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• W1995822626 abstract "We tested the hypothesis that increased Sarcoplasmic reticulum (SR) Ca content ([Ca]SRT) in phospholamban knockout mice (PLB-KO) is because of increased SR Ca pump efficiency defined by the steady-state SR [Ca] gradient. The time course of thapsigargin-sensitive ATP-dependent45Ca influx into and efflux out of cardiac SR vesicles from PLB-KO and wild-type (WT) mice was measured at 100 nm free [Ca]. We found that PLB decreased the initial SR Ca uptake rate (0.13versus 0.31 nmol/mg/s) and decreased steady-state45Ca content (0.9 versus 4.1 nmol/mg protein). Furthermore, at similar total SR [Ca], the pump-mediated Ca efflux rate was higher in WT (0.065 versus 0.037 nmol/mg/s). The pump-independent leak rate constant (k leak) was also measured at 100 nm free [Ca]. The results indicate thatk leak was <1% of pump-mediated backflux and was not different among nonpentameric mutant PLB (PLB-C41F), WT pentameric PLB (same expression level), and PLB-KO. Therefore differences in passive SR Ca leak cannot be the cause of the higher thapsigargin-sensitive Ca efflux from the WT membranes. We conclude that the decreased total SR [Ca] in WT mice is caused by decreased SR Ca influx rate, an increased Ca-pump backflux, and unaltered leak. Based upon both thermodynamic and kinetic analysis, we conclude that PLB decreases the energetic efficiency of the SR Ca pump. We tested the hypothesis that increased Sarcoplasmic reticulum (SR) Ca content ([Ca]SRT) in phospholamban knockout mice (PLB-KO) is because of increased SR Ca pump efficiency defined by the steady-state SR [Ca] gradient. The time course of thapsigargin-sensitive ATP-dependent45Ca influx into and efflux out of cardiac SR vesicles from PLB-KO and wild-type (WT) mice was measured at 100 nm free [Ca]. We found that PLB decreased the initial SR Ca uptake rate (0.13versus 0.31 nmol/mg/s) and decreased steady-state45Ca content (0.9 versus 4.1 nmol/mg protein). Furthermore, at similar total SR [Ca], the pump-mediated Ca efflux rate was higher in WT (0.065 versus 0.037 nmol/mg/s). The pump-independent leak rate constant (k leak) was also measured at 100 nm free [Ca]. The results indicate thatk leak was <1% of pump-mediated backflux and was not different among nonpentameric mutant PLB (PLB-C41F), WT pentameric PLB (same expression level), and PLB-KO. Therefore differences in passive SR Ca leak cannot be the cause of the higher thapsigargin-sensitive Ca efflux from the WT membranes. We conclude that the decreased total SR [Ca] in WT mice is caused by decreased SR Ca influx rate, an increased Ca-pump backflux, and unaltered leak. Based upon both thermodynamic and kinetic analysis, we conclude that PLB decreases the energetic efficiency of the SR Ca pump. sarcoplasmic reticulum free energy in the form of ATP maximal binding capacity maximal intra-SR Ca binding capacity [Ca] outside free SR [Ca] total SR [Ca] efflux buffer-sensitive [Ca]SRT efflux buffer-insensitive [Ca]SRT steady-state [Ca]SRT total [Ca] outside phospholamban nonpentameric mutant mice free energy used by the SR Ca pump to generate the free SR [Ca] gradient SR Ca pump efficiency time constant of45Ca efflux Hill coefficient Hill coefficient of unidirectional forward flux Hill coefficient of unidirectional backward flux time constant of45Ca influx SR leak flux net SR Ca pump flux forward SR Ca pump flux reverse SR Ca pump flux SR Ca flux ion binding affinity SR Ca binding affinity [Ca]c concentration at half-maximal net SR Ca pump rate [Ca]c at half-maximal forward SR Ca pump rate [Ca]c at half-maximal reverse SR Ca pump rate SR Ca leak rate constant wild-type mice phospholamban knockout mice mice expressing WT PLB against a PLB-KO background thapsigargin maximal net SR Ca pump rate maximal forward SR Ca pump rate maximal reverse SR Ca pump rate 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid 4-morpholinepropanesulfonic acid analysis of variance The SR1 Ca pump is an important cellular protein that transduces the energy stored in cytosolic ATP (ΔG ATP) into energy that is stored as a [Ca] gradient across the SR membrane (ΔG SRCa). The pump establishes this gradient at steady-state through a balance of the following 3 fluxes: an SR Ca pump-dependent influx balanced by a pump-dependent efflux (or “backflux”) and a passive leak flux (mediated by the ryanodine receptor or other pathways). Backflux is a unidirectional flux that results from reversal of the SR Ca pump in the forward mode. High Ca within the SR is transported through the pump back into the cytosol, and ATP can be made from ADP and inorganic phosphate as has been demonstrated directly in SR membrane vesicles (1Feher J.J. Briggs F.N. Biophys. J. 1984; 45: 1135-1144Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 2Takenaka H. Adler P.N. Katz A.M. J. Biol. Chem. 1982; 257: 12649-12656Abstract Full Text PDF PubMed Google Scholar, 3Weber, A., Herz, R., and Reis, I. (1966). 345, 329–369.Google Scholar, 4Makinose M. FEBS Lett. 1971; 12: 269-270Crossref PubMed Scopus (75) Google Scholar). Backflux has also been described in both digitonin-permeabilized isolated cardiac myocytes (5Shannon T.R. Ginsburg K.S. Bers D.M. Ann. N. Y. Acad. Sci. 1997; 853: 350-352Crossref Scopus (6) Google Scholar) and in isolated myocytes under whole-cell voltage clamp (6Shannon T.R. Ginsburg K.S. Bers D.M. Biophys. J. 2000; 78: 322-333Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). Phospholamban (PLB) is an important regulator of the SR Ca pump. In the unphosphorylated state it associates with the SR Ca pump to inhibit its activity. This inhibition can be relieved through phosphorylation by protein kinase A and/or Ca-calmodulin-dependent protein kinase (7Kranias E.G. Biochim. Biophys. Acta. 1985; 844: 193-199Crossref PubMed Scopus (91) Google Scholar, 8Tada M. Inui M. Yamada M. Kadoma M.A. Kuzuya T. Abe H. Kakiuchi S. J. Mol. Cell. Cardiol. 1983; 15: 335-346Abstract Full Text PDF PubMed Scopus (103) Google Scholar). Although it is well established that PLB inhibits the forward mode of SR Ca transport by increasing the forwardK Ca(K Ca-f) to higher [Ca], the effects of PLB on reverse SR Ca-ATPase andK Ca(K Ca-r) are unknown. This becomes an important issue, especially in the case where the Ca pump approaches thermodynamic equilibrium, as may be the case in intact myocytes under some conditions (9Ginsburg K.S. Weber C.R. Bers D.M. J. Gen. Physiol. 1998; 111: 491-504Crossref PubMed Scopus (48) Google Scholar). That is, the forward and reverse Ca flux can reach the point where they are equal and opposite, and the [Ca] gradient is maximal at about 7000:1 (10Shannon T.R. Bers D.M. Biophys. J. 1997; 73: 1524-1531Abstract Full Text PDF PubMed Scopus (91) Google Scholar). If PLB shiftsK Ca-f without shiftingK Ca-r by a comparable amount, the maximal [Ca] gradient that the SR Ca pump can generate may change, and this could alter the energetic efficiency of the pump. PLB coexists in the cardiac myocyte in both a monomeric and pentameric form. The pentameric form has been reported to form a channel that may mediate Ca leak from the SR (11Wegener A.D. Jones L.R. J. Biol. Chem. 1984; 259: 1834-1841Abstract Full Text PDF PubMed Google Scholar, 12Kovacs R.J. Nelson M.T. Simmerman H.K.B. Jones L.R. J. Biol. Chem. 1988; 263: 18364-18368Abstract Full Text PDF PubMed Google Scholar). In this report we measure forward and reverse Ca pump flux and also pump-independent leak in SR vesicles from wild-type (WT) mice, phospholamban knockout mice (PLB-KO), and mice that express equal amounts of either wild-type PLB (PLB-70) or nonpentamer-forming mutant PLB (PLB-C41F) upon a knockout background. Nonpump-mediated Ca leak was not different among these groups, which does not support any significant role for a PLB-mediated leak under our conditions. We hypothesize that PLB inhibits SR Ca pump influx at the same time that it stimulates SR Ca pump backflux. Such an effect would result in a decreased ΔG SRCa and decreased SR Ca pump energetic efficiency. The data in WT, PLB-KO, and PLB-70 membranes all support this hypothesis. All chemicals were from Sigma, except as indicated. Mathematical data manipulation was performed using Lotus 1–2-3 (Lotus Development Corp., Cambridge, MA) and Excel (Microsoft Corp., Seattle, WA). Nonlinear regression fits and statistics were done with GraphPad (iSi Software, Philadelphia, PA). For the purposes of this paper the efficiency of the SR Ca pump is defined as follows: Eftpump=ΔGSRCa·100ΔGATPEquation 1 where ΔG ATP is the free energy stored in the form of ATP in the cytosol or 59 kJ/mol (13Allen D.G. Morris P.G. Orchard C.H. Pirolo J.S. J. Physiol. 1985; 361: 185-204Crossref PubMed Scopus (182) Google Scholar) and ΔG SRCa is the free energy required by the pump to establish the SR [Ca] gradient. This is defined as follows: ΔGSRCa=2RT·ln[Ca]SR[Ca]cEquation 2 where [Ca]SR is free SR Ca and [Ca]SR/[Ca]cis the free SR Ca gradient. Therefore when the pump generates a higher SR [Ca] gradient, it is operating at a higher energetic efficiency. In this paper we measure [Ca]SRT(and relate it to [Ca]SR) in SR membrane vesicles at 100 nm[Ca]c as a measure of the SR [Ca] gradient. PLB-KO, PLB-70, and WT mice were anesthetized with 0.25 mg of pentobarbital/g of body weight. Thoracotomy was performed, and hearts were removed, cannulated via the aorta, and perfused with 5 ml of 10 mm caffeine, 10 mm EGTA, normal tyrode. Normal tyrode consisted of (in mm) 140 NaCl, 4 KCl, 10 glucose, 5 HEPES, and 1 MgCl2, pH 7.4, with NaOH. Hearts were then perfused with 15 ml of 0.5 mm BAPTA in (in mm) 140 sucrose, 70 KCl, 40 HEPES, pH 7.2 (membrane buffer). Subsequently, the ventricular tissue was separated from the rest of the heart and put into 0.5 BAPTA membrane buffer, minced, and homogenized with two 15-s pulses of a polytron homogenizer (Brinkmann Instruments, Inc., Westbury, NY). This procedure depletes the membranes of endogenous Ca. The membranes that resulted were centrifuged at 100,000 × g, resuspended in 1 ml 0.5 BAPTA, 100 μg/ml aprotinin, 100 μg/ml leupeptin, 0.5 ryanodine, and 20 μmdigitonin in membrane buffer, and glass-Teflon homogenized. The membranes were split into two portions. One was treated with 25 nmol/mg thapsigargin (Thg) and the other with Me2SO vehicle. The membranes were incubated for 20 min at room temperature to allow ryanodine and thapsigargin binding and were then put on ice. Triplicate incubates were set up for plus and minus Thg groups. Each incubate contained 86 μl of uptake buffer. MgATP (4 μl, 100 mm stock) and 10 μl of membranes (∼5 mg/ml) were beaded on the side of the test tube, and the assay for each time point was begun by vortexing, thus washing the ATP and the membranes into the incubate. The incubates contained (final concentration in mm during incubation) 0.69 MgCl2, 0.5 BAPTA, 0.151 CaCl2 (100 nm[Ca]c, 100–200 μCi of45Ca), 4 MgATP, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 14 sucrose, 127 KCl, 40 HEPES, pH 7.2. Phosphocreatine (12.5) and 5 units/ml creatine phosphokinase were present to regenerate ATP. Mitochondrial Ca uptake was inhibited by 2 μm ruthenium red and 4 μm oligomycin. Digitonin (20 μm) inhibited sarcolemmal Ca uptake, and ryanodine receptors were blocked by 0.5 mm ryanodine. Uptake was stopped with an ice-cold solution (in mm) of 1 EGTA, 200 KCl, 20 Mops, Tris to pH 7.4 (stop solution). The incubates were vacuum-filtered through Whatman GF/C glass fiber filters (Fisher Scientific, Pittsburgh, PA). The tubes were washed 3×, and the filter was washed an additional 2× with stop solution. Membranes were allowed to take up Ca for 0, 10, 20, 30, 60, and 90 s, thus forming the uptake part of the curve (see Fig. 2). The backflux part of the curve was constructed from incubates that were allowed to take up Ca for 90 s, at which point an efflux buffer was added. Efflux buffer consisted of (final concentration in mmduring incubation) 50 EGTA, 17.1 CaCl2 (100 nm[Ca]c), 40 HEPES, pH 7.2. This solution caused 45Ca efflux while holding [Ca]c at 100 nm and reducing extracellular 45Ca specific activity 100-fold. The indicated time points were taken after the addition of efflux buffer. Membrane protein concentration was determined using the Bio-Rad total protein assay reagent kit (Bio-Rad Laboratories, Hercules, CA). The influx part of the experiment was fit with a rising exponential function, [Ca]SRT=[Ca](SRT−SS)(1−exp(−kinf·t))Equation 3 where k inf is the rate constant and t is time. Steady-state [Ca]SRT([Ca]SRT-SS) was therefore the plateau of the relationship. Note that the described data are “efflux-sensitive” uptake. We found that a varying percentage of the total Ca taken up did not come out of the membranes upon addition of efflux buffer. We therefore characterized the kinetics of the SR Ca pump only in terms of Ca available for transport into and out of the membranes. The initial rate of uptake was determined as the derivative of this function at t = 0. JSR=d[Ca]SRTdt=kinf·[Ca](SRT−SS)·exp(−kinf·t)Equation 4 Similarly, the efflux part of the time course was described with an exponential decay, [Ca]SRT=[Ca](SRT+efl)·exp(−kefl·t)+[Ca](SRT−efl)Equation 5 where [Ca]SRT+efl is efflux buffer-sensitive [Ca]SRT, and [Ca]SRT-efl is efflux buffer-insensitive [Ca]SRT(i.e. see Fig. 2, bottom plateau of the curve).k inf is the rate constant, andt is the time from dilution. The rate of efflux at identical [Ca]SRT+efl (0.8 nmol/mg) in all groups was determined from the following derivative: JSR=−kefl·[Ca](SRT+efl)·exp(−kefl·t)Equation 6 where t is the time from dilution to [Ca]SRT-efl = 0.8 nmol/mg. The initial rate of 45Ca uptake when [Ca]SR = 0 is described by the classic Hill equation, Jpumpf=Vmax1+KCa­f[Ca]cnEquation 7 where J pumpf is the forward pump rate and V max,K Ca-f, and n are the maximal velocity, [Ca]c at half-maximal velocity, and the Hill coefficient in the forward direction, respectively. We set n to 2,K Ca-f to 0.25, and 0.14 for WT and PLB-KO, respectively (as measured by Frank, et al. (14Frank K. Tilgmann C. Shannon T.R. Bers D.M. Kranias E.G. Biochemistry. 2000; 39: 14176-14182Crossref PubMed Scopus (33) Google Scholar)). Given these parameters and J pumpf,V max was determined. The rate of backflux at steady-state was also determined by using Equation 6, where t = 0 (i.e. the rate when excess 40Ca is added; see Fig. 2). This rate is described by the following: Jpumpr=Vmax[Ca]SRK(Ca­r)n1+[Ca]cKCa­fn+[Ca]SRKCa­rnEquation 8 where J pumpr is the unidirectional reverse pump rate andK Ca-r has its usual meaning for the reverse pump rate. This is a special case of the generic reversible equation, Jpump=Vmaxf[Ca]cKCa­fnf−Vmaxr[Ca]SRKCa­rnr1+[Ca]cKCa­fnf+[Ca]SRKCa­rnrEquation 9 where V maxf =V maxr and nf =nr. For 45Ca efflux, the left term in the numerator is zero, giving Equation 8. GivenJ pumpr and theV max above, theK Ca-r can be inferred from Equation8. At steady-state J pump is zero, so the numerator of Equation 9 is zero, and this reduces to the Haldane relationship. [Ca]SR[Ca]c=Keq=KCa­rKCa­fEquation 10 Combining with Equation 2, we arrive at Equation 11. ΔGSRCa=2RT·lnKCa­rKCa­fEquation 11 Thus, if we know K Ca-f and derive K Ca-r from Equation 8 the ΔG SRCa can be inferred. Membranes from PLB-KO, PLB-70, and PLB-C41F mice were prepared as above except 30 μm EGTA, 10 μg/ml aprotinin, and 10 μg/ml leupeptin, 140 KCl, 40 HEPES, pH 7.4 was used for perfusion, homogenization, and resuspension instead of BAPTA. The protocol for measuring passive SR Ca leak from the membranes is illustrated in Fig. 1 A. Mouse membranes were added to a cuvette with stirring at a final concentration of ∼1–2 mg/ml. Also present were (final concentrations) EGTA (30 μm), MgCl2 (1 mm free), the protease inhibitors aprotinin and leupeptin (10 μg/ml), oligomycin (2 μm) and ruthenium red (2 μm) to inhibit mitochondrial uptake, and 0.5 mm ryanodine to block ryanodine receptors. [Ca]c was measured with 2 μm indo-1 (Molecular Probes, Eugene, OR). An 8100 series spectrofluorometer (Spectronic Instruments, Rochester, NY) was used to excite the indo-1 at 355 nm. Fluorescence emission at 400 and 470 nm was measured. The 400:470 ratio was converted to [Ca]c using the Grynkiewicz equation (9Ginsburg K.S. Weber C.R. Bers D.M. J. Gen. Physiol. 1998; 111: 491-504Crossref PubMed Scopus (48) Google Scholar, 15Grynkiewicz G. Poenie M. Tsien R.Y. J. Biol. Chem. 1985; 260: 3440-3450Abstract Full Text PDF PubMed Scopus (80) Google Scholar). Uptake was started by 4 mm ATP addition. ATP was regenerated with 5 units/ml creatine phosphokinase and 12.5 mm phosphocreatine. EGTA or Ca was added such that a plateau was reached at ∼100 nm[Ca]c. [Ca]c gradually rose after 10 nmol/mg Thg was added until the SR Ca pump was completely blocked. Passive leak continued until the SR was empty of Ca. The leak rate constant (see below) was used to characterize the leak in the different groups of membranes. [Ca]c was converted to total Ca ([Ca]T) using known Ca binding constants for all buffers within the cuvette (see Table I). These binding constants were collected from the literature, and nearly all of the endogenous affinity constants come from in vitromeasurements where physiological intracellular conditions were simulated (normal ionic strength and pH value), usually at room temperature.Table ICuvette Ca Buffering ParametersBufferB maxK dRef.μmSR Ca pump0.1576 μm0.639Feher J.J. Briggs F.N. J. Biol. Chem. 1982; 257: 10191-10199Abstract Full Text PDF PubMed Google ScholarSarcolemma0.14 μm1340Post J.A. Langer G.A. J. Membr. Biol. 1992; 129: 49-57Crossref PubMed Scopus (45) Google ScholarMembrane/high0.05 μm0.341Bers D.M. Allen L.-A.H. Kim Y.-J. Am. J. Physiol. 1986; 251: C861-C871Crossref PubMed Google ScholarEGTA · Ca≥30 μm0.17342Martell, A., and Smith, R. M. (eds) (1974) Critical Stability Constants, pp. 269-272, Plenum Publishing Corp., New YorkGoogle ScholarEGTA · Mg≥30 μm2330042Martell, A., and Smith, R. M. (eds) (1974) Critical Stability Constants, pp. 269-272, Plenum Publishing Corp., New YorkGoogle ScholarIndo-12 μm0.4543Jackson A. Timmerman M. Bagshaw C. Ashley C. FEBS Lett. 1987; 216: 35-39Crossref PubMed Scopus (91) Google ScholarAlthough all are accounted for, note that EGTA and indo-1 account for the majority of the buffering capacity. Open table in a new tab Although all are accounted for, note that EGTA and indo-1 account for the majority of the buffering capacity. Exogenous EGTA and indo-1 are overwhelmingly the dominant Ca buffering species accounting for >98% of the Ca bound (≥32 μm versus ∼0.3 μm). Fortunately these are the buffers that we know the most about and of which we can be most sure. Constants for EGTA in particular were fully corrected for ionic strength and pH value using the Maxchelator program (see Ref. 16Bers D.M. Patton C.W. Nuccitilli R. Methods Cell Biol. 1994; 40: 3-29Crossref PubMed Scopus (497) Google Scholar; free for download on the World Wide Web). Referenced values were converted from nmol/mg to μmol/liter cytosol using the conversion factors 0.4 liter of cell volume/kg of wet weight, 120 mg of homogenate protein/g of wet weight (17Hove-Madsen L. Bers D.M. Am. J. Physiol. 1993; 264: C677-C686Crossref PubMed Google Scholar) and a measured value of 0.312 mg of membranes/mg of homogenate. Therefore for each time point in Fig. 1 A, we know [Ca]c and [Ca]T. The difference between the [Ca]T before Thg and [Ca]T after all of the Ca has leaked out of the SR is the [Ca]SRTjust prior to Thg addition. Because we know how much Ca leaks out of the SR during the experiment, we can now calculate [Ca]SRT at each time point as the amount of Ca that hasn't leaked out yet. Given [Ca]SRT and the SR Ca buffering parameters, [Ca]SR can be calculated. SR volume was assumed to be 3% of cellular volume (18Page E. Mccallister L.P. Power B. Proc. Natl. Acad. Sci. 1971; 68: 1465-1466Crossref PubMed Scopus (121) Google Scholar,19Page E. Am. J. Physiol. 1978; 235: C147-C158Crossref PubMed Google Scholar). [Ca]SRT was converted to [Ca]SR using the following relationships. [Ca]SRT=[Ca·L]SR+[Ca]SREquation 12 [Ca·L]SR=Bmax−SR·[Ca]SRKd−SR+[Ca]SREquation 13 B max-SR andK d-SR have been previously determined to be 14 mmol/liter of SR and 638 μm, respectively (10Shannon T.R. Bers D.M. Biophys. J. 1997; 73: 1524-1531Abstract Full Text PDF PubMed Scopus (91) Google Scholar). Equation 13 was substituted into Equation 12, and [Ca]SR as a function of [Ca]SRT was calculated from the quadratic solution of the result. The leak rate (J leak) is the change in [Ca]T over time and is assumed to be proportional to the concentration difference ([Ca]SR − [Ca]c), Jleak=kleak([Ca]SR−[Ca]c)Equation 14 where k leak is a rate constant of leak flux, determined by linear regression of leak flux data (see Fig. 1 B; gray line). There are two primary routes of Ca efflux from the SR when the ryanodine receptors are blocked with high ryanodine (as is the case with all of the experiments here). These are backflux (1Feher J.J. Briggs F.N. Biophys. J. 1984; 45: 1135-1144Abstract Full Text PDF PubMed Scopus (12) Google Scholar, 2Takenaka H. Adler P.N. Katz A.M. J. Biol. Chem. 1982; 257: 12649-12656Abstract Full Text PDF PubMed Google Scholar, 3Weber, A., Herz, R., and Reis, I. (1966). 345, 329–369.Google Scholar, 4Makinose M. FEBS Lett. 1971; 12: 269-270Crossref PubMed Scopus (75) Google Scholar, 5Shannon T.R. Ginsburg K.S. Bers D.M. Ann. N. Y. Acad. Sci. 1997; 853: 350-352Crossref Scopus (6) Google Scholar, 6Shannon T.R. Ginsburg K.S. Bers D.M. Biophys. J. 2000; 78: 322-333Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar) and passive SR Ca leak across the membrane. To measure E pump with and without PLB we must first determine the extent to which passive leak rate contributes to SR Ca efflux in our system. Fig. 1 A shows the protocol that we used to measure this leak rate. When [Ca]c was ∼100 nm, Thg (25 nmol/mg) was added. Thg forms a dead-end complex with the SR Ca pump (20Sagara Y. Wade J.B. Inesi G. J. Biol. Chem. 1992; 267: 1286-1292Abstract Full Text PDF PubMed Google Scholar) thus inhibiting both SR Ca influx and backflux through the pump. [Ca]c gradually rose until the SR Ca pump was completely blocked. Passive leak continued until the SR was empty of Ca. [Ca]T was computed at each time point using known Ca binding constants (Table I). [Ca]SRT was computed as [Ca]T at the end of the leak minus [Ca]T before Thg, and both [Ca]SRT andd[Ca]SRT/dtcould be calculated for each time point. [Ca]SR was calculated using Equations 12 and 13 (10). The slope of the relationship between the leak flux (J leak) and the free Ca difference across the SR membrane isk leak (Fig. 1 B). The following three groups of mice were compared: 1) transgenic mice that expressed only mutant nonpentamer-forming PLB (Ref. 21Chu G. Li L. Sato Y. Harrer J.M. Kadambi V.J. Hoit B.D. Bers D.M. Kranias E.G. J. Biol. Chem. 1998; 273: 33674-33680Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar; PLB-C41F), 2) PLB-KO, and 3) PLB-70 (mice that express WT PLB at the same level as the mutant PLB is expressed in PLB-C41F). There was no significant difference in SR Ca leak between the PLB-C41F group, the PLB-70 group, and the PLB-KO group (0.058 ± 0.011 versus 0.061 ± 0.010 versus 0.045 ± 0.006/min, Student'st test, p > 0.05, n = 6). We conclude that passive SR Ca leak is unchanged in mice containing mutant or wild-type PLB or in PLB-KO. Furthermore, there was no evidence of SR Ca leak through a pentameric PLB channel under these conditions. Fig.2 A shows the protocol for measuring unidirectional SR Ca fluxes along with a typical experiment.45Ca uptake is started with the addition of ATP and ventricular membranes to the uptake medium as described under “Materials and Methods.” Incubates were filtered at the indicated times to measure [Ca]SRT. At 90 s excess nonradioactive 40Ca buffered with 50 mm EGTA was added. The high Ca/EGTA concentration diluted the 45Ca to negligible levels while maintaining a [Ca]c of 100 nm. Under this condition, unidirectional SR 45Ca efflux took place. Note that the actual [Ca]SRT and the fluxes at steady-state have not changed here. The backflux has only been “uncovered” by the dilution of 45Ca outside the vesicles. This is demonstrated in Fig. 2 B, where Ca flux rates were calculated from SR Ca content data as in Fig. 2 A. In this case mean kinetic parameters were taken from the mean PLB-KO data (see below). Fig. 2 A (dashed line) shows that although the [45Ca] has changed, [Ca]c and [Ca]SRT (and [Ca]SR) have not. The45Ca efflux gives a measure of the unidirectional backflux at steady-state, but the net total Ca flux is still zero. Also note the rapid loss of 45Ca compared with the leak in Fig.1 A (translated into the gray dashed line in Fig.2 A). Therefore nearly all of this 45Ca efflux is backflux through the SR Ca pump (Fig. 2 A). As can be seen in Fig. 3 A,45Ca began to accumulate in the SR immediately upon ATP and membrane addition in both WT and PLB-KO. [Ca]SRT came nearly to steady-state where Ca influx and efflux are equal by 90 s. [Ca]SRT-SS as determined by Equation 3 in WT was ∼25% of that in PLB-KO (0.9 versus4.1 nmol/mg of protein). This important result may reflect a lower SR [Ca] gradient and therefore a lower ΔG SRCa (Equation 2) at steady-state. Note that in some experiments, we found a varying percentage of the total Ca taken up did not come out of the membranes upon addition of efflux buffer (the nonzero plateau in Fig. 3 B). This Ca appears to be pump-insensitive. Characterization of the kinetics of the SR Ca pump is therefore only in terms of Ca that was available for both uptake and efflux from the membranes. [Ca]SRT-SS is therefore the plateau in Fig. 3 A minus the efflux-insensitive Ca (i.e.the plateau in Fig. 3 B). Note that this treatment is conservative in that it tends to minimize differences between WT and PLB-KO. From the higher [Ca]SRT in PLB-KO we conclude that the efficiency of SR Ca uptake is lower in the presence of PLB (Equation 1). The only alternatives would be (1Feher J.J. Briggs F.N. Biophys. J. 1984; 45: 1135-1144Abstract Full Text PDF PubMed Scopus (12) Google Scholar) a difference in leak flux (not the case, see above) or (2Takenaka H. Adler P.N. Katz A.M. J. Biol. Chem. 1982; 257: 12649-12656Abstract Full Text PDF PubMed Google Scholar) different intra-SR Ca buffering. The latter is highly unlikely, because the calsequestrin concentration is the same in PLB-KO and WT mice (22Luo W. Grupp I.L. Harrer J. Ponniah S. Grupp G. Duffy J.J. Doetschman T. Kranias E.G. Circ. Res. 1994; 75: 401-409Crossref PubMed Scopus (632) Google Scholar), and the concentration would have to be five times higher to explain the results. These results are also consistent with findings of much higher SR Ca load in PLB-KO intact myocytes (23Li L. Chu G. Kranias E.G. Bers D.M. Am. J. Physiol. 1998; 274: H1335-H1347Crossref PubMed Google Scholar). Having examined the raw data, we can now further characterize these experiments in a quantitative manner. Two different methods can be used to determine the SR Ca pump efficiency from the data. First previously determined SR Ca buffering characteristics (10Shannon T.R. Bers D.M. Biophys. J. 1997; 73: 1524-1531Abstract Full Text PDF PubMed Scopus (91) Google Scholar) were used to convert vesicular [Ca]SRT to [Ca]SR (using Equations 12 and 13; see also Table II). Note that these values are similar to the 7000:1 [Ca]SR:[Ca]cratio (∼700 μm[Ca]SR) expected from the results of Shannon and Bers (10Shannon T.R. Bers D.M. Biophys. J. 1997; 73: 1524-1531Abstract Full Text PDF PubMed Scopus (91) Google Scholar). From this ratio, the SR Ca pump efficiency was determined using Equations 1 and 2. We found a basal ΔG SRCa in WT mice of 33.5 kJ/mol (56.8% efficiency). PLB-KO had a much higher ΔG SRCa (43.9 kJ/mol; 74.5%). These results are summarized in the top half of Table II and graphically in Fig. 5.Table IISR Ca pump efficiency as determined from the SR [Ca] gradient and from the uptake kinetics, respectivelySteady-state [Ca] gradient analysisSR [Ca] gradientΔG SRCaEfficiencyPhenotype[Ca]SRT[Ca]SRT[Ca]SR(nmol/mg)(mmol/l SR )(mmol/l SR )([Ca]SR/[Ca]c)(kJ/mol)(%)WT0.91.820.0990033.556.8PLB-702.24.460.272272038.966.0PLB-KO4.18.310.75750043.974.5Kinetic analysisK Ca-fK Ca-rK eqΔG SRCaEfficiencyPhenotypeV max(nmol/mg/s)(μm)(mm)(KCa-r/KCa-f)(kJ/mol)(%)WT0.9430.250.298119334.959.1PLB-KO0.9430.141.401980045.376.7The analysis assumes SR Ca buffering parameters from Shannon and Bers (10Shannon T.R. Bers D.M. Biophys. J. 1997; 73: 1524-1531Abstract Full Text PDF PubMed Scopus (91) Google Scholar), ΔG ATP = 59 kJ/mol (13Allen D.G. Morris P.G. Orchard C.H. Pirolo J.S. J. Physiol. 1985; 361: 185-204Crossref PubMed Scopus (182) Google Scholar), and a Hill coefficient of 2. The K Ca-f values in the bottom part of the table are from Ref. 14Frank K. Tilgmann C. Shannon T.R. Bers D.M. Kranias E.G. Biochemistry. 2000; 39: 14176-14182Crossref PubMed Scopus (33) Google Scholar. V max is calculated from the initial forward uptake rate. This sameV max value is used to calculateK Ca-r from the rate of pump-mediated backflux. ΔG SRCa is calculated using Equation 11. Open table in a new tab The analysis assumes SR Ca buffering parameters from Shannon and Bers (10Shannon T.R. Bers D.M. Biophys. J. 1997; 73: 1524-1531Abstract Full Text PDF PubMed Scopus (91) Google Scholar), ΔG ATP = 59 kJ/mol (13Allen D.G. Morris P.G. Orchard C.H. Pirolo J.S. J. Physiol. 1985; 361: 185-204Crossref PubMed Scopus (182) Google Scholar), and a Hill coefficient of 2. The K Ca-f values in the bottom part of the table are from Ref. 14Frank K. Tilgmann C. Shannon T.R. Bers D.M. Kranias E.G. Biochemistry. 2000; 39: 14176-14182Crossref PubMed Scopus (33) Google Scholar. V max is calculated from the initi" @default.
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