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Expression Profiles of Na⁺,K⁺-ATPase during Acute and Chronic Hypo-osmotic Stress in the Blue Crab Callinectes sapidus

¹ Department of Biology, The College of New Jersey, Ewing, New Jersey 08628
² Mount Desert Island Biological Laboratory, Salisbury Cove, Maine 04672

^* To whom correspondence should be addressed. E-mail: lovett{at}tcnj.edu

	Abstract

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During acclimation to dilute seawater, the specific activity of Na⁺,K⁺-ATPase increases substantially in the posterior gills of the blue crab Callinectes sapidus. To determine whether this increase occurs through regulation of pre-existing enzyme or synthesis of new enzyme, mRNA and protein levels were measured over short (< 24 h) and long (18 days) time courses. Na⁺,K⁺-ATPase expression, both mRNA and protein, did not change during the initial 24-h exposure to dilute seawater (10 ppt salinity). Thus, osmoregulation in C. sapidus during acute exposure to low salinity likely involves either modulation of existing enzyme or mechanisms other than an increase in the amount of Na⁺,K⁺-ATPase enzyme. However, crabs exposed to dilute seawater over 18 days showed a 300% increase in Na⁺,K⁺-ATPase specific activity as well as a 200% increase in Na⁺,K⁺-ATPase protein levels. Thus, it appears that the increase in Na⁺,K⁺-ATPase activity during chronic exposure results from the synthesis of new enzyme. The relative amounts of mRNA for the -subunit increased substantially (by 150%) during the acclimation process, but once the crabs had fully acclimated to low salinity, the mRNA levels had decreased and were not different from levels in crabs fully acclimated to high salinity. Thus, there is transient induction of the Na⁺,K⁺-ATPase mRNA levels during acclimation to dilute seawater.

	Introduction

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The blue crab Callinectes sapidus is a strong hyperosmoregulator, maintaining relatively constant hemolymph osmolality across a wide range of dilute salinities in the estuarine environment. The primary site of osmoregulation is the posterior gills, which have the highest specific activity of Na⁺,K⁺-ATPase (reviewed in Mantel and Farmer, 1983). The gill epithelium consists of two basic cell types: gas exchange and ion-transporting cells (Copeland and Fitzjarrel, 1968). The ion-transporting cells, which are thought to be responsible for the blue crab’s ability to hyperosmoregulate in dilute seawater, contain a number of enzymes, cotransporters, antiporters, and channel proteins that facilitate the movement of ions across the gill epithelium. The Na⁺,K⁺-ATPase protein has been localized to the basolateral membrane of the epithelial cells, with the highest proportion occurring in the ion-transporting cells (Towle and Kays, 1986). The Na⁺,K⁺-ATPase enzyme is thought to provide the major driving force for ion transport, partly through establishment of an electrochemical gradient across epithelial cell membranes (reviewed in Lucu and Towle, 2003).

When crabs acclimated to full-strength seawater are transferred to dilute seawater below 27 ppt, a number of acute and chronic changes occur (reviewed in Lucu and Towle, 2003). During the acute response (within the first 24 h), hemolymph osmolality decreases but remains hyperosmotic to the seawater (Engel and Nichols, 1977). Most studies have found no significant increase in the specific activity of Na⁺,K⁺-ATPase in homogenates of posterior gills in C. sapidus during this acute response period (Neufeld et al., 1980; Henry et al., 2002; Lovett et al., 2006). An early study by Towle et al. (1976) reported a 50% increase in activity within 3 h of transfer, although the statistical significance of this increase was not tested. During acclimation to dilute seawater over long time periods, a substantial increase in the specific activity of Na⁺,K⁺-ATPase in homogenates of posterior gills and a substantial increase in the relative proportion of ion-transporting cells in the posterior gill lamellae both occur between days 1–6 after transfer to dilute salinity, although full acclimation to dilute seawater may take as long as 14–18 days to complete (Copeland and Fitzjarrell, 1968; Aldridge and Cameron, 1982; Neufeld et al., 1980; Henry and Cameron, 1982; Lovett et al., 2006).

We present here a detailed description of changes in the specific activity of Na⁺,K⁺-ATPase in relation to the relative concentration of -subunit protein and levels of its mRNA in posterior gill tissue during the time course of both the acute and chronic responses of C. sapidus to abrupt transfer to dilute seawater. These changes suggest that chronic hyperosmoregulation is accompanied by up-regulation of -subunit gene expression and apparent de novo synthesis of additional enzyme. Previous studies (Towle et al., 2001) compared crabs that had been fully acclimated to either full-strength or dilute seawater and found no difference in gene expression when using a manual semiquantitative PCR method. However, in the present study, transient changes in mRNA levels were detected using a more accurate method (quantitative real-time PCR) and by examining crabs at regular intervals throughout the acclimation process.

	Materials and Methods

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Animals
Intermolt blue crabs (Callinectes sapidus Rathbun) were maintained in recirculating tanks with biological filters and were acclimated to 32 ppt salinity (945 ± 10 mOsm/kg) artificial seawater (Instant Ocean, Aquarium Systems) for at least 18 days. Crabs were fed squid mantle ad libitum. Since male and female crabs respond differently to changes in salinity (Neufeld et al., 1980), only male crabs were used. Following acclimation, crabs were then transferred to 10 ppt salinity (315 ± 2 mOsm/kg) and collected at regular intervals for up to 18 days. Hemolymph samples were drawn from individual animals at the base of the fifth pereiopod. Crabs were anesthetized on ice and the sixth and seventh gill pairs (G6 and G7) were excised. Gill G7 from the left side was collected to measure mRNA levels; gill G7 from the right side and both gills from gill pair G6 were collected to measure Na⁺,K⁺-ATPase activity and protein levels. The salinity of the water in each tank was established using a refractometer, and its osmotic concentration was determined later with a vapor pressure osmometer (Wescor, Inc.). The osmolality of each hemolymph sample was measured with a vapor pressure osmometer.

Assay of Na⁺,K⁺-ATPase enzyme activity
To measure ATPase activity, gills were blotted, weighed, and homogenized in EIS buffer (2 mM di-sodium EDTA, 50 mM imidazole pH 7.2, 250 mM sucrose) with 0.15% deoxycholate, 0.1 mM AEBSF (4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride) and 0.1 mM PMSF (phenylmethanesulfonyl fluoride). Crude homogenates were partially purified by centrifugation at 10,000 x g to remove cellular debris and mitochondria, and were stored at –75 °C. Samples were subsequently thawed and assayed spectrophotometrically for ouabain-sensitive Na⁺,K⁺-ATPase with a linked pyruvate kinase/lactate dehydrogenase-NADH assay (Saintsing and Towle, 1978). Total protein in homogenates was measured using a BCA Protein Assay kit (Pierce Chemical Co.).

Measurement of Na⁺,K⁺-ATPase and actin protein
To determine whether the amount of Na⁺,K⁺-ATPase protein increases during acclimation to low salinity, Western blots of partially purified homogenates were prepared with 4%–15% Tris-HCl SDS-polyacrylamide gradient gels. Blots were probed with both a monoclonal antibody against the -5 subunit of chicken kidney Na⁺,K⁺-ATPase (Lebovitz et al., 1989) and a monoclonal antibody against mouse actin (Clone AC-40, Sigma Chemical Co.). Secondary antibody (peroxidase-linked sheep anti-mouse IgG, Amersham Pharmacia Biotech) was visualized with 3,3'-diaminobenzidine (Sigma Chemical Co.). Molecular weights were estimated using BioRad Kaleidoscope prestained molecular weight standards. Four different crabs were used for each treatment. Blots were analyzed densitometrically with a UVP EC3 bioimaging system with Labworks 4.5 software. Sample dilutions were normalized to total protein content of homogenates. Gels stained with Coomassie blue were used to confirm that protein content in each sample was equivalent.

Measurement of Na⁺,K⁺-ATPase and arginine kinase mRNA
Levels of mRNA were measured by quantitative real-time PCR. Total RNA was extracted from gills using Promega RNAgents Total RNA Isolation System. Poly(A) mRNA was reverse-transcribed into single-stranded cDNA using Invitrogen First-Strand Synthesis kit and oligo(dT) primer. Real-time quantitative PCR with gene-specific primers was used to measure the amount of specific mRNAs (-subunit of Na⁺,K⁺-ATPase and arginine kinase) in the total cDNA pool from each sample. Amplification with Stratagene Brilliant SYBR Green QPCR Master Mix was monitored using a Stratagene MX4000 Real-Time PCR instrument; cycles consisted of denaturation at 94 °C for 40 s, annealing at 50 °C for 40 s, and elongation at 72 °C for 1 min. The cDNA from 4–10 independent RNA preparations was analyzed in triplicate for each treatment. Standard curves were derived from analysis of threshold cycles (C_t) of serial dilutions of reference samples, and expression values were normalized to the lowest value for each transcript. The amount of cDNA for the -subunit of Na⁺,K⁺-ATPase was normalized to total RNA and to the relative amount of arginine kinase cDNA in each sample. Arginine kinase expression in gill tissues has been shown to not vary significantly with salinity (Kotylar et al., 2000; Towle et al., 2001).

Gene-specific primers described in Kotlyar et al. (2000) and Towle et al. (2001) were used for arginine kinase. Gene-specific primers for the -subunit of Na⁺,K⁺-ATPase (sense: 5'-GCC TCC GTG CCT CT ACC TCT-3'; antisense: 5'-TGG AGT TAC GGC GAG TCT TAC-3') were developed using Primer Premier. Primer specificity was confirmed by comparing sequences of amplification products with published sequences for arginine kinase [GenBank Accession Number AF233355] and Na⁺,K⁺-ATPase -subunit [AF327439] for C. sapidus.

Data analysis
Data are reported as means ± standard errors. Sample size for all means was 4–8 animals for acute studies and 6–10 animals for chronic studies, except for Western blots, where sample size was 4 animals for each mean. Measurement of each sample was made in triplicate. Results were analyzed by one-way analysis of variance followed by the Student-Newman-Keuls multiple comparisons test (InStat, ver. 2.04a, GraphPad Software).

	Results

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Osmoregulatory ability
When Callinectes sapidus was transferred to 10 ppt salinity (332 ± 5 mOsm/kg) seawater, its hemolymph osmolality decreased rapidly throughout the first 24 h, dropping to near its acclimation level (723 ± 12 mOsm/kg), and then did not change significantly (P > 0.05) thereafter (Fig. 1A, B). In contrast, the specific activity of Na⁺,K⁺-ATPase in the posterior gills did not increase (P > 0.05) during acute exposure (during the first 24 h after transfer) (Fig. 1A). However, during chronic exposure to 10 ppt seawater, Na⁺,K⁺-ATPase activity did increase from 7.8 ± 2.0 µmol P_i/h/mg protein (in crabs acclimated to 32 ppt), reaching the low-salinity acclimation value of 36.6 ± 7.8 µmol P_i/h/mg protein (more than a 300% increase) by day 8 (Fig. 1B). In an attempt to increase the magnitude of the response during acute exposure, a second set of crabs was transferred to 5 ppt salinity (167 ± 1 mOsm/kg) seawater; during the first 24 h there also was no significant change in the Na⁺,K⁺-ATPase activity in these crabs (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 1. Acute and chronic (acclimation) responses in the blue crab Callinectes sapidus acclimated to 32 ppt salinity and then transferred to 10 ppt salinity. (A) Acute response: n = 4–8. (B) Chronic response: n = 6–10. Open circles, hemolymph osmolality (Osm); closed circles, specific activity of Na+,K+-ATPase in partially purified homogenates of posterior gills (NaK). Mean ± SE indicated. Means with asterisks are significantly different from mean at t = 0 (*P < 0.05; ***P < 0.001).

View larger version (68K): [in this window] [in a new window]   Figure 2. Western blot of partially purified homogenates of posterior gills from the blue crab Callinectes sapidus acclimated to 32 ppt salinity and then transferred to 10 ppt salinity. Samples were normalized to total protein content of homogenates. Bands were visualized using antibodies to -subunit of Na+,K+-ATPase and to actin. Labels at top of lanes indicate the molecular weight standard (MW) and number of days that each crab had been exposed to dilute seawater. Labels to the left indicate molecular weights (kDa) of standards.

View larger version (12K): [in this window] [in a new window]   Figure 3. Protein concentration estimated from densitometry values for bands from Western blots of partially purified homogenates of posterior gills from the blue crab Callinectes sapidus acclimated to 32 ppt salinity and then transferred to 10 ppt salinity. Values reported were normalized to a value of 1.0 for the lowest amount for each protein. Open circles, band for -subunit of Na+,K+-ATPase with molecular weight estimated at 113 kDa (NaK 113); closed circles, band for -subunit of Na+,K+-ATPase with molecular weight of 131 kDa (NaK 13); triangles, band for actin. Mean ± SE indicated. Means with asterisks are significantly different from mean at t = 0 (*P < 0.05; **P < 0.01); n = 4.

Changes in mRNA levels
The relative amount of arginine kinase mRNA did not change (P > 0.05) throughout exposure to dilute seawater (Fig. 4). The relative amount of Na⁺,K⁺-ATPase -subunit mRNA also did not change (P > 0.05) during acute (24-h) exposure of crabs to dilute seawater (Fig. 5A). However, -subunit mRNA levels did increase (P < 0.01) after day 1, and continued to increase during chronic exposure to dilute seawater until day 8, at which time the amount of mRNA was 150% higher than that in crabs acclimated to 32 ppt seawater (Fig. 5B). After day 8, the relative amount of -subunit mRNA decreased until, upon acclimation to 10 ppt salinity at day 18, mRNA levels were not different (P > 0.05) from levels in crabs acclimated to 32 ppt. When these values were normalized to relative concentrations of arginine kinase mRNA in each sample, the pattern of change remained similar to that of samples that had been normalized to total RNA, but the maximum attained at day 8 represented only a 60% increase above levels in crabs acclimated to 32 ppt salinity (Fig. 5A, B). Thus, there was a transient, but substantial, increase in levels of -subunit mRNA during the acclimation of crabs to low salinity over the 18-day chronic exposure.

View larger version (7K): [in this window] [in a new window]   Figure 4. Relative amount of arginine kinase (ArK) mRNA in posterior gills of the blue crab Callinectes sapidus acclimated to 32 ppt salinity and then transferred to 10 ppt salinity. Amounts were estimated through quantitative real-time PCR. Values reported were normalized to a value of 1.0 for the lowest concentration of arginine kinase mRNA. Mean ± SE indicated. None of the means were significantly different from the mean at t = 0 (P > 0.05); n = 6–10.

View larger version (12K): [in this window] [in a new window]   Figure 5. Relative amount of -subunit of Na+,K+-ATPase mRNA in posterior gills during acute and chronic (acclimation) responses in the blue crab Callinectes sapidus acclimated to 32 ppt salinity and then transferred to 10 ppt salinity. Amounts were estimated through quantitative real-time PCR. (A) Acute response: n = 4–8. (B) Chronic response: n = 6–10. Closed circles, values normalized to a value of 1.0 for the lowest concentration of Na+,K+-ATPase mRNA (NaK); open circles, values normalized to the relative amount of arginine kinase mRNA, and then normalized to a value of 1.0 for the lowest relative value (NaK/ArK). Mean ± SE indicated. Means with asterisks are significantly different from mean at t = 0 (*P < 0.05).

	Discussion

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In the present study, no significant (P > 0.05) change in Na⁺,K⁺-ATPase activity was detected in vitro within 24 h after transfer to dilute seawater, even though stabilization of hemolymph osmotic concentrations by the end of this period would indicate that osmoregulation in C. sapidus had commenced. It is possible that no significant change in Na⁺,K⁺-ATPase activity was observed because transporters other than Na⁺,K⁺-ATPase may be more important in short-term control of ion uptake, as proposed by Lucu and Towle (2003). However, because Na⁺,K⁺-ATPase is considered to provide the primary driving force for hyperosmoregulation, its activity would be expected to increase in crabs that had begun to hyperosmoregulate. Many have proposed that existing Na⁺,K⁺-ATPase in crab gills may be activated or de-inhibited during acute exposure to dilute seawater (for example, Winkler et al., 1988; Lucu, 1990; Lucu and Flik, 1999; Henry et al., 2002). Although dramatic (300%–400%) increases in activity during chronic exposure are routinely reported, few studies of intact crabs have documented a significant increase in Na⁺,K⁺-ATPase activity during acute exposure to decreased salinity. Other experimental manipulations and in vitro studies of isolated gill tissue have provided evidence that Na⁺,K⁺-ATPase activity can be stimulated to increase in less than an hour (reviewed in Lucu and Towle, 2003; Mo et al., 2003; Halperin et al., 2004). But most of the studies that did measure an acute increase in Na⁺,K⁺-ATPase activity have examined intertidal species. As hypothesized by Henry et al., (2002), C. sapidus may not demonstrate the same rapid modulation of Na⁺,K⁺-ATPase activity as these other species because its subtidal habit would not expose it to the rapid fluctuations in salinity that intertidal species might experience.

Because the concentration of the -subunit protein in posterior gills of C. sapidus did not increase during the first 24 h after transfer, we conclude that the acute response involves mechanisms other than an increase in the amount of Na⁺,K⁺-ATPase enzyme. The decrease in the osmotic concentration of the hemolymph after exposure of crabs to dilute seawater may be impeded after 24 h either by an increase in Na⁺,K⁺-ATPase activity that is not detected through in vitro assays of gill homogenates or by other mechanisms such as mobilization of organic solutes or changes in gill permeability to water and ions (Gérard and Gilles, 1972; Cantelmo, 1977; Robinson, 1982, 1994; Henry and Watts, 2001). Henry et al. (2002) proposed that slight changes in enzyme activity may be sufficient to stabilize the osmotic concentration of the hemolymph until more permanent regulatory changes can occur. We suspect that substantial in vivo changes in Na⁺,K⁺-ATPase activity may occur within 24 h, but they are not detected by in vitro assays because tissue homogenization has disrupted intracellular compartmentalization. This disruption may cause the release of enzymes or compounds that affect Na⁺,K⁺-ATPase activity or it may cause the intermixing of cytoplasmic vesicles with plasma membrane vesicles formed during homogenization. Reversible phosphorylation of the -subunit modulates Na⁺,K⁺-ATPase activity in vertebrates (Ewart and Klip, 1995; Chibalin et al., 1999), and evidence has been presented to suggest that this mechanism also may modulate activity of existing Na⁺,K⁺-ATPase enzyme in crab gills (Péqueux, 1995; Lucu and Flik, 1999; Towle et al., 2001). Furthermore, addition of phosphatase inhibitors to the homogenization buffer increased the activity of Na⁺,K⁺-ATPase substantially above that measured in contralateral gill homogenates prepared without the inhibitors (Lovett et al., unpubl. data). On the other hand, Na⁺,K⁺-ATPase activity in the vertebrate kidney also is reduced through sequestration of the enzyme from the basolateral membrane into cytoplasmic vesicles (Chibalin et al., 1999; Dunbar and Caplan, 2001). If Na⁺,K⁺-ATPase were to be sequestered in a similar way by crabs in high salinity, the cytoplasmic vesicles could be trafficked back into the basolateral membrane during acute hypo-osmotic stress, reexposing the enzyme to the hemolymph and rapidly increasing the in vivo activity of the enzyme (Lucu and Flik, 1999; Parikh et al., 2005). When microsomal vesicles formed from each of these membrane pools are intermixed by the homogenization process, differences in the relative distribution of Na⁺,K⁺-ATPase enzyme between the two pools would go undetected in vitro.

Chronic response
Changes in Na⁺,K⁺-ATPase activity and amounts of -subunit.
An increase in Na⁺,K⁺-ATPase activity during chronic exposure of crabs to dilute seawater (with maximum enzyme activity reached by days 6–8 after transfer to low salinity) has been reported both for C. sapidus (Neufeld et al., 1980; Lovett et al., 2006) and for other euryhaline crabs (Siebers et al., 1983; Péqueux et al., 1984; D’Orazio and Holliday, 1985; Holliday, 1985; Holliday et al., 1990; Lucu and Flik, 1999; Henry et al., 2002). The present study also reports an increase in the concentration of Na⁺,K⁺-ATPase -subunit protein in gill tissue and demonstrates that the maximum concentration of the enzyme is attained within 8 days after transfer. Thus, it appears that the increase in Na⁺,K⁺-ATPase activity during long-term acclimation results from the synthesis of new enzyme. Masui et al. (2005) also reported a higher concentration of -subunit protein in posterior gills of Callinectes danae acclimated to dilute seawater than in gills of crabs acclimated to full-strength seawater. In contrast, Chung and Lin (2006) measured the concentration of -subunit protein in posterior gills of Scylla paramamosain, but no significant change was detected during acclimation to dilute seawater due to high variability among individual crabs.

Changes in amounts of mRNA.
Even though there was no significant difference in the relative amount of -subunit mRNA between animals fully acclimated to 32 ppt and animals acclimated to 10 ppt, mRNA levels changed significantly during the process of acclimation to dilute seawater. The relative amount of -subunit mRNA reached its maximum at day 8 during chronic exposure to reduced salinity and then decreased to initial levels. Studies of C. sapidus by Li et al. (2006), Scylla paramamosain by Chung and Lin (2006), and Chasmagnathus granulatus by Luquet et al. (2005) have each reported an increase in the amount of Na⁺,K⁺-ATPase -subunit mRNA in posterior gills following transfer to dilute seawater. However, none of these studies examined the response beyond 6 to 8 days following transfer, and, thus, none were able to report whether transcript quantity returned to initial levels after crabs had become fully acclimated to dilute seawater. The peaks in -subunit mRNA levels coincide with both the time at which Na⁺,K⁺-ATPase activity has reached its maximum and the time at which differentiation and hypertrophy of additional ion-regulatory cells in the gill epithelium occurs (Lovett et al., 2006). Thus, two distinct acclimation changes (changes in ion-transporting ability and changes in gill structure) appear to be initiated at about the same time (with measurable changes beginning some time after day 1). Neufeld et al. (1980) had originally hypothesized that acclimation changes in Na⁺,K⁺-ATPase activity in C. sapidus were due to de novo synthesis of enzyme, rather than modulation of existing enzyme, although the tools to verify the hypothesis were not available at the time. In studies of the acclimation response in Uca pugilator and Hemigrapsus nuda, initial increases in specific activity of Na⁺,K⁺-ATPase after 3 days were attributed to enzyme activation, while increases to the maximum activity 7 days after transfer were attributed to synthesis of new enzyme (D’Orazio and Holliday, 1985; Corotto and Holliday, 1996). The data presented here for C. sapidus suggest that increases in enzyme activity during chronic exposure to dilute seawater are due to synthesis of new enzyme. It was not possible to determine whether the increase in the amount of -subunit mRNA in C. sapidus occurred through induction (increased rate of transcription of the gene) or increased stability of the mRNA, but analysis of the 3' untranslated region of the -subunit cDNA has suggested that the mRNA for this subunit is long-lived and not subject to rapid degradation (Towle et al., 2001). Therefore, it is possible that there is up-regulation of both transcription and translation in response to sustained hypo-osmotic stress.

Na⁺,K⁺-ATPase isoforms
Only a single isoform of the -subunit has previously been reported from decapod crustaceans (Péqueux et al., 1984; Towle et al., 1997, 2001; Furriel et al., 2000), although Wanson et al. (1984) hypothesized the existence of two different Na⁺,K⁺-ATPase enzymes on the basis of differences in kinetic properties of homogenates from anterior and posterior gills of Uca minax. In contrast, two isoforms of the Na⁺,K⁺-ATPase -subunit have been reported from Artemia spp. (Peterson et al., 1982; Cortas et al., 1989). In the present study, two distinct electrophoretic bands for the -subunit of Na⁺,K⁺-ATPase were detected in C. sapidus gill homogenates, but only when a gradient gel was used. The relative concentration of the protein represented by the 131-kDa band increased substantially (P < 0.01) during the chronic response. The significance of the differential levels of the larger molecular weight species is not understood. In Artemia, mobility differences between the two forms of -subunit protein were attributed to differences in post-translational modification, particularly to differences in glycosylation of the proteins (Peterson et al., 1982). However, because Na⁺,K⁺-ATPase activity may be modulated by phosphorylation of the -subunit in C. sapidus, we are assessing whether differences in the mobility of the two bands in this species are the result of phosphorylation, glycosylation, or some other post-translational modification of the -subunit.

	Acknowledgments

 
This study was supported in part by MDIBL New Investigator Award to DLL and NSF grant DBI-0100394 to DWT. Antibody against Na⁺,K⁺-ATPase -subunit was generously provided by D. Fambrough, Johns Hopkins University. Amplification products were sequenced at the Mount Desert Island Biological Laboratory Molecular Sequencing Center.

	Footnotes

 
Received 24 August 2005; accepted 12 May 2006.

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