was correlated with Ca²⁺ influx; wholebody Ca content increased during larval development associated with ossification. Further incubating embryos in low-Ca FW caused induction of upregulation of Ca²⁺ influx and ECaC expression in gills and skin covering the yolk sac. Expression of both CaT1 and ECaC in duodenum and kidney have been studied in mouse development (Song et al.,2003). Intestinal CaT1 expression increased at weaning with induction of calbindin D_9K. Renal CaT and ECaC expression were equally expressed until weaning, when ECaC expression increased and CaT1 decreased. In rats, 1,25 dihydroxyvitamin D stimulated active intestinal Ca²⁺ absorption by increasing ECaC expression. Active reabsorption is also increased after feeding a low-Ca diet or under conditions of Ca²⁺ deficiency (van Abel et al., 2003). Duodenal expression of CaT1 is also vitamin D-dependent and expression of both CaT1 and ECaC are reduced in vitamin D receptor-knockout mice (van Cromphaut et al., 2001).

The logical next step in this research program is to generate homologous antibodies to ECaC so that the protein can be quantified and localized within the antennal gland. Identifying a cell system for functional expression will enable better understanding of the biophysical properties of this channel. Possible mechanisms for ECaC activation need to be addressed, such as de novo synthesis of ECaC, activation of existing ECaC channels by regulatory factors [including feedback inhibition of Ca concentration in the microdomain near the inner mouth of the channel(Hoenderop et al., 1999b),direct phosphorylation of the channel, membrane potential and interacting accessory proteins], and shuttling of ECaC between IC vesicles and apical membrane. Ultimately, we propose to study the ECaC promoter through reporter analysis in order to confirm transcriptional regulation of ECaC. Subsequently,promoter regions of apical entry and basolateral exit mechanisms will be studied to reveal regulatory relationships between genes enabling cellular Ca²⁺ homeostasis. We also propose to examine whether apical ECaC can serve as an exit mechanism in secretory epithelia (premolt digestive epithelium, postmolt cuticular hypodermis). Finally, it would be illuminating to study hormonal regulation of ECaC that is likely to involve both post-translational and transcriptional control mechanisms; possible candidate hormones in crustaceans are calcitonin, calcitonin gene-related peptide,vitamin D metabolites and ecdysterone(Flik et al., 1999).

The authors thank Dr Steven Berberich of the Center for Genomics Research at Wright State University for expert technical advice in real-time PCR, and Dr Mariana Morris and Mary Key of the Department of Pharmacology and Toxicology at Wright State University for help with the in situhybridization. This study was supported by the US National Science Foundation(Grants IBN 0076035 to M.G.W. and 0445202 to M.G.W. and Y.G.).