Low Temperature Molecular Adaptation of the Skeletal Muscle Sarco(endo)plasmic Reticulum Ca 2 1 -ATPase 1 (SERCA 1) in the Wood Frog ( Rana sylvatica )*

We have compared the primary sequence and enzymatic properties of the sarcoplasmic reticulum Ca 2 1 ATPases from a cold-tolerant frog Rana sylvatica with those of a closely related cold-intolerant frog, Rana clamitans . Sarcoplasmic reticulum isolated from leg muscles of both species contains a major protein ( ; 100 kDa) that reacts with a monoclonal antibody against sarco(endo)plasmic reticulum Ca 2 1 -ATPase type 1 (SERCA1). The apparent molecular mass of R. sylvatica SERCA1 is 115 kDa, whereas that of R. clamitans is 105 kDa. However, the deduced amino acid sequences obtained from cDNAs do not indicate a difference in molecular weight, thus suggesting post-translational protein modification of R. sylvatica SERCA1. Comparison of the temperature dependence of both ATP hydrolysis and Ca 2 1 transport indicates that R. sylvatica SERCA1 exhibits significantly lower activation energy below 20 °C and an ; 2-fold greater Ca 2 1 -ATPase activity near 0 °C. Furthermore, R. sylvatica SERCA1 exhibits simple Michaelis-Menten kinetics with ATP and Ca 2 1 as op-posed to the two-site ATP kinetics and positive cooperativity with Ca 2 1 observed for R.

There has been considerable interest in the mechanism by which the wood frog, Rana sylvatica, survives freezing. This frog is one of the few vertebrate species that can be frozen solid and revived upon thawing (1). R. sylvatica survives freezing by producing very high cryoprotectant concentrations of glucose in vital organs (2,3). Furthermore, R. sylvatica mates and lays eggs in water temperatures approaching 0°C. Because R. sylvatica muscle functions efficiently near 0°C, the Ca 2ϩ pump must also be functional at low temperatures. However, although the effects of freezing/thawing on several enzymes and physiological processes have been studied (4 -6), no studies on the wood frog skeletal muscle Ca 2ϩ -ATPase (SERCA1) 1 have been reported. The kinetic properties and amino acid sequence of SERCA1 from Rana esculenta (European green frog) have been reported (7,8) and are quite similar to those of the rabbit. However, R. sylvatica muscle could not function at near freezing temperatures if its Ca 2ϩ -ATPase had the same properties as the rabbit because rabbit SERCA1 is almost totally inactive at 0°C (9). Kossler and Kuchler (10) concluded that the main cause of severe inhibition of muscle contraction in the rat at 6°C is the temperature-dependent inhibition of Ca 2ϩ uptake and release. A similar conclusion was reached for another frog Rana temporaria at 10°C (11). Thus, SERCA1 is likely to have altered properties in the skeletal muscle of the cold-tolerant frog R. sylvatica.
There is a vast amount of knowledge concerning structure/ function relationships for SERCA1 because it is one of the most highly studied membrane-associated proteins. Structure/function relationships have recently been reviewed by MacLennan et al. (12). The protein was first purified in 1970 by MacLennan (13), and the sequence was deduced from the cDNA by MacLennan et al. (14). The complete three-dimensional structure has recently been obtained at 2.6-Å resolution by x-ray crystallographic analysis of protein crystals (15). This recent advance has confirmed the three-dimensional folding of the domains that had previously been proposed based on protein chemistry and analysis of two-dimensional arrays in membranes. An astonishing proportion of the amino acid residues predicted to be involved in SERCA1 function has been modified by site-directed mutagenesis and the effects on the function of the expressed protein were determined (12). However, there is no direct structural information regarding temperature dependence of the pump.
In the present study, we have compared structural and func-tional properties of SERCA1s from R. sylvatica with those of Rana clamitans, a closely related frog living in the same environment that is cold-sensitive and cannot function at 0°C. Our results indicate that there are significant differences in the kinetic properties of SERCA1 between these two species, including temperature dependence of ATP hydrolysis and Ca 2ϩ transport, kinetics with ATP, and cooperativity of Ca 2ϩ kinetics. We also report the complete amino acid sequences of these two SERCA1s.

EXPERIMENTAL PROCEDURES
Materials and Chemicals-R. sylvatica and R. clamitans were collected from local ponds in accordance with the guidelines provided by the Institutional Animal Care and Use Committee. The chemicals used for SDS-polyacrylamide gel electrophoresis and immunoblotting were obtained from Bio-Rad. Chemiluminescence reagents were purchased from PerkinElmer Life Sciences. Monoclonal anti-SERCA1, clone VE121G9 was from Affinity BioReagents, Inc. (Golden, CO) as were the monoclonal anti-SERCA2 (MA3-910) clone IID8 and polyclonal antipeptide anti-SERCA3 (PA1-910) antisera. All three antibodies exhibit cross-reactivity with several mammalian species. Restriction endonucleases were supplied by New England Biolabs, Inc. (Beverly, MA). The Expand ® Long Distance PCR kit, which uses a mixture of Taq and Pwo polymerases, was from Roche Molecular Biochemicals. ALF ® Express Sequencing kit and cyanin dATP-Labeling Mix as well as cyaninlabeled sequencing primers were purchased from Amersham Pharmacia Biotech. Plasmid preparations were carried out using Qiagen columns (Qiagen, Hilden, Germany). Restriction fragments used for subcloning procedures were gel-purified with the QIAEX II Gel Extraction kit (Qiagen). pGEM ® -T Easy vector (Promega, Madison, WI) was chosen for direct subcloning of all 5Ј and 3Ј RACE-PCR products. Gene-specific oligonucleotides were from Life Technologies, Inc. Other molecular biology grade chemicals were purchased from Sigma.
Preparation and Analysis of Sarcoplasmic Reticulum (SR)-Leg muscles were removed from euthanized R. sylvatica captured in late February and from R. clamitans captured in late March, both soon after emergence from hibernation. SR membranes were purified from the homogenized muscle by sucrose density centrifugation using the procedure described by Bull et al. (16). SDS-polyacrylamide gel electrophoresis was carried out according to Laemmli (17), and immunoblotting was done as described earlier (18). Ca 2ϩ -ATPase activity was assayed using a system with ATP hydrolysis coupled to NADH oxidation by pyruvate kinase and lactate dehydrogenase (19). In addition to the coupling components, the assay contained 10 mM Tes buffer at pH 7.4, 0.1 M KCl, and 5.0 mM MgCl 2 . ATP was prepared as a 1:1 MgATP complex. ATP and Ca 2ϩ dependence of Ca 2ϩ -ATPase activity were assayed at 30°C in a Bio-Tek Powerwave X 96-well plate reader, whereas the temperature dependence of Ca 2ϩ -ATPase activity was determined in a Cary 219 spectrophotometer. Ca 2ϩ -ATPase activity was calculated from the difference in the rate of ATP hydrolysis in the presence of various concentrations of Ca 2ϩ and 2 mM EGTA. For Ca 2ϩ dependence, free [Ca 2ϩ ] was calculated taking into account the concentrations of total Ca 2ϩ , EGTA, Mg 2ϩ , ATP, temperature and pH using the Winmaxc program available on the world wide web (Chris Patton, Stanford University). Ca 2ϩ transport was measured using the Ca 2ϩ -sensing dye, Rhod2, as described previously (20). The buffer components were the same as the components listed above for the Ca 2ϩ -ATPase assay except that MgCl 2 was eliminated, and 2.5 M Rhod2 was added. ATP (5 mM) was added as the MgATP complex. The assay was carried out at various temperatures in a PerkinElmer LS50B Luminescence Spectrometer with excitation at 553 nm and emission at 576 nm. Before the addition of membranes (a final concentration of 0.1 mg/ml), 0.5 nmol aliquots of CaCl 2 were added to calibrate the assay and allow for the determination of Ca 2ϩ transport rates. The addition of BrA23187 at the end of each assay released all sequestered Ca 2ϩ (20). Kinetic data were analyzed using Enzfitter© software (version 1.05), whereas curve fitting was accomplished using the Slide Write Plus© software (version 5.0). Protein concentration was estimated using bicinchoninic acid (BCA Protein Assay kit, Pierce) with bovine serum albumin as a standard.
Construction of Wood and Green Frog cDNA Libraries-Uncloned adaptor-ligated double-stranded (ds) cDNA libraries were constructed using the Marathon™ cDNA amplification kit from CLONTECH (Palo Alto, CA) according to manufacturer's instructions. Total RNA was isolated from either an entire wood frog (weighing 1 g) at an early age or a green frog skeletal muscle (weighing 1 g) using the guanidinium thiocyanate/CsCl purification method (21). 10 g of total RNA was used for each first-strand synthesis. The Marathon™ cDNA synthesis primer (52-mer) was extended with Moloney murine leukemia virus reverse transcriptase for 1 h at 42°C, after which second-strand synthesis was carried out by adding an enzyme mixture containing RNase H, Escherichia coli DNA polymerase I, and E. coli DNA ligase for 2 h at 16°C. A blunt ending of the ds-cDNAs was performed with T4 DNA polymerase at 16°C for 45 min. ds-cDNAs were purified by phenol/CHCl 3 / isoamyl alcohol (25:24:1) extraction precipitated by 95% ethanol in the presence of 4 M ammonium acetate. The pellet was washed with 80% ethanol and dissolved in sterile water. The Marathon™ cDNA adaptor was ligated to ds-cDNAs overnight at 16°C using T4 DNA ligase.
Rapid Amplification of 3Ј and 5Ј cDNA Ends by PCR (3Ј and 5Ј RACE-PCR)-3Ј and 5Ј RACE-PCR amplifications were performed using the adaptor-ligated ds-cDNAs as template DNAs, a mixture of Taq and Pwo (with a proofreading activity) polymerases, a common Marathon adaptor-primer 1, and an internal gene-specific primer. For the 3Ј RACE-PCR, a gene-specific primer PHϩ (5Ј-TCTGATAAGACTGG-TACTCTGACCACC-3Ј) was derived from the nucleotide sequence coding for the phosphorylation site ( 350 SDKTGTLTT) of the R. esculenta SERCA1a (7). The PHϩ primer corresponds to nucleotides 1275-1301 in the R. esculenta cDNA sequence (numbered according to the sequence deposited under GenBank TM accession number X63009) and to nucleotides 1251-1277 and 1254 -1280, respectively, in R. sylvatica and R. clamitans nucleotide sequences described in this study. However, note that the underlined base "T" of the R. esculenta is replaced by a "C" in both R. sylvatica and R. clamitans cDNA sequences. The 3Ј RACE-PCR amplifications with the PHϩ/adaptor primer 1 primer set were carried out for 30 cycles, each cycle consisting of 1 min of denaturation at 94°C, 1 min of annealing at 60°C, and 4 min of extension at 68°C. PCR products were size-fractionated by 1% agarose gel electrophoresis, visualized by ethidium bromide staining, gel-purified, and subcloned into the pGEM ® -T Easy vector. For each frog, several clones were partially sequenced. To perform the 5Ј RACE-PCR reactions, an antisense primer (5ЈRϪ) common for both frog cDNAs, was derived from the partially determined sequence 5Ј-GTGCACAGATTGTGGCCAACTC-CAG-3Ј. This primer corresponds to the inverse complement of nucleotides 1431-1458 and 1434 -1461, respectively, in R. sylvatica and R. clamitans sequences described in this study. 5Ј RACE-PCR amplifications with the adaptor primer 1/5ЈRϪ primer set were performed using the above mentioned cycle conditions. The 5Ј RACE-PCR products were also subcloned in pGEM ® -T Easy vector. The composite wood and green frog cDNA nucleotide sequences in both strands were determined completely using several gene-specific primers and the A.L.F. ® Express DNA Sequencer (Amersham Pharmacia Biotech). The sequencing strategy is shown in Fig. 5. For further applications, cDNA clones containing the entire coding regions were obtained by PCR amplifications using the following pair of primers: fcDNAϩ (5Ј-GGCTGAGAGGCGAGGAT-GGAGAACGCAC-3Ј) corresponding to nucleotides 189 -216 and 191-219, respectively, in wood and green frog cDNAs and fcDNAϪ (5Ј-TTATCCTTCAAGGTAGTTACGGGCAACA-3Ј) corresponding to the inverse complement of nucleotides 3161-3188 and 3164 -3191, respectively, in R. sylvatica and R. clamitans sequences. The cycle conditions were the same as above except for a longer extension time (5 min) per cycle at 72°C. The amplified cDNA products (3.2 kilobase pairs) were subcloned in either pCR ® 3.1 (for green frog cDNA) or NotI cut and dephosphorylated pcDNA3 (for R. sylvatica cDNA) expression vectors (Invitrogen, San Diego, CA. The accuracy of the nucleotide sequence was verified by complete sequencing of several clones for each frog SERCA cDNA. Reverse Transcriptase-PCR Analysis of R. sylvatica SERCA1 Splice Variants-The primers used to analyze the possibility of an alternative splicing event of the SERCA1 primary transcript are as follows: a 5Ј primer, SPLICEϩ (5Ј-GTAGAACAGTGGTTCGTTGTACTG-3Ј) corresponding to nucleotides 3093-3116 and a 3Ј primer, SPLICEϪ (5Ј-GCTGCATTCCTTTTACTGTGGTTTAT-3Ј) corresponding to the inverse complement of nucleotides 3225-3250 in wood frog SERCA cDNA. The adaptor-ligated ds-cDNA served as template DNA. PCR amplification was carried out for 35 cycles, each cycle consisting of 30 s at 94°C, 30 s at 55°C, and 30 s at 72°C. The PCR products were subcloned in the pGEM ® -T Easy vector, and several positive clones were sequenced.

Analysis of R. sylvatica and R. clamitans SR-SDS-poly-
acrylamide gel analysis of a Coomassie Blue-stained gel is shown in Fig. 1A. The major protein in the SR of both R. sylvatica and R. clamitans is ϳ100 kDa. Both preparations contain lesser amounts of several other proteins. Immunoblot analysis with monoclonal anti-SERCA1 reveals that the major 100-kDa proteins are indeed SERCA1 (Fig. 1B). Antibodies against SERCA2 and SERCA3 (see "Experimental Procedures" for details) did not recognize any frog proteins. SERCA1 in R. sylvatica exhibits a molecular mass of 115 kDa, whereas that of R. clamitans is ϳ105 kDa. Thus, the SERCA1s differ in apparent mass between the two frog species. This is not a general property because the other SR proteins evident in Fig. 1A exhibit the same molecular weights. This difference in apparent mass could indicate the presence of post-translational modifications that affect the enzymatic properties of the proteins.
Kinetic analyses were carried out to determine whether there are functional differences between the two SERCAs. The specific activities at 37°C of two preparations of R. sylvatica sarcoplasmic reticulum were 0.8 mol (used in Figs. 1 and 2A) and 3.0 mol of ATP hydrolyzed/min/mg (used in Figs. 2B, 3 and 4), whereas the specific activity of a single R. clamitans preparation was 2.7. These values are similar to those observed for rabbits (13).
Because we hypothesize that R. sylvatica SERCA1 is more active at 0°C than that of R. clamitans, we analyzed the temperature dependence of SERCA1 Ca 2ϩ -ATPase activity. ATP hydrolysis activity was determined at saturating ATP (10 mM) and Ca 2ϩ (100 M), and the results were analyzed using the Arrhenius plot after calculating the relative activities normalized to the maximum rate at 37°C. As shown in Fig. 2A, R. sylvatica SERCA1 has a higher relative activity at all temperatures tested, and activities for the two frogs at the two lowest temperatures (6 and 2°C corresponding to 3.584 and 3.636 ϫ 10 Ϫ3 1/K) are significantly different at the p Ͻ 0.005 level t test. Calculation of the activation energies by linear fitting below 20°C yields a significantly higher value for the green frog (28 Ϯ 1.8 kcal/mol) (mean Ϯ S.E.) than for the wood frog (23.7 Ϯ 0.6). Extrapolation to 0°C indicates that the wood frog will exhibit a 2.1-fold higher activity than the green frog. Thus, although the activity is low, the wood frog exhibits significantly higher activity. Fig. 2B shows a similar analysis for Ca 2ϩ transport data. Between 13 and 30°C, the activation energy for Ca 2ϩ transport is again higher for the green frog, 21.7 Ϯ 0.6 versus 19.0 Ϯ 0.1 kcal/mol (mean Ϯ S.E.), yielding an extrapolated value for Ca 2ϩ transport in the wood frog at 0°C that is 1.8-fold greater than in the green frog. This result suggests a similar coupling of ATP hydrolysis to Ca 2ϩ transport over a wide range of temperatures.
Next, the dependence of ATP hydrolytic rate on ATP concentration was determined. Fitted plots of wood and green frog kinetics are shown in Fig. 3. The R. clamitans SERCA1 data fit a two-site model with K m of 0.005 and 0.34 mM. The correlation coefficient (r 2 ) for the two-site model is 0.999, whereas attempting to fit the data to Michaelis-Menten kinetics yields a relatively poor correlation coefficient of 0.77. Thus, the green frog exhibits ATP kinetics quite similar to that described for rabbit SERCA1 (19). This behavior has been ascribed to half of the site reactivity in a SERCA1 dimer or additional ATP binding sites on a single SERCA1 monomer (22). Interestingly, the R. sylvatica SERCA1 data can be fitted with a simple single-site Michaelis-Menten model yielding a K m of 0.12 Ϯ 0.03 mM (mean Ϯ S.E.) (r 2 ϭ 0.94). Dean and Tanford (19) demonstrated that the multiple ATP site behavior was lost upon monomer formation of rabbit SERCA1 in C 12 E 8 , thus suggesting the possibility that protein-protein interactions are altered in R. sylvatica SR. The combination of higher activity at a low temperature and lower apparent K m could result in an enhanced ability of R. sylvatica SERCA1 to function at low temperature, especially in the physiological range of ATP concentrations (0.5-5 mM).
The kinetics of stimulation of ATP hydrolysis by Ca 2ϩ was also analyzed, as shown in Fig. 4. As indicated by the error bars in the plot, there is a significant difference in the Ca 2ϩ dependence of Ca 2ϩ -ATPase activity for the two frogs at both low and high Ca 2ϩ concentrations. The similarity in activities near the center of the plot indicate similar apparent affinities for Ca 2ϩ (0.5 M). Fitting the plots to a model of positive cooperativity yields a Hill coefficient of 1.5 Ϯ 0.2 (mean Ϯ S.D.) for the green frog indicating a positive cooperativity, a model that has been well established for rabbit SERCA1 (23). In contrast, the wood frog data yield a Hill coefficient of 0.9 Ϯ 0.2 (mean Ϯ S.D.) indicating a lack of cooperativity. This result suggests a loss of protein-protein interactions in R. sylvatica SERCA1 because the positive cooperativity of Ca 2ϩ binding to rabbit SERCA1 has been shown to be lost when the protein is dissociated to monomers in C 12 E 8 (23).
Characterization of cDNA Clones Coding for Wood and Green Frog SERCAs-To obtain the nucleotide sequence of the two frog SERCA cDNAs, we have employed the RACE-PCR strategy (24), which allows the amplification of both 3Ј and 5Ј cDNA ends. To conduct the 3Ј RACE reactions, a gene-specific oligonucleotide (PHϩ) was designed from the phosphorylation site of the known sequence of the previously cloned cDNA of the European frog (R. esculenta). For both frog SERCA cDNAs, 3Ј RACE-PCR products (2.3 kilobase pairs) were obtained and sequenced. From the obtained sequence, a new primer (5ЈRϪ) was generated to amplify the 5Ј cDNA ends. The resulting PCR products (1.5 kilobase pairs) were subcloned and sequenced. Using several other sequencing primers, the 3Ј and 5Ј overlapping PCR products were completely sequenced in both strands. The composite nucleotide sequences of R. sylvatica and R. clamitans SERCA cDNAs were determined (data not shown) (GenBank TM accession numbers AJ298901 and AJ298902). The sequencing strategy is shown in Fig. 5. Wood and green   FIG. 2. Temperature dependence of R. sylvatica and R. clamitans SR Ca 2؉ -ATPase and Ca 2؉ transport activities. SR membranes were assayed as described under "Experimental Procedures." The specific activity of the green frog SR preparation was 2.7 mol of ATP hydrolyzed/min/mg at 37°C, whereas the specific activity of the wood frog SR preparations was 0.8 in A and 3.0 in B. A, temperature dependence of Ca 2ϩ -ATPase activity of wood and green frogs with 10 mM MgATP and 10 M Ca 2ϩ plotted in the Arrhenius format. Error bars indicate the mean Ϯ S.D. from the mean for triplicate assays at each temperature. *, indicates significant differences at the p Ͻ 0.005 level with a paired t test. B, temperature dependence of Ca 2ϩ transport activity of wood and green frog SR plotted in the Arrhenius format. Each point is the average of two determinations at 5 mM MgATP and 5 M Ca 2ϩ . frog SERCA cDNAs are, respectively, 3790 and 3793 nucleotides in length. The nucleotide difference is due to the presence of three more nucleotides in the 5Ј-untranslated region of R. clamitans cDNA (206 versus 203 nucleotides in the wood frog sequence). The nucleotide sequence (AGGATGG) surrounding the translation initiation codon (ATG) is found in an acceptable context characteristic of initiation of eukaryotic translation (25). Two adjacent consensus polyadenylation signals (AT-TAAA) (26) in the 605-nucleotide 3Ј-untranslated region of both frog SERCA cDNAs are located, respectively, 18 and 12 nucleotides upstream of the beginning of the poly(A) tract.
Amino Acid Sequence Analysis and Comparison with Other SERCA Pumps-The primary translation products deduced from the two composite nucleotide sequences are 994 amino acids in length. The predicted molecular masses of R. sylvatica and R. clamitans SERCA proteins are, respectively, 109.7 and 109.3 kDa. In Fig. 6, the deduced amino acid sequence of R. sylvatica SERCA is compared with the sequences of other SERCA pumps expressed in skeletal muscle from R. clamitans, R. esculenta, and rabbit. In pairwise comparisons, R. sylvatica SERCA exhibits 98% (982 of 994 amino acids) overall identity with green frog SERCA and 95% (948 of 994 amino acids) and 88% (879 of 994 amino acids) identity with R. esculenta and rabbit SERCA1a, respectively. The deduced R. sylvatica SERCA sequence clearly matches that of rabbit fast-twitch skeletal muscle isoform (SERCA1a) at two characteristic points of deviation between rabbit SERCA1a and SERCA2a (expressed in cardiac and slow-twitch muscles) isoforms. One of them is represented by the COOH terminus (ARNYLEG 994 ), whereas the other consists of a 5-amino acid stretch (AAVGN 510 ) located COOH-terminal to Arg 505 . Therefore, based on these considerations and the overall sequence iden-tity, we suggest that the nomenclature used for R. sylvatica and R. clamitans Ca 2ϩ -ATPases that are encoded by the cloned cDNAs be SERCA1a as it was proposed earlier for R. esculenta (7). The hydrophobic putative transmembrane domains (M1-M10), the phosphorylation site (residues 349 -355), as well as sites known to bind fluorescein isothiocyanate or the ATP analogues FSBA and CIRATP in SERCA1 (27)(28)(29) are conserved in R. sylvatica SERCA1a. There are five nonconservative amino acid substitutions (Asn, Gln, Ala, Ser, and Leu) characteristic for R. sylvatica (Fig. 6 and Table I) Alternative Processing of the Wood Frog SERCA1 Primary Transcript-So far, no information suggesting the alternative splicing of SERCA1 primary transcript in frog species has been reported. We are now providing the first evidence for the expression of two SERCA1 splice variants in a frog species, R. sylvatica, by means of the PCR strategy using SPLICEϩ and SPLICEϪ as primers and the adaptor-ligated ds-cDNA as template. After amplification, two products of 159 and 117 base pairs were detected by 1.8% agarose gel electrophoresis (data not shown). Subcloning and subsequent sequencing confirmed that the 159-base pair PCR fragment corresponded to SERCA1a, whereas the second smaller fragment represented a novel splice variant, SERCA1b. The retention or exclusion of a 42-base pair optional exon (Fig. 7) gives rise, respectively, to SERCA1a and SERCA1b splice variants. The insertion of the optional exon in wood frog SERCA1a occurs immediately after nucleotide 2980 (relative to the ATG codon), which, interestingly, represents the point of divergence between the different splice variants in the related SERCA1, SERCA2, and SERCA3 genes. In turn, the nucleotide sequence divergence in SERCA1a splice variants is reflected at the protein level by a generation of SERCA1 isoforms with different COOH termini. Both isoforms present identical amino acid sequences up to amino acid 993 (Glu), after which the last residue (Gly 994 ) in SERCA1a is replaced in SERCA1b by a tetrapeptide stretch (DKPQ 997 ). The significance of the different COOH termini has still to be investigated. DISCUSSION The results presented herein may provide at least a partial explanation for the unusual low temperature muscle contractility of the wood frog R. sylvatica. R. sylvatica SERCA1 at saturating levels of ATP and Ca 2ϩ exhibits a 2-fold higher Ca 2ϩ -ATPase activity and an estimated 1.5-fold higher Ca 2ϩ transport rate at 0°C than R. clamitans (Fig. 2, A and B). Furthermore, R. sylvatica SERCA1 at 0.5 mM ATP has an activity 1.6-fold higher than that of R. clamitans (Fig. 3). Together, these differences in enzymatic activity may enable R.  (2), and rabbit (RAB SERCA1a) (10). Residues of the green frog, European frog, and rabbit SERCA1a that are identical with the corresponding residues of wood frog SERCA1a are represented by dashes. The amino acid numbers for wood frog SERCA1a are shown at the right. The hydrophobic putative transmembrane domains (M1-M10), phosphorylation site (Phos Site), the fluorescein isothiocyanate (FITC) binding site (7), and the FSBA/CIRATP (FSBA/CIRATP) binding region (8,9) are labeled and indicated by bars above the sequence. Positions of the amino acid residues unique to R. sylvatica enzyme are indicated by arrowheads. sylvatica SR to maintain muscle contractility near 0°C in contrast to R. clamitans. The fact that excitation/contraction coupling is maintained in the wood frog at 0°C signifies that very low Ca 2ϩ -ATPase activity at this temperature (1.5% of the activity at 37°C) is sufficient for SR function. The published physiological experiments indicate that in both rat and frog, loss of muscle function at low temperature results from the diminution of Ca 2ϩ release and re-uptake (10, 11). However, it is possible that the lower activity of green frog SERCA1 at 0°C (0.7% of the activity at 37°C) is not the rate-limiting step that prevents muscle function at this temperature in this frog.
Measurements of temperature dependence of rabbit SERCA1 activity in native SR membranes indicate an apparent discontinuity at ϳ20°C (32,33), and the data shown in Fig.  2A can also be fit with two linear segments and a break at 20°C. It was originally thought that this discontinuity in temperature dependence of ATP hydrolysis was a direct indicator of an abrupt change in the fluidity of the SR membrane (33). Subsequent work indicated that a lipid phase transition does not occur at 20°C, and it was proposed that the discontinuity was a result of changes in lipid cluster formation (34) or in a tightly associated lipid annulus (35). In contrast, Dean and Tanford (19) showed that the discontinuity at 20°C was maintained in the absence of phospholipid, which argues against any specific effect of lipid on SERCA1 at 20°C. Nevertheless, it is clear that the activation energy for Ca 2ϩ -stimulated ATP hydrolysis is higher for R. clamitans SERCA1 than for R. sylvatica below 20°C (28 versus 23.7 kcal/mol). These values are in the published range for rabbit, lobster, scallop, and winter flounder (32, 36 -38). The Arrhenius plots of ATPase activity versus temperature for the cold-tolerant scallop, lobster, flounder, and trout are similar to that of the rabbit, but they show modest shifts to lower activation energies below 20°C as is reported here for R. sylvatica. Interestingly, trout SERCA1 showed a nearly constant activation energy from 4 to 37°C for phosphoenzyme formation from phosphate resulting in much higher levels of this activity at 4°C than that observed for rabbit. The temperature dependence of phosphoenzyme formation in lobster, scallop, winter flounder, or frog has not been reported. Similarly, the cooperativity of kinetics with Ca 2ϩ and ATP observed to be different in R. sylvatica compared with R. clamitans or rabbit has not been described for lobster, scallop, or winter flounder. Kalabokis and Hardwicke (36) concluded that the lipid environment contributed to the differences in properties observed between rabbit and scallop SERCA1 because scallop SERCA1 solubilized in C 12 E 8 exhibited an Arrhenius plot similar to that of solubilized rabbit SERCA1. However, in the trout it was implied that differences in the temperature dependence of phosphoenzyme formation reside in the protein itself and not in the lipid environment due to the inability of C 12 E 8 to alter the temperature dependence of phosphoenzyme formation (37). Thus, the role of the phospholipid environment in altering temperature dependence of SERCA1 in the SR of cold-tolerant species is not established. This is an area for future work on R. sylvatica SR.
Several lines of evidence indicate that SERCA1 is aggregated in its native SR and that this interaction has a functional significance. Intrinsic fluorescence, fluorescence energy transfer, chemical cross-linking, and analytical ultracentrifugation all indicate a propensity for aggregation of SERCA1 (30). The positive cooperativity of Ca 2ϩ binding has been proposed to be a result of protein-protein interactions (23,30). Similarly, Dean et al. (19,31) have shown that solubilization of SERCA1 with the nonionic detergent C 12 E 8 results in the formation of an FIG. 7. Alternative splicing of the SERCA1 pre-mRNA in R. sylvatica. The nucleotide sequences of the PCR fragments corresponding to SERCA1a and SERCA1b splice variants are compared. Nucleotides are numbered at the right of the sequence. The PCR primers used (SPLICEϩ and SPLICEϪ) are indicated by arrows above their corresponding sequence. Below the nucleotide sequence, the translation products representing the COOH-terminal parts of SERCA1a and SERCA1b are compared (amino acids are numbered at the right). The optional exon (42 nucleotides) flanked by two slashes is shown in bold. The stop codon, TAA, is indicated by three consecutive asterisks. active monomer but that multiple site ATP kinetics is lost in the monomer. Because cooperativity of Ca 2ϩ activation and multiple site ATP kinetics are lost in R. sylvatica, it is possible that there is a change in protein-protein interactions in comparison with R. clamitans that results from amino acid differences between the two frogs. As a first step in reaching a molecular understanding of the enzymatic differences between R. sylvatica and R. clamitans SERCA1s, we report the complete amino acid sequences for the two proteins. Comparisons between R. sylvatica, R. clamitans, and R. esculenta indicate that there are seven amino acids in SERCA1 that are unique to R. sylvatica and, therefore, may be related to the functional differences noted above (Table I). Three of the seven amino acid substitutions (Asn 546 , Ala 547 , and Ser 643 ) that are unique to R. sylvatica when compared with the two other frogs are located in the ATP binding domain. Because the ATP kinetics are different in R. sylvatica in that Michaelis-Menten kinetics are exhibited, it appears likely that these mutations contribute to different enzymatic behavior exhibited by R. sylvatica SERCA1. None of these three amino acids has been examined by site-directed mutagenesis (12). It is possible that site-directed mutagenesis of single amino acid residues will result in changes in ATPase kinetics or that more than one concomitant change is required to achieve this effect.
Three of the remaining four amino acid changes in R. sylvatica are not in regions of the SERCA1 sequence with predicted functional roles. Gln 26 is located at the beginning of an NH 2 -terminal ␣-helix that has no known functional role. Leu 838 is located in the M7 transmembrane segment that is thought to be located on the outer surface of the proposed Ca 2ϩ channel and to have no direct role in Ca 2ϩ transport (15). Ser 877 is located in the extracellular loop between M7 and M8. Mutagenesis of this residue has no effect on activity (12). Ser 650 (Thr 650 in R. sylvatica) was originally proposed to be in the nucleotide binding domain (14), but the recent determination of the structure of SERCA1 at 2.6-Å resolution indicates that this residue is in the phosphorylation domain (15). This position has not been examined by site-directed mutagenesis.
Three distinct genes code for SERCA pumps in higher vertebrates: SERCA1 (ATP2A1), SERCA2 (ATP2A2), and SERCA3 (ATP2A3) (12). More isoform diversity is brought about by alternative processing of the SERCA primary transcripts as follows: adult SERCA1a and neonatal SERCA1b (both expressed in fast-twitch skeletal muscle) (39); SERCA2a (cardiac/slow-twitch skeletal muscle) and SERCA2b (ubiquitously expressed) (12); and SERCA3a, SERCA3b, and SERCA3c (broad cellular expression) (40). The alternative splicing mechanism governing the processing of rabbit SERCA1 pre-mRNA appears to be regulated developmentally . Both isoforms present identical amino acid sequences up to amino acid 993, after which the last amino acid (Gly 994 ) in SERCA1a is replaced by a highly charged octapeptide stretch, DPEDERRK, in SERCA1b (39). Previous investigations in R. esculenta (7) provided no evidence for alternative splicing of the frog SERCA1 pre-mRNA. One possible explanation for this lack of evidence may reside in the use of RNA isolated from adult frog tissues (7). In contrast, we used RNA isolated from an immediate post-metamorphic R. sylvatica frog. This enabled us to detect the neonatal SERCA1b splice variant. In the present report, we show that R. sylvatica SERCA1 pre-mRNA can also be spliced alternatively in the same manner as in rabbit, i.e. the last residue (Gly 994 ) in SERCA1a is replaced by a charged tetrapeptide, DKPQ, in SERCA1b isoform. Remarkably, this finding demonstrates that the alternative splicing of the SERCA1 gene is conserved among amphibians and mammals. The physiological significance of the two splice variants is not clear yet. Moreover, COS-1 cell expression studies revealed no functional differences between rabbit SERCA1a and SERCA1b isoforms (41). Furthermore, it is surprising that there are no reports in the literature indicating the expression of a SERCA2 gene in frogs, because the expression of a unique SERCA gene resembling the SERCA2 gene was also documented in invertebrates such as the crustacean Artemia franciscana (42) and the insect Drosophila melanogaster (43). Despite our efforts (Western blot analyses using anti-SERCA2 antibodies and reverse transcriptase-PCR studies using primers flanking nonconserved nucleotide sequences among invertebrate and higher vertebrate SERCAs), we were not able to demonstrate the existence of a SERCA2-like gene in R. sylvatica. However, we cannot rule out the possibility that the epitope for the anti-SERCA2 antibody is modified in the frog protein or that the SERCA2 gene may still be expressed but at much lower mRNA levels than SERCA1.
In conclusion, the molecular adaptation of SERCA1 in the skeletal muscle of R. sylvatica allows this frog to exploit a unique habitat for amphibians in freshwater temporary ponds in late winter. The unusual kinetic properties of R. sylvatica SERCA1 almost certainly result from the seven amino acid residues that are unique to this species. Future studies will shed light on the role of these amino acids in temperature dependence, kinetics with ATP, Ca 2ϩ cooperativity, and protein-protein interactions.