Ca2+/Calmodulin-dependent Protein Kinase II Is an Essential Mediator in the Coordinated Regulation of Electrocyte Ca2+-ATPase by Calmodulin and Protein Kinase A

The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (≈2-fold). The addition of cAMP (5-50 pm), forskolin (10 nm), or cholera toxin (10 or 100 nm) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA α-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA. The aim of this study was to investigate (a) whether Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) participates in the regulation of plasma membrane Ca2+-ATPase and (b) its possible cross-talk with other kinase-mediated modulatory pathways of the pump. Using isolated innervated membranes of the electrocytes from Electrophorus electricus L., we found that stimulation of endogenous protein kinase A (PKA) strongly phosphorylated membrane-bound CaM kinase II with simultaneous substantial activation of the Ca2+ pump (≈2-fold). The addition of cAMP (5-50 pm), forskolin (10 nm), or cholera toxin (10 or 100 nm) stimulated both CaM kinase II phosphorylation and Ca2+-ATPase activity, whereas these activation processes were cancelled by an inhibitor of the PKA α-catalytic subunit. When CaM kinase II was blocked by its specific inhibitor KN-93, the Ca2+-ATPase activity decreased to the levels measured in the absence of calmodulin; the unusually high Ca2+ affinity dropped 2-fold; and the PKA-mediated stimulation of Ca2+-ATPase was no longer seen. Hydroxylamine-resistant phosphorylation of the Ca2+-ATPase strongly increased when the PKA pathway was activated, and this phosphorylation was suppressed by inhibition of CaM kinase II. We conclude that CaM kinase II is an intermediate in a complex regulatory network of the electrocyte Ca2+ pump, which also involves calmodulin and PKA. The electrocytes of the electric organs from Electrophorus electricus L. are specialized structures that generate electric potentials and produce electric discharges similar to those of nerve and muscle (1Schoffeniels E. Chagas C. Paes de Carvalho A. Bioelectrogenesis: A Comparative Survey of Its Mechanisms with Particular Emphasis on Electric Fishes. Elsevier Science Publishers B. V., Amsterdam1961: 147-165Google Scholar). It is generally accepted that passive Ca2+ translocation across the membranes of the electrocytes plays a crucial role in the transmission of impulses within the electric organ (2Esquibel M.A. Effect of Calcium and Magnesium on the Neuroelectric Synapses. Federal University of Rio de Janeiro, Rio de Janeiro, Brazil1970Google Scholar) and that Ca2+ fluxes from the extracellular milieu toward the cytosolic compartment require a huge inwardly directed Ca2+ electrochemical gradient to be maintained (3Carafoli E. Physiol. Rev. 1991; 71: 129-153Crossref PubMed Scopus (572) Google Scholar). Among different mechanisms, the plasma membrane Ca2+-ATPase is responsible for fine-tuning of that Ca2+ disequilibrium (3Carafoli E. Physiol. Rev. 1991; 71: 129-153Crossref PubMed Scopus (572) Google Scholar, 4Penniston J.T. Enyedi A. J. Membr. Biol. 1998; 165: 101-109Crossref PubMed Scopus (157) Google Scholar, 5Shull G.E. Eur. J. Biochem. 2000; 267: 5284-5290Crossref PubMed Scopus (74) Google Scholar, 6Carafoli E. Brini M. Curr. Opin. Chem. Biol. 2000; 4: 152-161Crossref PubMed Scopus (131) Google Scholar, 7Gromadzinska E. Lachowicz L. Walkowiak B. Zylinska L. Biochim. Biophys. 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The smaller functional units of the electric organ present in E. electricus L. are the asymmetrically shaped cells called electrocytes, stacked one after another along the axis of the fish (13Gotter A.L. Kaetzel M.A. Dedman J.R. Comp. Biochem. Physiol. A. 1998; 119: 225-241Crossref PubMed Scopus (55) Google Scholar). One face of the electrocyte is innervated, whereas the other is not (14Almeida D.F. Castro G.O. Miranda M. Chagas C. Exp. Cell Res. 1963; 29: 42-49Crossref PubMed Scopus (7) Google Scholar), and electrocytes are organized in a capacitor-like structure that allows current flow during discharges (13Gotter A.L. Kaetzel M.A. Dedman J.R. Comp. Biochem. Physiol. A. 1998; 119: 225-241Crossref PubMed Scopus (55) Google Scholar). Exclusively localized in the innervated face, acetylcholine receptors generate endplate potentials, triggering action potentials (15Keynes R.D. Martins-Ferreira H. J. Physiol. 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Teixeira-Ferreira A. Somló C. de Souza W. Machado R.D. Vieyra A. J. Histochem. Cytochem. 1989; 37: 953-959Crossref PubMed Scopus (5) Google Scholar) not involved in Ca2+ transport. The plasma membrane high affinity Ca2+-ATPase is a member of the P-type class of ATPases (20Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (414) Google Scholar) and operates as an electrogenic Ca2+/H+ exchanger, pumping cytosolic Ca2+ to the extracellular space and internalizing H+ at a 1:1 stoichiometry (21Hao L. Rigaud J.L. Inesi G. J. Biol. Chem. 1994; 269: 14268-14275Abstract Full Text PDF PubMed Google Scholar). The different isoforms of Ca2+-ATPase (8Strehler E.E. Zacharias D.A. Physiol. Rev. 2001; 81: 21-50Crossref PubMed Scopus (474) Google Scholar, 22Guerini D. Cell Tissue Res. 1998; 292: 191-197Crossref PubMed Scopus (82) Google Scholar) are exquisitely regulated by a great variety of naturally existing mechanisms such as calmodulin, acidic phospholipids, limited proteolysis, protein G subunits, oligomerization, and phosphorylation/dephosphorylation by protein kinases on the C-terminal domain of the pump molecule (3Carafoli E. Physiol. Rev. 1991; 71: 129-153Crossref PubMed Scopus (572) Google Scholar, 4Penniston J.T. Enyedi A. J. Membr. Biol. 1998; 165: 101-109Crossref PubMed Scopus (157) Google Scholar, 7Gromadzinska E. Lachowicz L. Walkowiak B. Zylinska L. Biochim. Biophys. Acta. 2001; 1549: 19-31Crossref PubMed Scopus (11) Google Scholar, 9Carafoli E. Santella L. Branca D. Brini M. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 107-260Crossref PubMed Scopus (424) Google Scholar, 23Niggli V. Adunyah E.S. Penniston J.T. Carafoli E. J. Biol. Chem. 1981; 256: 395-401Abstract Full Text PDF PubMed Google Scholar, 24Benaim G. Zurini M. Carafoli E. J. 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The content of calmodulin (CaM) 1The abbreviations used are: CaM, calmodulin; CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; PKA, protein kinase A; TFP, trifluoperazine dihydrochloride; CTx, cholera toxin; PKIα, protein kinase A α-catalytic subunit inhibitor; NF, non-innervated face; IF, innervated face; BisTris propane, 1,3-bis(tris(hydroxymethyl)methylamino)propane; CCM, calmodulin-containing membranes; CDM, calmodulin-depleted membranes. 1The abbreviations used are: CaM, calmodulin; CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; PKA, protein kinase A; TFP, trifluoperazine dihydrochloride; CTx, cholera toxin; PKIα, protein kinase A α-catalytic subunit inhibitor; NF, non-innervated face; IF, innervated face; BisTris propane, 1,3-bis(tris(hydroxymethyl)methylamino)propane; CCM, calmodulin-containing membranes; CDM, calmodulin-depleted membranes. is especially high in the electric eel (≈1-2% of the total mass) (29Munjaal R.P. Connor C.G. Turner R. Dedman J.R. Mol. Cell. Biol. 1986; 6: 950-954Crossref PubMed Scopus (6) Google Scholar), and immunofluorescence studies have demonstrated that this regulatory peptide is concentrated and organized in both membranes of the electrocytes (13Gotter A.L. Kaetzel M.A. Dedman J.R. Comp. Biochem. Physiol. A. 1998; 119: 225-241Crossref PubMed Scopus (55) Google Scholar), thus allowing strong interactions with the membrane transporters, including the Ca2+ pump. Interestingly, the electrocytes are very rich in another regulatory Ca2+-binding protein, Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) (30Gotter A.L. Kaetzel M.A. Dedman J.R. Comp. Biochem. Physiol. A. 1997; 118: 81-91Crossref PubMed Scopus (4) Google Scholar). This multifunctional kinase, which appears to play a pivotal role in Ca2+ signaling and homeostasis (31Means A.R. Mol. Endocrinol. 2000; 14: 4-13Crossref PubMed Scopus (159) Google Scholar, 32Hook S.S. Means A.R. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 471-505Crossref PubMed Scopus (405) Google Scholar, 33Hudmon A. Schulman H. Biochem. J. 2002; 364: 593-611Crossref PubMed Scopus (461) Google Scholar) acting as a sensor of Ca2+ fluctuations (9Carafoli E. Santella L. Branca D. Brini M. Crit. Rev. Biochem. Mol. Biol. 2001; 36: 107-260Crossref PubMed Scopus (424) Google Scholar), is homogeneously spread within the electrocyte (30Gotter A.L. Kaetzel M.A. Dedman J.R. Comp. Biochem. Physiol. A. 1997; 118: 81-91Crossref PubMed Scopus (4) Google Scholar). The aim of this work was to investigate whether CaM and CaM kinase II interact in a regulatory fashion in the electrocyte Ca2+-ATPase and also their possible interaction with protein kinase A (PKA). High levels of cAMP have been demonstrated in the electric tissue (34Chagas C. Nesralla-Lobão H. Rebello M.A. C. R. Acad. Sci. (Paris). 1972; 274: 1526-1529Google Scholar), where phosphorylation events involving PKA appear to be implicated in the modulation of channel-mediated ion fluxes in the electric membranes (35Emerick M.C. Agnew W.S. Biochemistry. 1989; 28: 8367-8380Crossref PubMed Scopus (26) Google Scholar). Evidence for a coupling between CaM and CaM kinase II in regulating Ca2+-ATPase is described, as well as their interplay with a cAMP stimulatory pathway. This complex regulation of the Ca2+-ATPase (which was found asymmetrically concentrated in the excitable face of the cell) might be indicative of its important role in modulating the electric and chemical activities of the electroplax from E. electricus L. Materials—ATP, cAMP, ouabain, dl-dithiothreitol, β-mercaptoethanol, trifluoperazine dihydrochloride (TFP), cholera toxin (CTx), molecular mass markers, and bovine serum albumin were purchased from Sigma. The CaM kinase II inhibitor KN-93 (N-(2-(N-(chlorocinnamyl)-N-methylaminomethyl)phenyl)-N-(2-hydroxyethyl)-4-methoxybenzene-sulfonamide) was from Calbiochem-Novabiochem. The PKA α-catalytic subunit inhibitors (PKIα), peptide 5-24 and the heat-stable inhibitor (equally efficient at nanomolar concentrations), were from Sigma and Calbiochem-Novabiochem, respectively. Forskolin was from Alomone Labs (Jerusalem, Israel). Deionized water was obtained from a Nanopure II system (Sybron-Barnstead, Newton, MA). All other reagents were of the highest purity available. Preparation of CaM-containing Electrocyte Membranes—E. electricus L. specimens were supplied by the Museu Paraense Emílio Goeldi (Belém, Para, Brazil) and were transported and kept in an aquarium. They were anesthetized with ice-cold water containing 2% urethane and killed by decapitation. Plasma membrane fractions were obtained by differential centrifugation of homogenates of the main electric organ using a sucrose gradient (16Lowe J. Araujo G.M. Pedrenho A.R. Nunes-Tavares N. Ribeiro M.G. Hassón-Voloch A. Biochim. Biophys. Acta. 2004; 1661: 40-46Crossref PubMed Scopus (5) Google Scholar). The main electric organs of electric fishes (200-500 g of tissue) were dissected and minced in 250 mm sucrose, 100 mm NaI, 1 mm phenylmethylsulfonyl fluoride, 1 mm β-mercaptoethanol, and 50 mm Tris-HCl (pH 7.4). The fragments were then homogenized in a Virtis apparatus for 1 min at its maximal speed and left overnight in a cold room under gentle stirring. The next day, the homogenate was centrifuged at 5000 × g for 20 min. The pellet was resuspended in a solution containing 250 mm sucrose, 1 mm phenylmethylsulfonyl fluoride, and 50 mm Tris-HCl (pH 7.4); rehomogenized in a blender (three periods of 20 s); and left again overnight. The suspension was filtered with gauze and centrifuged at 5000 × g for 30 min, and the recovered supernatant was centrifuged again at 10,000 × g for 30 min. The sediment containing a fraction enriched with membranes of the anterior non-innervated face (NF) of the electrocyte (14Almeida D.F. Castro G.O. Miranda M. Chagas C. Exp. Cell Res. 1963; 29: 42-49Crossref PubMed Scopus (7) Google Scholar) was resuspended in 250 mm sucrose. The corresponding supernatant was centrifuged at 100,000 × g for 2 h, and the final sediment containing the membranes of the posterior innervated face (IF) of the electrocyte (14Almeida D.F. Castro G.O. Miranda M. Chagas C. Exp. Cell Res. 1963; 29: 42-49Crossref PubMed Scopus (7) Google Scholar) was also resuspended in 250 mm sucrose. These membrane fractions were stored in small flasks under liquid N2. In the IF fraction used in most of the experiments of this work, cytochemical and freeze-fracture studies have shown vesicles together with membrane sheets (36de Souza W. Benchimol M. Somló C. Machado R.D. Hassón-Voloch A. Cell Tissue Res. 1979; 202: 275-281Crossref PubMed Scopus (5) Google Scholar). Even though there is a larger number of right-side-out vesicles, as in other plasma membrane preparations (37Boumendil-Podevin E.F. Podevin R.A. Biochim. Biophys. Acta. 1983; 735: 86-94Crossref PubMed Scopus (84) Google Scholar), there is also a population of sealed inside-out vesicles that allows measurement of ATP-dependent 45Ca2+ accumulation in their lumen. Protein determination was carried out using the Folin-phenol method (38Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) with bovine serum albumin as a standard. Preparation of CaM-depleted Membranes—CaM-depleted innervated membranes were obtained after incubation in mild alkaline (pH 7.8) hypotonic buffer (10 mm Tris-HCl and 2 mm EDTA) according to Niggli et al. (23Niggli V. Adunyah E.S. Penniston J.T. Carafoli E. J. Biol. Chem. 1981; 256: 395-401Abstract Full Text PDF PubMed Google Scholar) with slight modifications. Briefly, the membranes (0.2 mg/ml) were preincubated for 10 min in Eppendorf tubes in ice-cold hypotonic buffer. The samples were then centrifuged at 18,000 × g for 15 min, and the sediment was recovered for protein determination and Ca2+-ATPase activity as described (12Tortelote G.G. Valverde R.H.F. Lemos T. Guilherme A. Einicker-Lamas M. Vieyra A. FEBS Lett. 2004; 576: 31-35Crossref PubMed Scopus (23) Google Scholar). Ca2+-ATPase Activity Determination—Except when indicated otherwise, the reaction medium for the Ca2+-ATPase activity assay contained 50 mm BisTris propane buffer (pH 7.4), 125 mm KCl, 5 mm MgCl2, 5 mm ATP, 10 mm NaN3, 200 μm EGTA, and the CaCl2 concentrations needed for the desired free Ca2+ concentrations. Except when the Ca2+ concentration dependence was studied, free Ca2+ was kept at 0.5 μm. Free Ca2+ was calculated using a computer program that took into account the different species involved in the equilibrium between EGTA, Ca2+, ATP, Mg2+, H+, and K+ (39Inesi G. Kurzmack M. Coan C. Lewis D.E. J. Biol. Chem. 1980; 255: 3025-3031Abstract Full Text PDF PubMed Google Scholar, 40Sorenson M.M. Coelho H.S. Reuben J.P. J. Membr. Biol. 1986; 90: 219-230Crossref PubMed Scopus (72) Google Scholar). The temperature of the assay was 37 °C. The membranes (0.2 mg/ml) were preincubated for 30 min at room temperature in the presence of 3 mm ouabain and the compounds indicated in the corresponding figures and figure legends. The reaction was started by the addition of reaction medium to the preincubated membranes and was arrested after 10 min with 1 volume of activated charcoal in 0.1 n HCl. After centrifugation, aliquots of the supernatants were removed to measure the amount of released Pi following the method of Taussky and Shorr (41Taussky H.H. Shorr E. J. Biol. Chem. 1953; 202: 675-685Abstract Full Text PDF PubMed Google Scholar). The Ca2+-ATPase activity was calculated after subtraction of the activity measured in the presence of 2 mm EGTA. Calculation of Kinetic Parameters for Ca2+-ATPase Activity—Data for Ca2+-ATPase activity at a Ca2+ concentration range from 5 nm to 200 μm were simulated with Kinsim (42Barshop B.A. Wrenn R.F. Frieden C. Anal. Biochem. 1983; 130: 134-145Crossref PubMed Scopus (666) Google Scholar), assuming a simplified four-step catalytic cycle with 1:1 stoichiometry of Ca2+ to ATP (3Carafoli E. Physiol. Rev. 1991; 71: 129-153Crossref PubMed Scopus (572) Google Scholar) (Scheme 1), where k1Cacyt, k2ATP, k3, and k4 are the rate constants of steps 1-4 in the forward direction of the cycle (ATP consumption), respectively, and k-1, k-2ADP, k-3Caext, and k-4Pi are the corresponding rate constants in the backward direction (ATP synthesis). The equilibrium constants K1 = k-1/k1 and K3 = k3/k-3 define the Ca2+ dissociation constants at steps 1 (high affinity stimulatory site) and 3 (low affinity inhibitory site), respectively. Simulations were performed by defining a set of values for the rate constants and setting [ATP] and [Ca2+] to the experimental values. The Ca2+-ATPase activity, calculated at steady state for each [Ca2+], displayed a bell shape dependence on [Ca2+] as the experimental curve. Because various sets of rate constants (Scheme 1) could simulate the same experimental curve, we defined overall parameters that could be directly evaluated from the activity measurements. These parameters are K, the apparent dissociation constant for Ca2+ at the high affinity site; K′, the apparent dissociation constant for Ca2+ at the low affinity site; and Vmax, the maximal activity. It was assumed for simplification that (a) [ATP] and [Ca2+] are constant during the assays (hydrolysis of the nucleotide was <2%; free Ca2+ ≪ total Ca2+, and therefore, it can be considered constant even though there was a small parcel of sealed inside-out vesicles) (see Fig. 2); and (b) the reverse phosphoryl transfer reactions are slow, so that k-2[ADP] (ADP binding and ATP synthesis) and k-4[Pi] (phosphorylation by Pi) are set to zero and k2[ATP] is replaced by k2′. According to the cycle described above (Scheme 1), Ca2+-ATPase can be viewed as a transporter of cytosolic Ca2+, which is inhibited by extracellular Ca2+, and its ATPase activity is as follows ((Eq. 1), (Eq. 2), (Eq. 3), (Eq. 4), (Eq. 5)). V/Eo=Vmax×[Ca2+]cyt/([Ca2+]ext[Ca2+]cyt/K'+[Ca2+]cyt+K)(Eq. 1) Eo=E+CaE+CaE∼P+E-P(Eq. 2) K=K1(k4/k2'+k4/k-1)/(1+k4/k3+k4/k2')(Eq. 3) K'=K3(1+k4/k3+k4/k2')(Eq. 4) Vmax=k4/(1+k4/k3+k4/k2')(Eq. 5) Considering again that, in our experiments, there was only a small parcel of sealed inside-out vesicles (36de Souza W. Benchimol M. Somló C. Machado R.D. Hassón-Voloch A. Cell Tissue Res. 1979; 202: 275-281Crossref PubMed Scopus (5) Google Scholar), it can be also assumed for simplification that there is no Ca2+ gradient across most of the Ca2+-ATPase molecules (extracellular [Ca2+] = cytosolic [Ca2+]), and thus, Equation 1 becomes Equation 6. V/Eo=Vmax×[Ca2+]/([Ca2+]2/K'+[Ca2+]+K)(Eq. 6) ATP-dependent 45Ca2+ Accumulation—Active Ca2+ transport was measured by filtration using 0.45-μm pore size filters and by liquid scintillation counting. The experiments were started by the addition of the membranes (0.2 mg/ml) to a reaction medium containing 50 mm BisTris propane buffer (pH 7.4), 125 mm K+ (Cl- plus phosphate salts), 5 mm MgCl2, 5 mm ATP, 10 mm NaN3, 40 mm phosphate, 3 mm phosphoenolpyruvate plus 20 units/ml pyruvate kinase (to avoid ADP accumulation), 21.1 μm EGTA, and 15 μm 45CaCl2 (5 × 104 cpm/nmol; 0.5 μm free Ca2+). This combination of EGTA/calcium buffer was chosen to keep total 45CaCl2 as low as possible, thus avoiding high blank values. After 45Ca accumulation attained a plateau, concentrated A23187 (in Me2SO; final concentration of 10 μg/ml) was added to visualize the rapid 45Ca2+ release from the vesicular lumen. Active 45Ca2+ uptake was calculated after subtraction of the radioactivity found in the filters in the absence of ATP. SDS-PAGE and Immunoblotting—After SDS-PAGE (15% acrylamide) of the innervated membranes, the separated proteins were transferred to a nitrocellulose membrane (Protran™, Schleicher & Schüll) and incubated with the desired antibodies. The antibodies against plasma membrane Ca2+-ATPase (clone 5f10) and N-terminal CaM kinase II (amino acids 7-20) were from Affinity BioReagents (Golden, CO) and Calbiochem-Novabiochem, respectively. The rabbit phospho-Ser/Thr PKA substrate polyclonal antibody was from Cell Signaling Technology (Beverly, MA). The bound antibodies were detected using the ECL™ system (Amersham Biosciences, Buckinghamshire, UK) and X-Omat™ diagnostic film (Eastman Kodak Co., Resende, Brazil). Gels of the SDS-treated membranes and molecular mass standards were run in parallel and were then stained with Coomassie Brilliant Blue. Determination of CaM content in the IF was carried out by densitometric analysis of gels stained with Coomassie Brilliant Blue using purified bovine brain CaM as a standard. Hydroxylamine-resistant Phosphorylation of Ca2+-ATPase—Direct regulatory phosphorylation of the Ca2+ pump was assayed as described previously for the Na+ pump (43Caruso-Neves C. Rangel L.B. Vives D. Vieyra A. Coka-Guevara S. Lopes A.G. Biochim. Biophys. Acta. 2000; 1468: 107-114Crossref PubMed Scopus (28) Google Scholar) with slight modifications. Briefly, the membranes were preincubated for 15 min with the reaction medium employed to measure ATPase activity (0.5 μm Ca2+; no ATP) in the presence of CTx, KN-93, and PKIα in the combinations described in the legend to Fig. 9. The reaction was started by adding 2 mm [γ-32P]ATP (5 × 107 cpm/μmol), and 5 min later, the assays were mixed with a concentrated solution of hydroxylamine in 0.5 m citrate buffer (pH 5.5) to give a final concentration of 1 m. After vortexing for 5 min, the reaction was stopped with ice-cold 30% trichloroacetic acid. The samples were centrifuged at 2500 rpm for 25 min in a clinical centrifuge, and the precipitates were washed three times with citrate buffer and immediately used for protein determination and SDS-PAGE (7.5% acrylamide). The gels were exposed for 6 h to a phosphor screen and analyzed using a PhosphorImager to measure the intensity of the 140-kDa 32P-phosphorylated bands recognized by antibody 5f10. The Ca2+-ATPase activity was measured in membranes derived from both the IFs and NFs of polarized electrocytes. As shown in Fig. 1A, the activity was 10-fold higher in the IF compared with that in the NF. Because the acetylcholinesterase activity (the marker of the IF) is 10-fold lower in the NF (16Lowe J. Araujo G.M. Pedrenho A.R. Nunes-Tavares N. Ribeiro M.G. Hassón-Voloch A. Biochim. Biophys. Acta. 2004; 1661: 40-46Crossref PubMed Scopus (5) Google Scholar), the Ca2+-ATPase activity of this fraction probably reflects contamination with contralateral membranes. Thus, in this work, we examined the regulation of the Ca2+-ATPase resident in the IF. Fig. 1A (inset) shows the immunodetection of a band of 140 kDa recognized by monoclonal antibody 5f10, raised against a common epitope between amino acids 724 and 783 of the human erythrocyte Ca2+-ATPase, which are conserved in all isoforms of the plasma membrane Ca2+ pump (22Guerini D. Cell Tissue Res. 1998; 292: 191-197Crossref PubMed Scopus (82) Google Scholar, 44Borke J.L. Caride A. Verma A.K. Penniston J.T. Kumar R. Am. J. Physiol. 1989; 257: R842-R849Crossref PubMed Google Scholar), indicating that the enzyme of electrocyte origin shares structural properties with other Ca2+-ATPases of the P2-type ATPase family (20Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (414) Google Scholar). To investigate Ca2+ affinities of the pump in this especially CaM- and CaM kinase II-rich system (30Gotter A.L. Kaetzel M.A. Dedman J.R. Comp. Biochem. Physiol. A. 1997; 118: 81-91Crossref PubMed Scopus (4) Google Scholar), the dependence of ATPase activity on the free Ca2+ concentration was studied. The biphasic variation of the activity in the presence of increasing free calcium concentrations ranging from 5 nm to 200 μm is shown in Fig. 1B. Fitting Equation 6 (see "Experimental Procedures") to the experimental data allows to determine the values for the three global parameters defined above (Equations 3-5) as K = 21 nm, K′= 12 μm, and Vmax = 16 nmol of Pi/mg/min, therefore revealing an enzyme with an extremely high affinity for Ca2+. Fig. 1C shows that there was no difference in Ca2+-ATPase activity in either the absence or presence of thapsigargin, indicating that contamination of the preparation with reticular membranes is negligible (45Sagara Y. Inesi G. J. Biol. Chem. 1991; 266: 13503-13506Abstract Full Text PDF PubMed Google Scholar). Moreover, the sealed inside-out vesicles present in the preparation (36de Souza W. Benchimol M. Somló C. Machado R.D. Hassón-Voloch A. Cell Tissue Res. 1979; 202: 275-281Crossref PubMed Scopus (5) Google Scholar), as in others (37Boumendil-Podevin E.F. Podevin R.A. Biochim. Biophys. Acta. 1983; 735: 86-94Crossref PubMed Scopus (84) Google Scholar), were able to accumulate 45Ca2+ in their lumen in the presence of 0.5 μm free Ca2+ (Fig. 2). This capacity to transport Ca2+ at a concentration that also stimulated hydrolytic activity (Fig. 1B) and at comparable initial rates (Fig. 2, inset) gives strong support to the view that the Ca2+-ATPase activity is due to the electrocyte Ca2+ pump. Fig. 3A shows the response of electrocyte Ca2+-ATPase to CaM depletion and CaM inactivation. When the membranes were depleted of their endogenous CaM, Ca2+-ATPase activity decreased to 50% of the control value measured in the untreated preparation (calmodulin-containing membranes (CCM)), a maximal activity that could be restored after the addition of 50 nm CaM. The same 50% inhibition was seen in the presence of the well known CaM antagonist TFP (46Yamaotsu N. Suga M. Hirono S. Biopolymers. 2000; 58: 410-421Crossref Scopus (13) Google Scholar), which had no effect on calmodulin-depleted membranes (CDM). In addition, the Ca2+ curve of ATPase activity in the presence of 50 μm TFP showed an apparent >10-fold lower Ca2+ affinity (data not shown), reaching a value similar to that found for the non-poisoned plasma membrane Ca2+ pump from other different sources in the presence of CaM (3Carafoli E. Physiol. Rev. 1991; 71: 129-153Crossref PubMed Scopus (572) Google Scholar). Densitometric comparison of the CaM content of the IF (40 μg of the total protein) with a purified standard (2.5 μg) allowed us to estimate that CaM corresponds to ≈5% of the total IF proteins (Fig. 3B, inset), in agr