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First published online May 8, 2007
Journal of Experimental Biology 210, 1813-1824 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02761

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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

View larger version (65K): [in this window] [in a new window]   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.

View larger version (75K): [in this window] [in a new window]   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.

View larger version (24K): [in this window] [in a new window]   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).

View larger version (15K): [in this window] [in a new window]   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).

View larger version (21K): [in this window] [in a new window]   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.

View larger version (15K): [in this window] [in a new window]   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.

View larger version (68K): [in this window] [in a new window]   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 µm), including bladder (B), coelomosac (C), labyrinth (L) and nephridial canal (NC).

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