1813
Introduction 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,
This study describes the cloning, sequencing and functional
characterization of an epithelial Ca2+ channel (ECaC)-like gene
isolated from antennal gland (kidney) of the freshwater crayfish
Procambarus clarkii. The full- length cDNA consisted of 2687·bp
with an open reading frame of 2169·bp encoding a protein of 722
amino acids with a predicted molecular mass of 81.7·kDa. Crayfish
ECaC had 76–78% identity at the mRNA level (80–82% amino acid
identity) with published fish sequences and 56–62% identity at the
mRNA level (52–60% amino acid identity) with mammalian ECaCs.
Secondary structure of the crayfish ECaC closely resembled that of
cloned ECaCs. Postmolt ECaC expression was exclusively restricted
to epithelia associated with Ca2+ influx and was virtually
undetectable in non-epithelial tissues (eggs, muscle). Compared
with expression levels in
hepatopancreas, expression in gill was 10-fold greater and
expression was highest in antennal gland (15-fold greater than in
hepatopancreas). Compared with baseline expression levels in
intermolt stage, expression of ECaC in antennal gland increased
7.4- and 23.8-fold, respectively, in pre- and postmolt stages of
the molting cycle. This increase was localized primarily in the
labyrinth and nephridial canal, regions of the antennal gland
associated with renal Ca2+ reabsorption. The ECaC in crayfish
appears to be expressed in epithelia associated with unidirectional
Ca2+ influx and relative expression is correlated with rate of Ca2+
influx.
Key words: crayfish, Procambarus clarkii, antennal gland, gill,
hepatopancreas, epithelial Ca2+ channel, ECaC, mRNA expression and
localization, molting cycle.
Summary
The Journal of Experimental Biology 210, 1813-1824 Published by The
Company of Biologists 2007 doi:10.1242/jeb.02761
Molecular characterization of an epithelial Ca2+ channel-like gene
from crayfish Procambarus clarkii
Yongping Gao and Michele G. Wheatly* Department of Biological
Sciences, Wright State University, Dayton, OH 45435, USA
*Author for correspondence (e-mail:
[email protected])
Accepted 22 February 2007
1814
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.
Materials and methods Experimental animals
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 situ hybridization.
Isolation of total RNA and mRNA
After dissection, tissues were frozen immediately in liquid N2 and
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·g and 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).
Cloning of crayfish ECaC by RT-PCR and RACE
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·l) included 2·l 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·mol·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·g·ml–1 of ethidium
bromide in 1 TAE buffer [40·mmol·l–1 Tris,
Y. Gao and M. G. Wheatly
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1815Cloning and characterization of crayfish EcaC
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-CACCTCG CTGA - CCCTGAACACG-3 and
5-ACGCACAGCAGCACCAC - CAG-3, and a nested primer, 5-CAGGGAGGCATAGGT
- GATAAGGAT-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.
DNA sequencing and analysis
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 and quantitative RT-PCR assays
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·g 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·g) 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 ExpressTM 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
TGTGCACCTATTCTCAAGTGTGCTCGACATTTTCTGTTCCCCCAGTACCTCATTGGTCTGG 61
AAG ATGTCCCCGTCTCTGGCCAGGTCGGCTCCAAGTGAGCTCAACCACAGGTGGAGTCAGTTTAGG
187
ACTATCAGGGATAGTGGAATACTACTTTTGCCTTTTAAGGTGGAATCCCCAGGTGTCCGG
124
M S P S L A R S A P S E L N H R W S Q F R 21
TTTCGCCTCCATAACAGGAAAGGATGGAAAGAGATGTTGGATGAAACTTTTCTGCTCGATAAC 250
F R L H N R K G W K E M L D E T F L L D N 42
AAGAAGATAAACAACATTCCTCTCTTCTATGCGGCCCCAGAGAATAATGCAGGTTGCATCAAG 313
K K I N N I P L F Y A A P E N N A G C I K 63
AAACTTCTAGGCTGCTCCTCCACTAACATATTTGAAAGAGGGGCTCTGGGGGAGACGGCGCTA 376
K L L G C S S T N I F E R G A L G E T A L 84
CACGTCGCAGTTATGAATGACAACGTGGAAGCCGCTCTGGTTCTGATGGAGGGGGCACCTGAA 439
H V A V M N D N V E A A L V L M E G A P E 105
CTCATCAATGAGCCCATGACCTCTGAGCTTTTCCTAGGTGTGACTCCTCTCCATATTGCCGTG 502
L I N E P M T S E L F L G V T P L H I A V 126 Real-time sense
primer
GTGAATCAGAACGTTAACTTGGTCCACGACCTGATTAGCCGTGGGGCCGATGTAGCTACGCCC 565
V N Q N V N L V H D L I S R G A D V A T P 147
AGGGTCACAGGGCTGTATTTCAGGAAGAGAATCGGAGCGCTGCTTTACTACGGTGACCACATC 628
R V T G L Y F R K R I G A L L Y Y G D H I 168
CTGGCATTTGCTGCCTGTGTGGGGAATCAGGACATCATCTCCCTGCTCATCGAGGGAGGAGCC 691
L A F A A C V G N Q D I I S L L I E G G A 189
Real-time PCR antisense primer
AGCACCAGGGCCCAGGATTCCCTTGGTAACACAGTGCTCCATATTCTGGTCCTGCAGGCCAAC 754
S T R A Q D S L G N T V L H I L V L Q A N 210
AAGACTATAGCATGCCAGGTGTTGGACCTGCTGCTGGCACGTGATGGAGAGCTGGACCAGCCG 817
K T I A C Q V L D L L L A R D G E L D Q P 231
RT-PCR sense primer
GTGCCCCTGGACATGGTGCCCAACCACCAGGGTCTCACCCCCTTCAAACTGGCTGCCAAGGAG 880
V P L D M V P N H Q G L T P F K L A A K E 252
GGTAACCCTGTGGCGTTCCAGCACCTGGTGAATAAGAGGCGGCTCATCCAGTGGACGCTGGGA
943
G N P V A F Q H L V N K R R L I Q W T L G 273
CCCCTGACCTCCAACCTCTATGACCTCACAGAGATCGACTCCTGGGCTGACGACATGTCTGGC
1006 P L T S N L Y D L T E I D S W A D D M S G 294
CTGGAGCTCGTCGTGGGCAGTAAGAAGAGAGAGGCTAGGCGGATTCTGGAACTGACTCCTGTG
1069 L E L V V G S K K R E A R R I L E L T P V 315
AAGCAGCTGGTCAGTTTCAAGTGGAACCTGTATGGCCGGCAGTACTTCAGGATGCTGGGGTTG
1132 K Q L V S F K W N L Y G R Q Y F R M L G L 336
CTGTACCTCCTGTACATTGGCACCTTCACACTGTGTTGTGTATATCGCCCCCTTAAGGACACT
1195 L Y L L Y I G T F T L C C V Y R P L K D T 357 Transmembrane
domain 1
CCTGAGAACTACACTGAATCTGAGCTGGACAACACCATCCGCGTGCAGAAAACGCTGCAGGAG
1528 P E N Y T E S E L D N T I R V Q K T L Q E 378
AGTTATGTGACACGTGACGACAACTTGCGGCTGGTGGGAGAGCTGATCAGCGTCCTGGGGGCT
1321 S Y V T R D D N L R L V G E L I S V L G A 399
GTGGTCATCCTGCTCCTGGAGATCCCAGATATCCTAAGAGTTGGGGCCAAGCGTTATTTTGGA
1384 V V I L L L E I P D I L R V G A K R Y F G 420
Transmembrane domain 2 In situ hybridization probe
CAGACGGCCCTGGGGGGGCCCTTCCATGTCATCCTTATCACCTATGCCTCCCTGGTGGTGCTG
1447 Q T A L G G P F H V I L I T Y A S L V V L 441 Transmembrane
domain 3
CTGTGCGTGTTCAGGGTCAGCGAGGTGCAGGGGGAGGCGGTCCTCATGGCCCTGGCCCTGGTG
1510 L C V F R V S E V Q G E A V L M A L A L V 462
CTGGGCTGGAGCAATGTCATGTTCTTCGCTCGAGGATTCCAGATGCTGGGCCCCTACGTCATC
1573 L G W S N V M F F A R G F Q M L G P Y V I 483 Transmembrane
domain 4
ATGATACAGAAGATTATATTTGGAGACCTGACGAAGTTCATGTGGCTGAGGTTCGTGGTGCTC
1636 M I Q K I I F G D L T K F M W L R F V V L 504 Transmembrane
domain 5
ATGGGATTTTCCTCCGCCTTGTGGGTGGTGTACATGACAGAGGACCCAGACTCTCTGCCGCCA
1699 M G F S S A L W V V Y M T E D P D S L P P 525
TTCGGGTACTTCCCCATCACACTGTTCTCCAAGTTTGAGCTGTGTGTGAGTCTGATAGACCTC
1762 F G Y F P I T L F S K F E L C V S L I D L 546 Pore region
CCTGTGGACCACAGCATCAACATGCCCTTCATGGTCAGCGTGGTGAACTGCACCTTCTCTATG
1825 P V D H S I N M P F M V S V V N C T F S M 567
RT-PCR antisense primer
GTCTCCAACATGCTCCTGCTCAACCTGTTCATAGCCATGATGGGCGACACCCACTGGAGGGTG
1888 V S N M L L L N L F I A M M G D T H W R V 588
Transmembrane domain 6
GCCCAGGAGAGGGACGAGCTCTGGAGGGCCCAGGTTGTGGCCACCACAGTGATGCTGGAGAGG
1951 A Q E R D E L W R A Q V V A T T V M L E R 609
AAGCTGCCTCGCTGCCTGTGGCCTCGACTCGGGGTGTGTGGCCTGGAGTACGGTCTGGGGGAG
2014 K L P R C L W P R L G V C G L E Y G L G E 630
CGCTGGTTCCTGCGGGTTGAGGACCGAAATGACCCGCTGGTTCAGAAGATGCGTCGCTACGTG
2077 R W F L R V E D R N D P L V Q K M R R Y V 651
AAAGCCTTCTCTAACAAGGAGGATGACCACGAACAACCGGCGGAGAAGGAGGCCATGAGGTGT
2140 K A F S N K E D D H E Q P A E K E A M R C 672
CAGGGTCTAGAACCTATCTGGTTCGAACTTAAAACGACAGGATCTCCAAACAGGAAGTCCCTG
2203 Q G L E P I W F E L K T T G S P N R K S L 693
ACAGGCTGGCAGATGATTCGCCACAGCACCCTGGGGTTAGATCTGGGACAGGAGGAGCCAGAA
2266 T G W Q M I R H S T L G L D L G Q E E P E 714
GATGACCAGGAAGTCAAGTATCTGTAGCAACAAATCCTGTGACTGGAGAACTGAGTGGCTCAG
2329 D D Q E V K Y L . 722
ATTGTAAGGACAAAAGCTGTTCCTGTGATGTTATTATGCATAGAAAAGGGTATTATTTTTGGA
2392
TAACAAATGTGGAAAGTAGATTAGCTAGTACACGCATTCCAAATTGGGTTGAAATGTTAATAA
2455
TATCTGTTTAATTGACTTATGTGGCTTTCAAGTAGATTCCACGGCAATGTTTTTTACACTCTA
2518
AGAGATTTACGAGACAGAATATGATCATTAAAGTGTTAGTGGAAACGGTTTTTTTCTTGATGG
2581
TAATATCCTGACAGAAAACAAAATGGCCATTTGATGGTAATGGTCATTGTATAGAATGGTCTC
2644 GAATGGAGAAGAAATACCGAAGCTAAAAAAAAAAAAAAAAAAA 2687
Fig.·1. The complete nucleotide and deduced amino acid sequence of
crayfish Procambarus clarkii antennal gland (kidney) epithelial
Ca2+
channel (ECaC) cDNA (GenBank accession number AY452713).
Nucleotides and amino acids are numbered to the right of the
sequence. The start and stop codons are indicated in bold letters.
Transmembrane domains and putative pore-forming region are in bold
and underlined. The gray boxes indicate the primers used for
real-time PCR, RT-PCR and the probe used for in situ
hybridization.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1816
Procambarus clarkii 18s rRNA gene (accession number AF436001). The
reaction mixture (20·l) contained 2·l of 10 SYBR Green PCR buffer,
3·l of 25·mmol·l–1 MgCl2, 2·l 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·l
of AmpliTaq Gold (5·U·l–1), 0.25·l of AmpErase UNG 91·U·l–1), 2·l
of template cDNA and 4.08·l of each 5·mol·l–1 primers in
water.
Real-time PCR reactions were performed in a 96-well microtiter
plate using the relative quantification Ct 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·l of each 1·mol·l–1 primers and 2·l 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 Ct 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 (Ct), and was
finally calculated using Ct of the control tissue/condition as
calibrator. Ct corresponds to the difference between the Ct of the
gene of interest and the Ct of the endogenous control 18s rRNA.
Fold-change in expression was calculated as RQ=2Ct. 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-GGCTG - CCAAGGAGGGTAA-3
(sense) and 5-CTCTCCTGGGC - CA CCCT-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·l) included 2·l 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·mol–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·l) were analyzed by electrophoresis on a 1.0% agarose gel with
0.5·g·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 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·m
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-GAACACGCAC - AGCAGCACCACCAGGGAGGCATAGGT-3, and the sense of
this probe sequence (used as a negative control for non- specific
hybridization) was 5-ACCTATGCCTCCCTGGTG - GTGCTGCTGTGCGTGTTC-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·l of 4·pmol·l–1 probe,
4·l of ddH2O, 5·l of 5 terminal transferase buffer, 5·l of CoCl2,
2·l of TdT (400·U) and 4·l of 35S-dATP (1250·Ci·mmol·l–1; NEN Life
Sciences, Boston, MA, USA). Then, 50·l 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·g·ml–1 yeast tRNA, 10%
dextran sulfate (v/v), 10·mmol·l–1 DTT, 500·g·ml–1 boiled salmon
sperm DNA] to yield approximately 0.5106·cpm 100·l–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·l 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–1 PBS 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
Y. Gao and M. G. Wheatly
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1817Cloning and characterization of crayfish EcaC
with a BAS-IIIs imaging plate along with high and low standards to
convert intensity to·Ci. 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.
Results Cloning of crayfish ECaC
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
Crayfish MS-----PSLARSAPSELNHRW-SQFRFRLHNRKGWKEMLDETFLLDNKKI 45
Pufferfish **-----************W**-T*L****Q*K***N*********YT*** 45
Rainbow trout *A-----*A******G****W*-*******Q***************Q**RT
45 Human *--------------------------------------------*QQ*R* 7
Rabbit *GACPPKAKGPWAQLQKLLIS*PV-------GEQD*EQYR*RVNM*QQER* 44 Rat
*G-----VKKPWIQLQKRLNW*-V-------REQD*NQHV*QLHM*QQ*S*
Crayfish NNIPLFYAAPENNAGCIKKLLGCSSTNIFERGALGETALHVAVMNDNVEAA 96
Pufferfish ***********S********************************S**LD** 96
Rainbow trout *GV***F**K*SS********D*A*******R***************M***
96 Human LES**LR**L**DLMVLRQ**LDCTCDVRQ************ALY**L*** 58
Rabbit RDS**LQ**K*SDLRLL*I**LNQ*CD*QQ***V********ALY**L*** 95 Rat
WES**LR**K**DMCTL*R*QHDQNCDFRQ************ALY**LD** 89 Ankyrin
repeat Crayfish LVLMEGAPELINEPMTSELFLGVTPLHIAVVNQNVNLVHDLISRGADVATP
147 Pufferfish V*******************Q*************I***QH*****G*A***
147 Rainbow trout
*A********************MK**********F***RS**GK******* 147 Human
IM***T**Y*VT*STLC*P*V*Q*A****IM*******RA*LA***SASA- 109 Rabbit
TL***A****AK**ALC*P*V*Q*A*****M*******RA*LA***S*SA- 147 Rat
IM***T**Y*VT*STLC*P*V*Q*A****IM*******RA*LA***SASA- 140 domain
Ankyrin repeat domain Crayfish
RVTGLYFRKRIGGLIYYGEHILSFAACVGNEDIISLIIDAGASTRAQDSLG 198 Pufferfish
**************************A*****************V***YR* 198 Rainbow
trout **********R*******************Q****MV*NV*********I* 198 Human
*A**AA**RSPHN*******P*******NS*E*VR*L*EH**DI******* 159 Rabbit
*A**AA**RSPHN*******P********S*E*VR*L*EH**DI******* 196 Rat
*A**SA**RSSHN*******P********S*E*VR*L*EH**DI******* 188 Ankyrin
repeat domain Crayfish
NTVLHILVLQPNKTIACQVLDLLLARDGELDQPVPLDMVPNHQGLTPFKLA 249 Pufferfish
*****V************AM**IM***A****S********SR******** 249 Rainbow
trout *****************LVL*******I****A********YH******** 249 Human
*******I******F***MYN***SY**HG*HLQ***L************* 209 Rabbit
*******I******F***MYN***SY*EHS*HLQS**************** 246 Rat
**************F***MYN***SH**-G*HLKS*EL***N********* 240
Crayfish AKEGNPVAFQHLVNKRRLIQWTLGPLTSNLYDLTEIDSWADDM--SGLELV 298
Pufferfish *****R***********VV**S******Y************G*--*V***I 298
Rainbow trout *****L********R**IN**N************G***LV***DC*V**HI
300 Human GV***T******MQ***H****Y*****I**********GEEL--*F**** 259
Rabbit GV***T******MQ**KHV***C*****T**********GEEL--*F**** 296 Rat
GV***T******MQ**KHI**S******SI*********GE*L--*F**** 289
Crayfish VGSKKREARRILELTPVKQLVSFKWNLYGRQYFRMLGLLYLLYIGTFTLCC 349
Pufferfish ***QQK***G*******MR***L******KH***L*L************** 349
Rainbow trout ********KR***V***R***********KH***L*L**************
351 Human *S*D*****Q***Q****E*******K***P**CI*AA*****M****T** 310
Rabbit *S*K*****Q***Q****E******KK***P**CV*AS*****MIC**T** 347 Rat
*S*K*K***Q***Q****E***L**KK**QP**CL**M**IF*MIC**T** 340
◊◊ ◊ Transmembrane domain 1 Crayfish
VYRPLKDTPENYTESELDNTIRVQKTLQESYVTRDDNLRLVGELISVLGAV 400 Pufferfish
T******IS****K****H***************E*S******V******L 400 Rainbow
trout *******A*****V*DM*K********K*****YG*****A**M******L 402 Human
******FRGG*R*H*R-*I**LQ**L***A*E**E*II******V*IV*** 360 Rabbit
I*****LRDD*R*DPR-*I**LQ**L***A***HQ**I******VT*T*** 397 Rat
******FRDA*R*HVR-***VLE**P***A***YQ*KV******VT*I*** 390
Transmembrane domain 2 Crayfish
VILLLEIPDILRVGAKRYFGQTALGGPFHVILITYASLVVLLCVFRVSEVQ 399 Pufferfish
**********I**********************S************AC*** 451 Rainbow
trout ******V**M******H****************A**F***********G** 453 Human
I*********F****S****K*I********I*******LVTM*M*LTNTN 411 Rabbit
I*********F****S******I********I*******L*TM*M*LTNMN 448 Rat
**********F****S****H*V********I*******L*IM*M*LTSMN 441
Transmembrane domain 3 Crayfish
GEAVLMALALVLGWSNVMFFARGFQMLGPYVIMIQKIIFGDLTKFMWLRFV 502 Pufferfish
****V***S***************E**********************FS*I 502 Rainbow
trout **T*V**VC***************************************S*I 504 Human
**V*PMSF******CS**Y*T*********T*****M*****MR****MA* 462 Rabbit
**V*PLSF******CS**Y**********FT*****M*****MR****MA* 499 Rat
**V*PISM******CS**Y*S********FT*****M*****LR****MAM 492
Transmembrane domain 4 Transmembrane domain 5 Crayfish
VLMGFSSALWVVYMTEDPDSLPPFGYFPITLFSKFELCVSLIDLPVDHSIN 553 Pufferfish
**I*Y*T********Q****M***RS*******E***S*G********R*M 553 Rainbow
trout **I***TS*******Q******AYRS*******Q***S*G*******T*TT 555 Human
**L**A**FYIIFQ****T**GQ*YDY*MA***T***FLTV**A*ANYDVD 513 Rabbit
**L**A**FHITFQ****NN*GE*SDY*TA***T***FLTI**GP*NY*VD 550 Rat
**L**A**FYIIFQ****E**GE*SDY*TAM**T***FLTI**GP*NY*VD 543 Pore region
Crayfish MPFMVSVVNCTFSMVSNMLLLNLFIAMMGDTHWRVAQERDELWRAQVVATT 604
Pufferfish T*PI*H*LH****L**YI*****L****S********************** 604
Rainbow trout ***I*H*LH****V**YI*****LT***S*Q********************
606 Human L***F*I*NFA*AIIATL********************************* 564
Rabbit L***YCITYAA*AIIATL*M******************************* 601 Rat
L-**Y*LTYFA*AIIATL*M******************************* 594
Transmembrane domain 6 Crayfish
VMLERKLPRCLWPRLGVCGLEYGLGERWFLRVEDRNDPLVQKMRRYVKAF- 655 Pufferfish
L****R******************K***Y**********F******I***- 655 Rainbow
trout L****R**************L*******Y******************Q**- 657 Human
**************S*I**C*F***D*******NH**QNPLRVL***EV*K 605 Rabbit
******M**F****S*I**Y*****D*******NHH*QNPLRVL***E**K 652 Rat
******M**F****S*I**C*****D*******HHQEQNPLRVL***E**K 645
Crayfish -SNKEDDHEQP------AEKE---------AMRC—QGLEPIWFELKTTGSP 686
Pufferfish -*K---EDDTD------*KTD---------TTKDFIP-**TQ*--G****E 686
Rainbow trout -*K---*ED*SKEREEMENTD---------MSK---P*S*L*--RT*HR*G
692 Human N*D****Q*HP------SE*QPSGAESGTL*RAS--LA**TS--S*SR*A* 658
Rabbit C*D***GQ**L------SE*RPSTVESGMLSRAS—-VAFQTP--S*SR*T* 695 Rat
S*D**EVQ**L------SE*QPSGTETGTL*RGS--VV*QTP--P*SR*T* 688
◊
Fig.·2. The alignment of the deduced crayfish Procambarus clarkii
ECaC protein sequence with pufferfish ECaC (GenBank accession
number AY232821), rainbow trout ECaC (AY256348), human ECAC1
(AJ271207), rabbit ECaC (AJ133128) and rat CaT1 (AF160798). Amino
acids are numbered on the right and significant residue identities
are indicated by an asterisk (*). Light-gray boxes indicate ankyrin
repeat domains, dark-gray boxes are predicted transmembrane domains
and the black box is the putative pore- forming region. The
putative protein kinase A and C phosphorylation sites are also
indicated by closed and open diamonds, respectively.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1818
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].
Tissue-specific ECaC mRNA expression
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 and localization in antennal gland during molt
stages
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.
Discussion Cloning and sequencing of crayfish ECaC
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
Y. Gao and M. G. Wheatly
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1819Cloning and characterization of crayfish EcaC
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 Km 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).
Tissue-specific ECaC expression
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.
1 2 3
9 723
)
Fig.·3. Hydrophobicity plot for crayfish Procambarus clarkii ECaC
(GenBank accession number AY452713) in comparison with rainbow
trout ECaC (AY256348), pufferfish ECaC (AY232821), human ECAC1
(AJ271207), rabbit ECaC (AJ133128) and rat CaT1 (AF160798)
sequences. Transmembrane domains are numbered from 1-6 and the
putative pore- forming region is indicated by the letter ‘P’.
Hydrophobicity values were determined by the method of Kyte and
Doolittle (Kyte and Doolittle, 1982), using a window of 19 residues
(http://arbl.cvmbs.colostate.edu/molkit/
hydropathy/index.html).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1820
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-D28K (the
Y. Gao and M. G. Wheatly
Mouse CaT (TRPV6)
Mouse ECaC (TRPV5)
Rat CaT1 (TRPV6)
Human CaT1 (TRPV6)
Human CaT2 (TRPV5)
Human ECaC1 (TRPV5)
Rabbit ECaC (TRPV5)
Rat ECaC1 (TRPV5)
Rainbow ECaC (TRPV5)
Pufferfish ECaC (TRPV5)
Crayfish ECaC (TRPV5)
Human TRP3 (TRPC3)
Human TRP4 (TRPC4)
Chicken VR-OAC (TRPV4)
Fig.·4. Phylogram based on full-length sequences of crayfish ECaC
(GenBank accession number AY452713), rainbow trout ECaC (AY256348),
pufferfish ECaC (AY232821), rabbit ECaC (AJ133128), human CaT1
(AF365928), human CaT2 (AF209196), human ECAC1 (AJ271207), human
VR1 (AAG43466), human TRP3 (NP_003296), human TRP4 (XP-027181),
mouse ECaC (336378), mouse CaT (AB037373), mouse TRP2 (AF111107),
mouse TRP5 (AF029983), mouse TRP6 (U49069), mouse GRC (AB0216650),
rat CaT1 (AF160798), rat ECaC1 (NP_446239), rat VR1 (AF029310), rat
VRT (AF1291130) and chicken VR-OAC (AAG28026).
H ep
at op
an cr
ea s
A nt
en na
n
A
B
Fig.·5. (A) Real-time PCR assay for the relative expression of
crayfish Procambarus clarkii ECaC mRNA in postmolt hepatopancreas,
gill and antennal gland. Expression in postmolt cardiac and axial
muscle was undetectably low relative to expression in epithelial
tissues and so data were not included. Relative quantification (RQ
expressed as mean ± s.d. from three different samples with 4-5
crayfish in each sample) was performed by normalizing the Ct value
of each sample with the Ct value of the endogenous control (18s
rRNA gene, Ct), and finally calculated using Ct of control
(hepatopancreas) as calibrator. (B) Quantitative RT-PCR showing
distribution of Procambarus clarkii ECaC (upper panel) and 18s rRNA
(lower panel) in a range of postmolt crayfish tissues.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1821Cloning and characterization of crayfish EcaC
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-D9K 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 (D9K versus D28K, respectively), and so it is
not surprising that different ECaC proteins are involved (Peng et
al., 1999).
Relative expression of ECaC in antennal gland in different molt
stages
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·equiv·kg–1·h–1) (Wheatly and Toop,
1989), which exceeded unidirectional influx at the gill (Wheatly,
1999). In
0.0
10.0
20.0
30.0
B
Fig.·6. (A) Real-time PCR assay for the expression of crayfish
Procambarus clarkii ECaC mRNA in antennal gland at different
molting stages. Relative quantification (RQ expressed as mean ±
s.d. from three different samples with 4-5 crayfish in each sample)
was performed by normalizing the Ct value of each sample with the
Ct value of the endogenous control (18s rRNA gene, Ct) and finally
calculated using Ct of control (intermolt) as calibrator. (B)
Quantitative RT-PCR assay of crayfish Procambarus clarkii ECaC
(upper panel) and 18s rRNA (lower panel) in antennal gland at
different stages of molting cycle.
Fig.·7. Upper: representative digitized computer images of in situ
hybridization of ECaC antisense probe in Procambarus clarkii
antennal gland sections in various molting stages. Abundance of
mRNA is illustrated by increasing yellow/orange intensity compared
with control (blue). Lower: representative digitized computer
images of in situ hybridization of ECaC sense probe in Procambarus
clarkii antennal gland sections in various molting stages. Middle:
structural regions of the antennal gland (scale bar, 100·m),
including bladder (B), coelomosac (C), labyrinth (L) and nephridial
canal (NC).
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1822
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 D9K. 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).
Directions for future research
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).
Y. Gao and M. G. Wheatly
THE JOURNAL OF EXPERIMENTAL BIOLOGY
1823Cloning and characterization of crayfish EcaC
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 situ hybridization. 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.).
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Y. Gao and M. G. Wheatly
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