Phenylephrine hypertrophy, Ca2+-ATPase (SERCA2), and Ca2+ signaling in neonatal rat cardiac myocytes.

We endeavored to use a basic and well-controlled experimental system to characterize the extent and time sequence of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) involvement in the development of cardiac hypertrophy, including transcription, protein expression, Ca(2+) transport, and cytoplasmic Ca(2+) signaling. To this end, hypertrophy of neonatal rat cardiac myocytes in culture was obtained after adrenergic activation with phenylephrine (PE). Micrographic assessment of myocyte size, rise of [(14)C]phenylalanine incorporation and total protein expression, and increased transcription of atrial natriuretic factor demonstrated unambiguously the occurrence of hypertrophy. An early and prominent feature of hypertrophy was a reduction of the SERCA2 transcript, as determined by RT-PCR with reference to a stable marker such as glyceraldehyde-3-phosphate dehydrogenase. Reduction of Ca(2+)-ATPase protein levels and Ca(2+) transport activity to approximately 50% of control values followed with some delay, evidently as a consequence of a primary effect on transcription. Cytosolic Ca(2+) signaling kinetics, measured with a Ca(2+)-sensitive dye after electrical stimuli, were significantly altered in hypertrophic myocytes. However, the effect of PE hypertrophy on cytosolic Ca(2+) signaling kinetics was less prominent than observed in myocytes subjected to drastic SERCA2 downregulation with small interfering RNA or inhibition with thapsigargin (10 nM). We conclude that SERCA2 undergoes significant downregulation after hypertrophic stimuli, possibly due to lack of SERCA gene involvement by the hypertrophy transcriptional program. The consequence of SERCA2 downregulation on Ca(2+) signaling is partially compensated by alternate Ca(2+) transport mechanisms. These alterations may contribute to a gradual onset of functional failure in long-term hypertrophy.

Method-related effects of adenovirus-mediated LacZ and SERCA1 gene transfer on contractile behavior of cultured failing human cardiomyocytes.

INTRODUCTION: Adenovirus-mediated gene transfer into cardiomyocytes has emerged as an interesting tool to study functional effects of single proteins. However, the functional consequences of cell isolation, cell culture per se and adenovirus-mediated transfer of the LacZ or SERCA1 gene in failing human cardiomyocytes warrant further investigation. METHODS: Primary cell culture was performed without or after adenovirus-mediated gene transfer of LacZ or SERCA1. Functional behavior of myocytes was assessed under basal conditions (field stimulation, 0.5 Hz, 37 degrees C), and during inotropic stimulation with isoproterenol (ISO; 10(-9)-10(-5) M), [Ca(2+)](o) (1.5-15 mM) or increasing stimulation rates (0.25-2.5 Hz). Results were compared to trabeculae from the same hearts. RESULTS: Freshly isolated myocytes showed full inotropic competence as compared to multicellular preparations. The response to stimulation with ISO and [Ca(2+)](o), as well as changes in stimulation rate resulted in a maximal increase in fractional cell shortening (FS) to 215+/-24% and 291+/-34%, and a frequency-dependent decline in FS to 46+/-5% of the basal value, respectively. After 48 h of cell culture, basal FS did not change significantly compared to fresh cells but both time to peak shortening and time to 50% relengthening were prolonged. After culture, the concentration-response curve for ISO was significantly shifted to the left (EC(50) 5.16 x 10(-8) vs. 1.12 x 10(-8) M, p<0.05). LacZ gene transfer caused efficient beta-Gal expression without affecting the inotropic responses to ISO or stimulation rate but impaired the contractile amplitude. SERCA1 gene transfer increased FS by 68% vs. LacZ and accelerated relengthening kinetics (+dL/dt 93+/-13 vs. 61+/-8 mum/s, p<0.05 vs. LacZ). DISCUSSION: Contractile responses of isolated human myocytes are comparable to multicellular preparations. The use of primary cell culture and adenovirus infection with CMV-promoter-mediated LacZ expression per se modulates contractile behavior in failing human myocytes. SERCA1 expression markedly improves contractile function. The method-related changes in contractile behavior observed here need to be taken into account in further studies.

Sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) gene silencing and remodeling of the Ca2+ signaling mechanism in cardiac myocytes.

Transient elevations of cytosolic Ca2+ are a common mechanism of cellular signaling. In striated muscle, the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) plays an important role in terminating Ca2+ transients by returning cytosolic Ca2+ to intracellular stores. Stored Ca2+ can then be released again for subsequent signaling. We down-regulated SERCA2 gene expression in cultured cardiac myocytes by means of endogenous transcription of small interfering RNA encoded by an exogenous cDNA template. The cDNA template was delivered by adenovirus vector. Reduction of SERCA expression in all myocytes in culture was documented by immunochemistry, real-time RT-PCR, and determination of ATP-dependent Ca2+ transport. The reduction of SERCA2 expression was associated with the up-regulation of transient receptor potential (TRP) channel proteins (TRPC4 and TRPC5) and Na+/Ca2+ exchanger, indicating that intracellular store deficiency was compensated for by Ca2+ fluxes through the plasma membrane. In fact, SERCA silencing was followed by increased transcription of Na+/Ca2+ exchanger, TRPC4, TRPC5, and related transcriptional factors, such as stimulating protein 1, myocyte enhancer factor 2, and nuclear factor of activated cells 4, through activation of calcineurin. This finding demonstrates that the observed compensation occurs through transcriptional crosstalk and the remodeling of Ca2+ signaling pathways. The wide significance of this regulatory mechanism is related to its general involvement in Ca2+ signaling dynamics and in cardiac development and hypertrophy.

The role of the M6-M7 loop (L67) in stabilization of the phosphorylation and Ca(2+) binding domains of the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA).

The amino acid sequence (L67) intervening between the M6 and M7 transmembrane segments of the Ca(2+) transport ATPase was subjected to mutational analysis. Mutation of Pro(820) to Ala interferes with protein expression even though transcription occurs at normal levels. Single mutations of Lys(819) or Arg(822) to Ala, Phe, or Glu allow good expression, but produce strong inhibition of ATPase activity. The main defect produced by these mutations is strong interference with enzyme phosphorylation by ATP in the presence of Ca(2+), and also by P(i) in the absence of Ca(2+). The Lys(819) and Arg(822) mutants undergo slight and moderate reduction of Ca(2+) binding affinity, respectively. Reduction of overall steady state ATPase velocity is then due to inhibition of phosphorylated intermediate formation. On the other hand, a cluster of conservative mutations of Asp(813), Asp(815), and Asp(818) to Asn interferes strongly with enzyme activation by Ca(2+) binding and formation of phosphorylated enzyme intermediate by utilization of ATP. Enzyme phosphorylation by Pi in the absence of Ca(2+) undergoes slight or no inhibition by the triple aspartate mutation. Therefore, the triple mutation interferes mainly with the calcium-dependent activation of the ATPase. The effect of the triple mutation can be to a large extent reproduced by single mutation of Asp(813) (but not of Asp(815) or Asp(818)) to Asn. Functional and structural analysis of the experimental data demonstrates that the L67 loop plays an important role in protein folding and function. This role is sustained by linking the cytosolic catalytic domain and the transmembrane Ca(2+) binding domain through a network of hydrogen bonds.

Tight control of exogenous SERCA expression is required to obtain acceleration of calcium transients with minimal cytotoxic effects in cardiac myocytes.

Collateral effects of exogenous sarcoendoplasmic reticulum Ca(2+) ATPase (SERCA) expression were characterized in neonatal rat and chicken embryo cardiac myocytes, and the conditions required to produce acceleration of Ca(2+) transients with minimal toxicity were established. Cultured myocytes were infected with adenovirus vector carrying the cDNA of wild-type SERCA1, an inactive SERCA1 mutant, or enhanced green fluorescence protein under control of the cytomegalovirus promoter. Controls were exposed to empty virus vector. Each group was tested with and without phenylephrine (PHE) treatment. Under conditions of limited calf-serum exposure, the infected rat myocytes manifested a more rapid increase in size, protein content, and rate of protein synthesis relative to noninfected controls. These changes were not accompanied by reversal to fetal transcriptional pattern (as observed in hypertrophy triggered by PHE) and may be attributable to facilitated exchange with serum factors. SERCA virus titers >5 to 6 plaque-forming units per cell produced overcrowding of ATPase molecules on intracellular membranes, followed by apoptotic death of a significant number of rat but not chicken myocytes. Enhanced green fluorescence protein virus and empty virus also produced cytotoxic effects but at higher titers than SERCA. Expression of exogenous SERCA and enhancement of Ca(2+) transient kinetics could be obtained with minimal cell damage in rat myocytes if the SERCA virus titer were maintained within 1 to 4 plaque-forming units per cell. Expression of endogenous SERCA was unchanged, but expression of exogenous SERCA was higher in myocytes rendered hypertrophic by treatment with PHE than in nontreated controls.

Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes.

1. Sarco-endoplasmic reticulum Ca2+-ATPase from fast skeletal (SERCA1) or cardiac muscle (SERCA2a) was expressed in embryonic chicken and neonatal rat cardiac myocytes by adenovirus vectors, with c-myc tags on both constructs to compare expression and distinguish exogenous from endogenous SERCA2a in myocytes. 2. Expression of the two isoforms was similar (approximately 3-fold higher than endogenous SERCA). However, SERCA1 activity was 2-fold greater than SERCA2a activity, due to intrinsic differences in turnover rates. Activation of both exogenous SERCA isoforms by Ca2+ was displaced to slightly lower [Ca2+], suggesting that the overexpressed isoforms were independent of phospholamban. In fact, phospholamban and calsequestrin expression were unchanged. 3. Decay time constants of cytosolic Ca2+ transients from cells overexpressing SERCA1 were reduced by 30-40 % and half-widths by 10-15 % compared to controls. SERCA2a overexpression produced much less acceleration of transients in chick than in rat, and less acceleration than SERCA1 overexpression in either species. There was no significant change in resting [Ca2+], peak amplitudes, or in the amount of Ca2+ releasable by caffeine from overexpression of either SERCA isoform. However, the amplitudes of the transients increased with SERCA1 overexpression when pacing frequency limited refilling of the sarcoplasmic reticulum. 4. It is concluded that total SERCA transport velocity has a primary effect on the decay phase of transients. Transport velocity is affected by SERCA isoform turnover rate, temperature, and/or SERCA copy number.

Comparison of SERCA1 and SERCA2a expressed in COS-1 cells and cardiac myocytes.

Cultured COS-1 cells, as well as chicken embryonic and neonatal rat cardiac myocytes, were infected with recombinant adenovirus vectors to define limiting factors in the expression and Ca2+ transport function of recombinant sarcoplasmic-endoplasmic reticulum Ca(2+) (SERCA) isoforms. Titration experiments showed that all COS-1 cells and myocytes in culture could be infected by an adenovirus titer of 10 plaque-forming units (pfu) per seeded cell. Raising the adenovirus titer further yielded higher protein expression up to an asymptotic limit for functional, membrane-bound SERCA protein. The asymptotic behavior of SERCA expression was not transcription related but was due to posttranscriptional events. The minimal (-268) cardiac troponin T (cTnT) promoter was a convenient size for adenovirus vector construction and manifested tight muscle specificity. However, its efficiency was lower than that of the nonspecific cytomegalovirus (CMV) promoter. At any rate, identical maximal levels of SERCA expression were obtained with the CMV and the cTnT promoter, as long as the viral titer was adjusted to compensate for transcription efficiency. A maximal threefold increase of total SERCA protein expression over the level of the endogenous SERCA of control myocytes was reached (a sevenfold increase compared with the endogenous SERCA of the same infected myocytes due to reduction of endogenous SERCA after infection). In contrast with previous reports [Ji et al. Am. J. Physiol. 276 (Heart Circ. Physiol. 45): H89-H97, 1999], a higher kinetic turnover was demonstrated for the SERCA1 compared with the SERCA2a isoform as shown by a 5.0- versus 2.6-fold increase in calcium uptake rate accompanying maximal expression of recombinant SERCA1 or SERCA2a, respectively. This information is deemed necessary for studies attempting to modify myocardial cell function by manipulation of SERCA expression.

Direct demonstration of Ca2+ binding defects in sarco-endoplasmic reticulum Ca2+ ATPase mutants overexpressed in COS-1 cells transfected with adenovirus vectors.

Single mutations of specific amino acids within the membrane-bound region of the sarco-endoplasmic reticulum Ca2+ (SERCA)-1 ATPase interfere with Ca2+ inhibition of ATPase phosphorylation by Pi (1), suggesting that these residues may be involved in complexation of two Ca2+ that are known to bind to the enzyme. However, direct measurements of Ca2+ binding in the absence of ATP have been limited by the low quantities of available mutant protein. We have improved the transfection efficiency by means of recombinant adenovirus vectors, yielding sufficient expression of wild type and mutant SERCA-1 ATPase for measurements of Ca2+ binding to the microsomal fraction of the transfected cells. We find that in the presence of 20 microM Ca2+ and in the absence of ATP, the Glu771 --> Gln, Thr799 --> Ala, Asp800 --> Asn, and Glu908 --> Ala mutants exhibit negligible binding, indicating that the oxygen functions of Glu771, Thr799, Asp800, and Glu908 are involved in interactions whose single disruption causes major changes in the highly cooperative "duplex" binding. Total loss of Ca2+ binding is accompanied by loss of Ca2+ inhibition of the Pi reaction. We also find that, at pH 7.0, the Glu309 --> Gln and the Asn796 --> Ala mutants bind approximately half as much Ca2+ as the wild type ATPase and do not interfere with Ca2+ inhibition of the Pi reaction. At pH 6.2, the Glu309 --> Gln mutant does not bind any Ca2+, and its phosphorylation by Pi is not inhibited by Ca2+. On the contrary, the Asn796 --> Ala mutant retains the behavior displayed at pH 7.0. This suggests that in the Glu309 --> Gln mutant, ionization of acidic functions in other amino acids (e.g. Glu771 and Asp800) occurs as the pH is shifted, thereby rendering Ca2+ binding possible. In the Asn796 --> Ala mutant, on the other hand, the Glu309 carboxylic function allows binding of inhibitory Ca2+ even at pH 6.2. In all cases mutational interference with the inhibition of the Pi reaction by Ca2+ can be overcome by raising the Ca2+ concentration to the mM range, consistent with a general effect of mutations on the affinity of the ATPase for Ca2+.

Cell-specific promoter in adenovirus vector for transgenic expression of SERCA1 ATPase in cardiac myocytes.

Adenovirus-mediated transfer of cDNA encoding the chicken skeletal muscle sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA1) yielded selective expression in cultured chick embryo cardiac myocytes under control of a segment (-268 base pair) of the cell-specific cardiac troponin T (cTnT) promoter or nonselective expression in myocytes and fibroblasts under control of a constitutive viral [cytomegalovirus (CMV)] promoter. Under optimal conditions nearly all cardiac myocytes in culture were shown to express transgenic SERCA1 ATPase. Expression was targeted to intracellular membranes and was recovered in subcellular fractions with a pattern identical to that of the endogenous SERCA2a ATPase. Relative to control myocytes, transgenic SERCA1 expression increased up to four times the rates of ATP-dependent (and thapsigargin-sensitive) Ca2+ transport activity of cell homogenates. Although the CMV promoter was more active than the cTnT promoter, an upper limit for transgenic expression of functional enzyme was reached under control of either promoter by adjustment of the adenovirus plaque-forming unit titer of infection media. Cytosolic Ca2+ concentration transients and tension development of whole myocytes were also influenced to a similar limit by transgenic expression of SERCA1 under control of either promoter. Our experiments demonstrate that a cell-specific protein promoter in recombinant adenovirus vectors yields highly efficient and selective transgene expression of a membrane-bound and functional enzyme in cardiac myocytes.

ATPase gene transfer and mutational analysis of the cation translocation mechanism.

The peptide segment interposed between cation binding and phosphorylation domains retains a high degree of homology in all cation transport ATPases. Mutational analysis and chimeric replacements of Ca2+ ATPase components with corresponding Na+,K(+)-ATPase components indicate that this segment is utilized by various cation ATPases as a common structural device for a long-range functional linkage of enzyme phosphorylation and cation transport. Vectorial displacement of bound cation is rendered possible by a transmembrane channel formed by four clustered helices (M4, M5, M6, and M8). Originating from the four helices, the oxygen functions of Glu309, Glu771, Thr799, Asp800, and Glu908 form a duplex Ca2+ binding site in the middle of the channel, while Lys297 seals the luminal end of the channel with its positively charged side chain. The perturbation triggered by enzyme phosphorylation is apparently transmitted through the linkage segment to produce rotational displacement of the M4 helix with minimal change of secondary structure. The cation binding site is thereby disrupted and the Lys297 side chain removed, permitting Ca2+ to dissociate in exchange for H+ and to flow through the luminal end of the channel.

Energy transduction and kinetic regulation by the peptide segment connecting phosphorylation and cation binding domains in transport ATPases.

The sarcoplasmic reticulum ATPase segment (Thr316-Leu356) connecting the extramembranous phosphorylation domain to the preceding transmembrane helix M4 (which is an integral component of the Ca2+ binding domain) retains a high degree of sequence homology with other cation transport ATPases. Single, non conservative mutations of homologous residues in this segment produces enzyme inhibition (Zhang et al., 1995). We have now produced single and multiple mutations of non-homologous residues in this segment of the Ca2+ ATPase to match the corresponding residues of the Na+, K+ ATPase. We find that the main characteristics of the ATPase mechanism (i.e., Ca2+ dependent phosphoenzyme formation and thapsigargin sensitivity) are retained even when the entire 41-amino acid (Thr316-Leu356) segment of the Ca2+ ATPase is rendered identical to the corresponding segment of the Na+, K+ ATPase by sequential mutations of the 14 non-homologous amino acids. However, the phosphoenzyme turnover (likely rate limited by the "Ca2.E1-P-->Ca.E2-P transition") is progressively reduced if four or more Ca2+ ATPase residues are mutated to the corresponding residues of the Na+, K+ ATPase. The time course of enzyme inactivation by EGTA (likely rate limited by the "E1 to E2 transition") is also prolonged. Our findings suggest that an analogous peptide segment provides a functional linkage for energy transduction between phosphorylation and cation binding domains in various cation transport ATPase. However, its kinetic influence on rate-limiting conformational transitions is dependent on matching specific structures in each ATPase.

Short and long range functions of amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase. A mutational study.

Mutational analysis of several amino acids in the transmembrane region of the sarcoplasmic reticulum ATPase was performed by expressing wild type ATPase and 32 site-directed mutants in COS-1 cells followed by functional characterization of the microsomal fraction. Four different phenotype characteristics were observed in the mutants: (a) functions similar to those sustained by the wild type ATPase; (b) Ca2+ transport inhibited to a greater extent than ATPase hydrolytic activity; (c) inhibition of transport and hydrolytic activity in the presence of high levels of phosphorylated enzyme intermediate; and (d) total inhibition of ATP utilization by the enzyme while retaining the ability to form phosphoenzyme by utilization of P(i). Analysis of experimental observations and molecular models revealed short and long range functions of several amino acids within the transmembrane region. Short range functions include: (a) direct involvement of five amino acids in Ca2+ binding within a channel formed by clustered transmembrane helices M4, M5, M6, and M8; (b) roles of several amino acids in structural stabilization of the helical cluster for optimal channel function; and (c) a specific role of Lys297 in sealing the distal end of the channel, suggesting that the M4 helix rotates to allow vectorial flux of Ca2+ upon enzyme phosphorylation. Long range functions are related to the influence of several transmembrane amino acids on phosphorylation reactions with ATP or P(i), transmitted to the extramembranous region of the ATPase in the presence or in the absence of Ca2+.

Ca2+ binding and translocation by the sarcoplasmic reticulum ATPase: functional and structural considerations.

Three experimental systems are described including sarcoplasmic reticulum (SR) vesicles, reconstituted proteoliposomes, and recombinant protein obtained by gene transfer and expression in foreign cells. It is shown that the Ca(2+) ATPase of sarcoplasmic reticulum (SR) includes an extramembranous globular head which is connected through a stalk to a membrane bound region. Cooperative binding of two calcium ions occurs sequentially, within a channel formed by four clustered helices within the membrane bound region. Destabilization of the helical cluster is produced following enzyme phosphorylation by ATP at the catalytic site in the extramembranous region. The affinity and orientation of the Ca2+ binding site are thereby changed, permitting vectorial dissociation of bound Ca2+ against a concentration gradient. A long range linkage between phosphorylation and Ca2+ binding sites is provided by an intervening peptide segment that retains high homology in cation transport ATPases, and whose function is highly sensitive to mutational perturbations.

Mutational analysis of the peptide segment linking phosphorylation and Ca(2+)-binding domains in the sarcoplasmic reticulum Ca(2+)-ATPase.

The sarcoplasmic reticulum ATPase segment extending from the phosphorylation site (Asp-351) to the preceding transmembrane helix M4 (which is involved in Ca2+ binding in conjunction with transmembrane helices M5, M6, and M8) retains a marked sequence homology to the corresponding segments of other cation ATPases. We made 26 point mutations in this segment and found that nonconservative mutations of residues that are homologous in various cation ATPases result in strong inhibition of catalytic and transport function. Mutations of nonhomologous residues to match the corresponding residues of other cation ATPases are not inhibitory and, in some cases, produce higher activity. The inhibitory mutations affect the phosphorylated intermediate turnover, which is associated with the vectorial translocation of bound Ca2+. The same mutations do not affect the kinetics of ATPase activation by Ca2+ following enzyme preincubation with EGTA. This suggests that activation of the phosphoryl transfer reaction by Ca2+ binding and vectorial displacement of bound Ca2+ by enzyme phosphorylation do not occur simply as the forward and reverse directions of the same process, but are linked to distinct structural features of the enzyme. The peptide segment extending from the phosphorylation site in the enzyme extramembranous headpiece through the M4 helix in the membrane-bound region sustains a prominent role in transmission of the phosphorylation signal for displacement of bound Ca2+. A critical structural role of this segment is also demonstrated by the interference of specific mutations with membrane assembly of the expressed protein.

High sensitivity to site directed mutagenesis of the peptide segment connecting phosphorylation and Ca2+ binding domains in the Ca2+ transport ATPase.

Nine residues (Leu321, Lys329, Asn330, Val333, Arg334, Leu336, Pro337, Val339 and Glu340), within the peptide segment intervening between the catalytic domain and the Ca2+ binding domain of the sarcoplasmic reticulum (SERCA 1) ATPase, were individually mutated to Ala. The mutated proteins were recovered in the microsomal fraction of COS-1 cells following transient expression, and exhibited inhibition of Ca2+ uptake and ATPase hydrolytic activity, while forming discernable levels of phosphorylated intermediate. Mutation of Glu340 to Gln (rather than to Ala) was much less effective, suggesting that the functional consequence of the mutation is related to structural perturbation, rather than loss of the acidic side chain. The high sensitivity of this peptide segment to single mutations suggests that its structural integrity is required for functional linkage of the phosphorylation and Ca2+ binding domains.

Ca(2+)-dependent and thapsigargin-inhibited phosphorylation of Na+,K(+)-ATPase catalytic domain following chimeric recombination with Ca(2+)-ATPase.

Two chimeric proteins comprising the Na,K-ATPase catalytic domain (large cytosolic loop) and the two flanking regions of the Ca-ATPase were obtained by transient or stable expression in mammalian cells transfected with recombinant DNA. In the first chimera (CpNC), a large portion (containing the nucleotide-binding site) of the cytosolic loop between putative membrane spans M4 and M5 of the sarcoendoplasmic reticulum Ca2+ (SERCA) 1 (fast muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. In the second chimera (CNpC), an even larger portion (containing the nucleotide-binding site and the phosphorylation site) of the analogous cytosolic loop of the SERCA2 (cardiac muscle) ATPase was replaced by the corresponding portion of the Na,K-ATPase alpha 1 subunit. Steady state Ca2+ transport and coupled ATP hydrolysis by the chimeric proteins were negligible as compared to those obtained with SERCA enzymes. Nevertheless, the chimeric proteins were able to utilize ATP to form phosphoenzyme levels equal to those formed by SERCA ATPases. Chimeric and SERCA enzymes exhibited an identical Ca2+ requirement for ATP utilization and sensitivity to thapsigargin (TG) which is a specific inhibitor of SERCA ATPase and not of Na,K-ATPase. Furthermore, both SERCA and chimeric enzymes could be phosphorylated with P(i), and this reaction required removal of Ca2+. In comparative experiments, the functional pattern of seemingly unaffected phosphoenzyme formation and inhibited Ca2+ transport was produced in the SERCA ATPase even by single mutation of Pro337 to Ala, evidently due to defective protein conformation. Retention of Ca2+ and TG sensitivity by the chimeric proteins demonstrates that the Ca(2+)- and TG-binding domains do not reside within the cytosolic loop replaced by chimeric substitution and strongly support previous studies suggesting that binding of calcium required for enzyme activation occurs within the membrane-bound region of the SERCA ATPases (Clarke et al., 1989a; Sumbilla et al., 1991).

Coupled expression of Ca2+ transport ATPase and a dihydrofolate reductase selectable marker in a mammalian cell system.

Stable expression of a full-length cDNA encoding chicken fast muscle Ca2+ transport ATPase was obtained in a Chinese hamster lung cell line (DC-3F), using a dual-promoter expression vector (pH beta FCaA3) in which the ATPase was cloned downstream of a human beta-actin gene promoter, and a mutant dihydrofolate reductase cDNA (A3/DHFR) was cloned downstream of an SV40 promoter-enhancer. Owing to its essentially normal catalytic activity and modest (20-fold) resistance to the antifolate methotrexate (MTX), the A3/DHFR mutant enzyme served as an efficient dominant selection marker in transfected cell populations challenged with MTX and, within a broad range of drug concentrations, allowed subsequent amplification and overexpression of vector sequences. In stable transfectants, the expressed ATPase was targeted to intracellular membranes, and the microsomal fractions from those cells exhibited high rates of Ca2+ transport. In comparative experiments using transient expression in COS1 cells, the level of ATPase per transfected cell was greater, but less than 5% of the transfected population exhibited ATPase expression. Furthermore, as opposed to the stable lines, the transiently expressing cells could not be propagated. Overall, the yield of ATPase was 12-16 and 4-6 micrograms per milligram of microsomal protein in the stable and the transient expression systems, respectively. The advantages of the stably transfected cell lines therefore lie in the homogeneity of ATPase expression and its distribution in cells and microsomes, in the large yield of microsomes obtained by continuous cell propagation, and in the reproducible functional characteristics of the microsomes. Moreover, the microsomes derived from stably transfected cell lines provide a convenient system for studies of Ca2+ transport and ATPase partial reaction, eliminating the need to conduct repetitive transient transfections to obtain sufficient amounts of enzyme for functional studies.

Structural perturbation of the transmembrane region interferes with calcium binding by the Ca2+ transport ATPase.

The Ca(2+)-ATPase of sarcoplasmic reticulum reacts with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl) carbodiimide (NCD4) yielding a fluorescence labeling that interferes with calcium binding to activating and transport sites of the enzyme and, thereby, with Ca(2+)-dependent ATPase activity. On the other hand, the catalytic site does not appear altered, as revealed by the normal occurrence of Ca(2+)-independent reactions, such as enzyme phosphorylation with Pi in the reverse direction of the catalytic cycle. This reaction is not inhibited by Ca2+ in the labeled enzyme, while it is inhibited in the native enzyme. The NCD4 reaction which is involved in functional inactivation occurs in the membrane-bound portion of the ATPase. Sodium dodecyl sulfate solubilization of hydrophobic peptides, electrophoresis, and microsequencing of transblotted electrophoretic bands revealed that the fluorescent NCD4 label resides in a segment of tryptic fragment A1, intervening between Glu231 and Glu309. This segment includes two transmembrane helices, and does not include the domain involved in the phosphoryl transfer reaction during catalytic activity. This specific labeling does not occur when the NCD4 derivatization procedure is carried out in the presence of Ca2+ concentrations that also prevent functional inactivation. Fluorescence characterization by steady state and intensity decay measurements shows only negligible energy transfer between the NCD4 label and fluorescein isothiocyanate label of Lys515, indicating that the NCD4 label is unlikely to reside within the extramembranous region of the ATPase. On the other hand, the fluorescence emission of intrinsic tryptophan residues clustered within or near the transmembrane region of the ATPase, is distinctly affected by NCD4 label specifically bound to the ATPase, and NCD4 label nonspecifically bound to the sarcoplasmic reticulum membrane. The combined sequencing and spectroscopic observations indicate that derivatization with NCD4 induces a perturbation within or near the transmembrane region of the ATPase (at a relatively large distance from the catalytic site) that interferes with specific calcium binding. This is in agreement with experiments (Clarke et al., 1989) demonstrating that mutations of any of six amino acids within the transmembrane region of the ATPase interfere with enzyme activation by Ca2+.

Specific association of calmodulin-dependent protein kinase and related substrates with the junctional sarcoplasmic reticulum of skeletal muscle.

A systematic study of protein kinase activity and phosphorylation of membrane proteins by ATP was carried out with vesicular fragments of longitudinal tubules (light SR) and junctional terminal cisternae (JTC) derived from skeletal muscle sarcoplasmic reticulum (SR). Following incubation of JTC with ATP, a 170,000-Da glycoprotein, a 97,500-Da protein (glycogen phosphorylase), and a 55,000-60,000-Da doublet (containing calmodulin-dependent protein kinase subunit) underwent phosphorylation. Addition of calmodulin in the presence of Ca2+ (with no added protein kinase) produced a 10-fold increase of phosphorylation involving numerous JTC proteins, including the large (approximately 450,000 Da) ryanodine receptor protein. Calmodulin-dependent phosphorylation of the ryanodine receptor protein was unambiguously demonstrated by Western blot analysis. The specificity of these findings was demonstrated by much lower levels of calmodulin-dependent phosphorylation in light SR as compared to JTC, and by much lower cyclic AMP dependent kinase activity in both JTC and light SR. These observations indicate that the purified JTC contain membrane-bound calmodulin-dependent protein kinase that undergoes autophosphorylation and catalyzes phosphorylation of various membrane proteins. Protein dephosphorylation was very slow in the absence of added phosphatases, but was accelerated by the addition of phosphatase 1 and 2A (catalytic subunit) in the absence of Ca2+, and calcineurin in the presence of Ca2+. Therefore, in the muscle fiber, dephosphorylation of SR proteins relies on cytoplasmic phosphatases. No significant effect of protein phosphorylation was detected on the Ca2(+)-induced Ca2+ release exhibited by isolated JTC vesicles. However, the selective and prominent association of calmodulin-dependent protein kinase and related substrates with junctional membranes, its Ca2+ sensitivity, and its close proximity to the ryanodine and dihydropyridine receptor Ca2+ channels suggest that this phosphorylation system is involved in regulation of functions linked to these structures.