Yongping Gao

Characterization and expression of plasma membrane Ca2+ ATPase (PMCA3) in the crayfish Procambarus clarkii antennal gland during molting

SUMMARY The discontinuous pattern of crustacean cuticular mineralization (the molting cycle) has emerged as a model system to study the spatial and temporal regulation of genes that code for Ca2+-transporting proteins including pumps, channels and exchangers. The plasma membrane Ca2+-ATPase (PMCA) is potentially of significant interest due to its role in the active transport of Ca2+ across the basolateral membrane, which is required for routine maintenance of intracellular Ca2+ as well as unidirectional Ca2+ influx. Prior research has suggested that PMCA expression is upregulated during periods of elevated Ca2+ influx associated with postmolt cuticular mineralization. This paper describes the cloning, sequencing and functional characterization of a novel PMCA3 gene from the antennal gland (kidney) of the crayfish Procambarus clarkii. The complete sequence, the first obtained from a non-genetic invertebrate species, was obtained through reverse transcription-polymerase chain reaction (RTPCR) and rapid amplification of cDNA ends (RACE) techniques. Crayfish PMCA3 consists of 4148 bp with a 3546 bp open reading frame coding for 1182 amino acid residues with a molecular mass of 130 kDa. It exhibits 77.5-80.9% identity at the mRNA level and 85.3-86.9% identity at the protein level with PMCA3 from human, mouse and rat. Membrane topography was typical of published mammalian PMCAs. Northern blot analysis of total RNA from crayfish gill, antennal gland, cardiac muscle and axial abdominal muscle revealed that a 7.5 kb species was ubiquitous. The level of PMCA3 mRNA expression in all tissues (transporting epithelia and muscle) increased significantly in pre/postmolt stages compared with relatively low abundance in intermolt. Western analysis confirmed corresponding changes in PMCA protein expression (130 kDa).

Characterization of sarcoplasmic calcium binding protein (SCP) variants from freshwater crayfish Procambarus clarkii

Sarcoplasmic calcium binding protein (SCP) is an invertebrate EF-hand calcium buffering protein that has been proposed to fulfill a similar function in muscle relaxation as vertebrate parvalbumin. We have identified three SCP variants in the freshwater crayfish Procambarus clarkii. The variants (pcSCP1a, pcSCP1b, and pcSCP1c) differ across a 37 amino acid region that lies mainly between the second and third EF-hand calcium binding domains. We evaluated tissue distribution and response of the variants to cold exposure, a stress known to affect expression of parvalbumin. Expression patterns of the variants were not different and therefore do not provide a functional rationale for the polymorphism of pcSCP1. Compared to hepatopancreas, expression of pcSCP1 variants was 100,000-fold greater in axial abdominal muscle and 10-fold greater in cardiac muscle. Expression was 10-100 greater in fast-twitch deep flexor and extensor muscles compared to slow-twitch superficial flexor and extensors. In axial muscle, no significant changes of pcSCP1, calmodulin (CaM), or sarcoplasmic/endoplasmic reticulum Ca-ATPase (SERCA) expression were measured after one week of 4°C exposure. In contrast, large decreases of pcSCP1 were measured in cardiac muscle, with no changes in CaM or SERCA. Knockdown of pcSCP1 by dsRNA led to reduced muscle activity and decreased expression of SERCA. In summary, the pattern of pcSCP1 tissue expression is similar to parvalbumin, supporting a role in muscle contraction. However, the response of pcSCP1 to cold exposure differs from parvalbumin, suggesting possible functional divergence between the two proteins.

Cloning and characterization of a calmodulin gene (CaM) in crayfish Procambarus clarkii and expression during molting.

Calmodulin (CaM) is a highly conserved calcium (Ca(2+)) binding protein that transduces Ca(2+) signals into downstream effects influencing a range of cellular processes, including Ca(2+) homeostasis. The present study explores CaM expression when Ca(2+) homeostasis is challenged during the mineralization cycle of the freshwater crayfish (Procambarus clarkii). In this paper we report the cloning of a CaM gene from axial abdominal crayfish muscle (referred to as pcCaM). The pcCaM mRNA is ubiquitously expressed but is far more abundant in excitable tissue (muscle, nerve) than in any epithelia (gill, antennal gland, digestive) suggesting that it plays a greater role in the biology of excitation than in epithelial ion transport. In muscle cells the pcCaM was colocalized on the plasma membrane with the Ca(2+) ATPase (PMCA) known to regulate intracellular Ca(2+) through basolateral efflux. While PMCA exhibits a greater upregulation in epithelia (than in non-epithelial tissues) during molting stages requiring transcellular Ca(2+) flux (pre- and postmolt compared with intermolt), expression of pcCaM exhibited a uniform increase in epithelial and non-epithelial tissues alike. The common increase in expression of CaM in all tissues during pre- and postmolt stages (compared with intermolt) suggests that the upregulation is systemically (hormonally) mediated. Colocalization of CaM with PMCA confirms physiological findings that their regulation is linked.

Roles of NCX and pmca in basolateral calcium export associated with mineralization cycles and cold acclimation in crayfish

Abstract:  Basolateral Na+/Ca2+ exchanger (NCX) and plasma membrane Ca2+ ATPase (PMCA) are the primary transmembrane proteins that export calcium (Ca2+) from cells. In our lab we use a nonmammalian animal model, the freshwater crayfish, to study cellular Ca2+ regulation. Two experimental conditions are employed to effect Ca2+ dyshomeostasis: (a) in the postmolt stage of the crustacean molting cycle increased unidirectional Ca2+ influx associated with cuticular mineralization is accompanied by elevated basolateral Ca2+ export (compared with intermolt Ca balance); and (b) exposure of the poikilothermic crayfish to cold acclimation (4°C) causes influx of Ca2+ into cells, which is compensated by increased basolateral Ca2+ export (compared with exposure to 23°C). This study compares expression of both NCX and PMCA mRNA (real‐time PCR) and protein (Western) in both epithelial (kidney) and nonepithelial tissue (tail muscle) during elevated basolateral Ca2+ export. Both experimental treatments produced increases in NCX and PMCA expression (mRNA and protein) in both tissues. Mineralization produced greater upregulation of mRNA in kidney than in tail, whereas cold acclimation yielded comparable increases in both tissues. Protein expression patterns were generally confirmatory of real‐time PCR data although expression changes were less pronounced. Both experimental treatments appear to increase basolateral Ca2+ export.

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

SUMMARY 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.

Upregulation of NCX Protein in Hepatopancreas and Antennal Gland of Freshwater Crayfish Associated with Elevated CA2+ Flux

The molting cycle of the freshwater crayfish Procambarus clarkii has emerged as an ideal model to study cellular/molecular mechanisms of Ca homeostasis because of its ability to maximally absorb Ca from a Ca deficient environment. Freshwater contains only 1 mM Ca as opposed to the 10 mM Ca afforded by marine environments. The model is based on a negligible net transepithelial Ca flux during intermolt that changes to vectorial Ca flux around ecdysis. During premolt, 20% of cuticular Ca is reabsorbed for deposition into CaCO3 disks and the remainder is excreted. Postmolt is characterized by net environmental Ca uptake rates of 2-10 mmol/kg/h and remobilization of stored Ca. Both sources are subsequently used for the calcification of a new exoskeleton. The Ca transporting epithelia are the gills, antennal gland (kidney), hepatopancreas (liver) and cuticular hypodermis. Arthropod molting is coordinated by the steroid ecdysone and unknown environmental cues. Previous studies have shown a marked increase in basolateral Ca pump expression during postmolt, suggesting that Ca transporting proteins are upregulated during periods of elevated transepithelial Ca uptake. Kinetic studies suggest that the NCX is the primary extrusion mechanism for basolateral Ca efflux. Therefore, we propose that the NCX may be upregulated during premolt and postmolt compared with intermolt in hepatopancreas and antennal gland.

Elongation factor 1Bγ (eEF1Bγ) expression during the molting cycle and cold acclimation in the crayfish Procambarus clarkii

Eukaryotic elongation factor 1Bgamma (eEF1Bgamma) is a subunit of elongation factor 1 (EF1), which regulates the recruitment of amino acyl-tRNAs to the ribosome during protein synthesis in eukaryotes. In addition to structural roles within eEF1, eEF1Bgamma has properties which suggest sensory or regulatory activities. We have cloned eEF1Bgamma from axial abdominal muscle of freshwater crayfish, Procambarus clarkii. The predicted amino acid sequence has 66% identity to Locusta migratoria eEF1Bgamma and 65% identity to Artemia salina eEF1Bgamma. We measured eEF1Bgamma expression by real-time PCR, using the relative quantification method with 18s ribosomal RNA as an internal calibrator. eEF1Bgamma expression was lowest in gill, axial abdominal muscle, and hepatopancreas, and was highest in the antennal gland (5.7-fold above hepatopancreas) and cardiac muscle (7.8-fold above hepatopancreas). In axial abdominal muscle, eEF1Bgamma expression was 4.4-fold higher in premolt and 11.9 higher in postmolt compared to intermolt. In contrast, eEF1Bgamma was decreased or unchanged in epithelial tissues during pre- and postmolt. eEF1Bgamma expression in the hepatopancreas was 3.5-fold higher during intermolt compared to premolt and was unchanged in gill and antennal gland. No significant differences in eEF1Bgamma were found after 1 week of acclimation to 4 degrees C. These results show that eEF1Bgamma is regulated at the mRNA level with tissue-specific differences in expression patterns.

Epithelial Calcium Transport in Crustaceans: Adaptation to Intrinsic and Extrinsic Stressors

Since the classical studies of Ussing employing a nonmammalian isolated epithelium (frog skin) to explore the basic principles of ion transport, physiologists have adopted increasingly reductionist approaches to dissect the biophysical mechanisms undergirding biological transport. In vitro characterization has employed isolated perfused organs, isolated epithelia, and reconstituted vesicle studies. Depth of resolution has been further enhanced by the emerging molecular revolution. Following years of deconstruction, physiologists are now engaging in reconstruction, namely putting the genes back into the organism. This contribution attempts such an integrative approach for a single electrolyte, calcium (Ca2+), in a nonmammalian epithelium, the crayfish antennal gland (kidney). Two collaborating laboratories have archived an inventory of Ca2+ associated proteins believed to play a role in transcellular Ca2+ movement. Using the basic building blocks (expression profiles of key Ca2+ associated proteins and their regulators), the authors attempt to reconstruct a whole cell model for Ca2+ regulation in transporting epithelium (compared with a nonepithelial tissue) under stressors that perturb Ca2+ homeostasis which originate either intrinsically (the postmolt stage of the molting cycle) or extrinsically (unanticipated cold acclimation). Through horizontal integration of expression profiles of seven target Ca2+ associated proteins in epithelial and nonepithelial tissue under two contrasting experimental conditions, emergent themes inform the physiological complexity of Ca2+ homeostasis. Integration at the next level will require placing the epithelium in the context of organismic Ca2+ balance. The unique Ca2+ handling capabilities of the freshwater crayfish make it an excellent nonmammalian model for those studies.

Expression of Plasma Membrane Ca2+ ATPase in Crayfish During Molting

Northern blot analysis indicated that the level of PMCA3 mRNA expression in all tissues increased significantly in pre/postmolt stages as compared to a very low level in the intermolt stage. Similar expression patterns were confirmed at the protein level by Western blotting. In situ hybridization confirmed that PMCA3 expression is increased in pre- and postmolt in kidney. Collectively these results indicate that the crayfish PMCA3 plays a crucial role in maintaining Ca2+ homeostasis during the fluctuations in Ca2+ flux associated with the crayfish molting cycle.

Salinity Affects Crayfish PMCA and NCX Expression

Exposure to elevated external Ca2+ resulted in upregulation of PMCA3 and NCX in both gill and kidney; however, expression in liver remained unchanged. Gill and kidney appear to respond to elevated apical Ca2+ entry with increased basolateral efflux. The liver may be pre-adapted to handle Ca2+ load in the diet and, as such, does not need to increased expression of basolateral export mechanisms when environmental levels are elevated. We will further explore these trends through protein analysis.