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Originally published In Press as doi:10.1074/jbc.M501880200 on June 29, 2005

J. Biol. Chem., Vol. 280, Issue 34, 30611-30618, August 26, 2005
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Ca2+/Calmodulin-dependent Protein Kinase II Is an Essential Mediator in the Coordinated Regulation of Electrocyte Ca2+-ATPase by Calmodulin and Protein Kinase A*

Rafael H. F. Valverde{ddagger}§, Giovane G. Tortelote{ddagger}§, Thiago Lemos{ddagger}, Elisabeth Mintz||, and Adalberto Vieyra{ddagger}**

From the {ddagger}Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro 21941-590, Brazil and the ||Laboratoire de Biophysique Moléculaire et Cellulaire, UMR 5090 Commissariat à l'Energie Atomique-CNRS-Université Joseph Fourier, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France

Received for publication, February 18, 2005 , and in revised form, June 10, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 ({approx}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 {alpha}-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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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 (1). 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 (2) and that Ca2+ fluxes from the extracellular milieu toward the cytosolic compartment require a huge inwardly directed Ca2+ electrochemical gradient to be maintained (3). Among different mechanisms, the plasma membrane Ca2+-ATPase is responsible for fine-tuning of that Ca2+ disequilibrium (3-9), which allows the generation of Ca2+ spikes, waves, and oscillations in most of the eukaryotic cells (10-12).

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 (13). One face of the electrocyte is innervated, whereas the other is not (14), and electrocytes are organized in a capacitor-like structure that allows current flow during discharges (13). Exclusively localized in the innervated face, acetylcholine receptors generate endplate potentials, triggering action potentials (15); the Na+ concentration is restored by the abundant (Na+ + K+)-ATPase resident in both faces (16). With respect to Ca2+ fluxes, an ATP-dependent Ca2+ uptake was described in membranes derived from the electroplax (17), and a high affinity Ca2+-ATPase was shown using a crude homogenate (18). Low affinity Ca2+- or Mg2+-ATPases found in electrocytes (18) are probably regulatory ecto-ATPases (19) not involved in Ca2+ transport.

The plasma membrane high affinity Ca2+-ATPase is a member of the P-type class of ATPases (20) and operates as an electrogenic Ca2+/H+ exchanger, pumping cytosolic Ca2+ to the extracellular space and internalizing H+ at a 1:1 stoichiometry (21). The different isoforms of Ca2+-ATPase (8, 22) 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 (3, 4, 7, 9, 23-28).

As far as we know, there is no information regarding the localization of the plasma membrane Ca2+-ATPase on the surface of the electrocyte and the mechanisms involved in its regulation. The content of calmodulin (CaM)1 is especially high in the electric eel ({approx}1-2% of the total mass) (29), and immunofluorescence studies have demonstrated that this regulatory peptide is concentrated and organized in both membranes of the electrocytes (13), 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) (30). This multifunctional kinase, which appears to play a pivotal role in Ca2+ signaling and homeostasis (31-33) acting as a sensor of Ca2+ fluctuations (9), is homogeneously spread within the electrocyte (30).

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 (34), where phosphorylation events involving PKA appear to be implicated in the modulation of channel-mediated ion fluxes in the electric membranes (35). 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.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—ATP, cAMP, ouabain, DL-dithiothreitol, {beta}-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 {alpha}-catalytic subunit inhibitors (PKI{alpha}), 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 (16). 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 {beta}-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 x 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 x g for 30 min, and the recovered supernatant was centrifuged again at 10,000 x g for 30 min. The sediment containing a fraction enriched with membranes of the anterior non-innervated face (NF) of the electrocyte (14) was resuspended in 250 mM sucrose. The corresponding supernatant was centrifuged at 100,000 x g for 2 h, and the final sediment containing the membranes of the posterior innervated face (IF) of the electrocyte (14) 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 (36). Even though there is a larger number of right-side-out vesicles, as in other plasma membrane preparations (37), 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 (38) 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. (23) 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 x g for 15 min, and the sediment was recovered for protein determination and Ca2+-ATPase activity as described (12).



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  		SCHEME 1
 
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+ (39, 40). 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 (41). 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 (42), assuming a simplified four-step catalytic cycle with 1:1 stoichiometry of Ca2+ to ATP (3) (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 (Equations 1-5).

(Eq. 1)

(Eq. 2)

(Eq. 3)

(Eq. 4)

(Eq. 5)

Considering again that, in our experiments, there was only a small parcel of sealed inside-out vesicles (36), 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.

(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 x 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 [GenBank] (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 (ProtranTM, 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 ECLTM system (Amersham Biosciences, Buckinghamshire, UK) and X-OmatTM 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 (43) 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 PKI{alpha} in the combinations described in the legend to Fig. 9. The reaction was started by adding 2 mM [{gamma}-32P]ATP (5 x 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
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 (16), 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 (22, 44), indicating that the enzyme of electrocyte origin shares structural properties with other Ca2+-ATPases of the P2-type ATPase family (20). To investigate Ca2+ affinities of the pump in this especially CaM- and CaM kinase II-rich system (30), 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+.



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  		FIG. 1.
Asymmetrical distribution of plasma membrane Ca2+-ATPase activity in the electrocyte. A, activities measured in the IF and NF of the electrocyte. Assays were carried out as described under "Experimental Procedures" in the presence of 0.5 µM free Ca2+. Data are means ± S.E. of at least five experiments performed in quadruplicate. The inset shows the immunodetection of plasma membrane Ca2+-ATPase using monoclonal antibody 5f10. B, Ca2+ concentration dependence of Ca2+-ATPase activity. Measurements were carried out in the presence of the free Ca2+ concentrations shown on the abscissa. The curve was adjusted to the experimental points using Equation 6 (see "Experimental Procedures"). C, Ca2+-ATPase activity (with 0.5 µM free Ca2+) in the absence (white bar) or presence (black bar) of 5 nM thapsigargin.

 
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 (45). Moreover, the sealed inside-out vesicles present in the preparation (36), as in others (37), 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 (46), 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 (3). 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 {approx}5% of the total IF proteins (Fig. 3B, inset), in agreement with the 1-2% shown in immunofluorescence studies of the whole electrocyte (13). Because there was no stimulation of Ca2+-ATPase when exogenous CaM was added to CCM (Fig. 3B), we concluded that the CaM content is sufficient to yield maximal Ca2+-ATPase rates. These control experiments support the view that the unusual, very high affinity of the electrocyte enzyme for Ca2+ (as well as the regulatory properties described below) is indeed due to the high amount of CaM present in the electric tissue, which is considered a "hyperexpressing CaM system" (29).



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  		FIG. 2.
Time course of ATP-dependent Ca2+ transport by sealed inside-out vesicles derived from innervated membranes of the electrocyte. Assays were carried out by filtration as described under "Experimental Procedures" in the presence of 0.5 µM free 45Ca2+. Results corresponding to the first 2 min can be better observed in the inset, where an amplified scale was used, thus allowing the rough evaluation of the linear velocity of the initial, predominantly unidirectional Ca2+ intake (VO, dotted line). Ionophore A23187 [GenBank] (10 µg/ml) was added at the arrow. Data are means ± S.E. of three experiments.

 
In addition to CaM, the high content of CaM kinase II is also a particular feature of the electrocyte, and this enzyme appears to be one of the most important mediators in the signaling cascade of the electric organ (30). Thus, the possible involvement of CaM kinase II in modulating Ca2+-ATPase activity was examined. To verify a possible regulatory role of this protein kinase in Ca2+-ATPase activity, its specific inhibitor KN-93 (31, 47) was used. Increasing concentrations of the inhibitor up to 1 µM led to a maximal decrease in enzyme activity to {approx}50% of the control value (Fig. 4A), as in the case of TFP (cf. Fig. 3A). Moreover, there was no additive effect of KN-93 and TFP (Fig. 4B), an indication that they converge to the same system, one that ultimately modulates the Ca2+ pump. Thus, the inhibition by KN-93 provides evidence for the first time (as far as we know) of the involvement of CaM kinase II in the stimulation of a plasma membrane Ca2+-ATPase. The presence of the kinase in the isolated membranes was confirmed by immunodetection of a 51-kDa protein using a polyclonal antibody (Fig. 4A, inset). When the concentration of KN-93 was fixed at 250 nM, sufficient for maximal inhibition of the Ca2+-ATPase (Fig. 4A), and the Ca2+ dependence was studied, a modification in the pattern of the Ca2+ activation curve was detected (Fig. 4C). Together with the {approx}50% decrease in Vmax (from 16 to 9 nmol of Pi/mg/min), an increase in the K for Ca2+ (from 21 to 37 nM) was detected compared with control conditions (cf. Fig. 1C).

As mentioned above, the formation of cAMP, one of the major second messengers in signaling systems, is very high in the electric tissue (34). In addition, there is evidence of Gs- and cAMP-mediated activation of the plasma membrane Ca2+ pump from different sources (3, 4, 8, 9), and there are three consensus sequences in the CaM kinase II molecule that can be phosphorylated by PKA. Therefore, the aim of the following experiments was to investigate whether or not cAMP participates in the route of activation of electrocyte Ca2+-ATPase by CaM and CaM kinase II. Fig. 5A shows that picomolar concentrations of cAMP resulted in maximal stimulation of Ca2+-ATPase activity, and Fig. 5B shows that activation of Gs by CTx or of adenylyl cyclase by forskolin mimicked the maximal stimulation obtained with cAMP. It should be mentioned that CTx alone stimulated Ca2+-ATPase in a concentration-dependent manner in the nanomolar range (data not shown).

To determine whether there was any interplay between the CaM kinase II and cAMP pathways, the nucleotide and KN-93 were added together. With this combination, the stimulus by cAMP was no longer seen, and Ca2+-ATPase remained inhibited as in the presence of KN-93 alone (Fig. 5C). Taken as a whole, these data confirm the existence of a Gs-, cAMP-, and CaM kinase II-mediated and sequential stimulatory pathway of the Ca2+ pump.

Fig. 6 shows that the cAMP activatory pathway of Ca2+-ATPase indeed involves a PKA, which in turn is able to compensate for inactivation of CaM: the inhibition promoted by TFP was cancelled by CTx. However, when 10 nM PKI{alpha} was present, the counteracting effect of CTx upon TFP-poisoned ATPase was lost (Fig. 6A). In contrast with the results shown in Fig. 6A for TFP, KN-93-inhibited Ca2+-ATPase was not reactivated by CTx (Fig. 6B), evidence that the cAMP/PKA compensatory pathway elicited by CTx is blocked at the level of CaM kinase II. That CTx can activate Ca2+-ATPase via a regulatory pathway with PKA and CaM kinase II as intermediates is confirmed by the experiments shown in Fig. 7. Fig. 7A shows that Ca2+-ATPase activity from CDM (a) recovered to the control values of CCM when 10-8 M CTx was added, an effect that was blocked by PKI{alpha}, and (b) became insensitive to KN-93. Conversely, when CDM were supplied with exogenous CaM (50 nM) (Fig. 7B), Ca2+-ATPase activity was fully restored (see also Fig. 3A); it again became sensitive to KN-93; and its stimulation by CTx was also blocked by PKI{alpha}.

The reversal by CTx of the inhibition of the Ca2+ pump by TFP (Fig. 6A) can be explained if PKA phosphorylates CaM kinase II, promoting its activation, with a subsequent and positive influence on the pump. Indeed, when anti-phospho-Ser/Thr PKA substrate antibody was used, it was possible to demonstrate that activation of Gs by CTx strongly increased the basal PKI{alpha}-sensitive phosphorylation of CaM kinase II, an effect that was mimicked by cAMP (Fig. 8). The correlation between activation of Ca2+-ATPase, phosphorylation of CaM kinase II, and regulatory phosphorylation of the Ca2+ pump itself under conditions in which the kinase pathways are stimulated or inhibited is revealed by the experiments presented in Fig. 9. Hydroxylamine-resistant phosphorylation of a band of 140 kDa (recognized by the monoclonal antibody against Ca2+-ATPase) increased {approx}100% when CTx was added, a phenomenon that was completely blocked by both the CaM kinase II and PKA inhibitors.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
This study has revealed that the synaptic-like face (IF) of the electrocytes of E. electricus L. has an asymmetrically localized, very high affinity plasma membrane Ca2+-ATPase (Fig. 1A). Upon SDS-PAGE, one band of 140 kDa was recognized by the antibody raised against the erythrocyte Ca2+-ATPase (Fig. 1A, inset), thus confirming the existence of a Ca2+ pump that is structurally related to others in the same family described so far (22, 44). The rate of active 45Ca2+ flux across the IF can be compared with that found in other plasma membrane preparations, e.g. red cells or kidney (48, 49), where the Ca2+-ATPase corresponds to only a small fraction of the membrane proteins (3). Moreover, the rate of unidirectional 45Ca2+ intake (6 nmol/mg/min) (Fig. 2, inset) can be reasonably compared with the initial velocity of Ca2+-ATPase activity (16 nmol of Pi/mg/min) because there is a significant population of non-vesicular membrane sheets in the preparation, as mentioned above (36).



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  		FIG. 3.
Response of electrocyte Ca2+-ATPase to CaM depletion and CaM inactivation. The Ca2+-ATPase activity was measured as described under "Experimental Procedures" in the presence of 0.5 µM free Ca2+. A, Ca2+-ATPase in CCM or CDM. Exogenous CaM or TFP was added as shown on the abscissa. *, statistically different from the control (CCM with no additions). B, Ca2+-ATPase activity in CCM at 0.5 µM free Ca2+ lacks sensitivity to exogenous CaM. Inset, measurement of CaM content in the IF (40 µg (left lane) and 20 µg (middle lane)) was carried out as described under "Experimental Procedures" using 2.5 µg of purified bovine brain CaM (right lane). Error bars in both A and B correspond to S.E. values calculated from the absolute activity values and converted to percent values.

 
Although the mechanism by which CaM directly activates the plasma membrane Ca2+-ATPase is well known (3, 8, 9, 24), the results presented in this work open up the possibility of a novel and additional mechanism by which CaM can modulate Ca2+ pumping activity: one that is mediated by CaM kinase II. An previous observation made in hippocampus neurons (where CaM kinase II is also abundant (50)) demonstrated that the kinase phosphorylates several receptors and ion channels, thereby regulating transport processes and intracellular ion concentrations (9). In vertebrate central nervous tissue, CaM kinase II is a major component of the postsynaptic membranes (51), which are functionally equivalent to the innervated membranes of the electrocytes. In these types of membranes, a stable interaction of CaM kinase II with CaM and Ca2+-ATPase would therefore be responsible for the kinetic and regulatory properties of the Ca2+ pump described in this work.



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  		FIG. 4.
Membrane-associated CaM kinase II is involved in the activation of the electrocyte Ca2+-ATPase by CaM. A, Ca2+-ATPase activity assayed in CCM as described under "Experimental Procedures" (0.5 µM free Ca2+) in the presence of increasing concentrations of the specific CaM kinase II inhibitor KN-93. The curve was drawn by eye. The inset shows the immunodetection of CaM kinase II in the IF using a rabbit polyclonal antibody raised against the CaM kinase II N terminus (amino acids 7-20). Lane a, 20 µg of rat cerebellum homogenate as a positive control; lane b, 15 µg of IF membrane protein; lane c, 30 µg of IF membrane protein (see also Fig. 8C). B, Ca2+-ATPase activity assayed in CCM in the presence of KN-93 and TFP in the combinations shown on the abscissa. Error bars correspond to S.E. values calculated from the absolute activity values and converted to percent values. *, statistically different from the control. C, Ca2+ concentration dependence of Ca2+-ATPase activity in the presence of 250 nM KN-93 (triangles). The curve was adjusted to the experimental points using a rectangular hyperbola, i.e. a simplified version of Equation 6 (see "Experimental Procedures"). The white circle shows the control (no KN-93 at 0.5 µM free Ca2+).

 
The initial hypothesis raised in this work that CaM kinase II may have an important role in regulating the electrocyte Ca2+ pump (and therefore, intracellular Ca2+ activity) is supported by the experiments of Fig. 4, which show a dose-dependent decrease in electrocyte Ca2+-ATPase by KN-93, a specific CaM kinase II inhibitor (31, 47). Although CaM kinase II has been purified from the soluble fraction of electric eel and although its association with subcellular structures appears to be reversible (52), Fig. 4A (inset) shows that CaM kinase II remains bound to the innervated membrane after cell fractionation.

The participation in the electrocyte innervated membranes of a PKA that stimulates active Ca2+ transport (as clearly demonstrated in many other cells (3, 4, 7-9, 53)) is confirmed by the observations presented in Figs. 5 and 6. Stimulation of a PKA pathway in different ways markedly increased Ca2+-ATPase activity (Fig. 5) and reactivated the pumping activity impaired by the CaM antagonist TFP (Fig. 6A). The addition of PKI{alpha} suppressed the reactivating influence of CTx in membranes containing inactivated CaM, a clear demonstration that a PKA-catalyzed step participates in the stimulatory compensatory cascade that reverts the effect of TFP on Ca2+-ATPase. Although reversal by PKA of the inhibition promoted by TFP does not represent a physiological condition, it offers a plausible role for the PKA route in compensating for inhibition of CaM binding or a low sensitivity to CaM, similar to that observed in some isoforms and alternative splice variants of Ca2+-ATPase (8, 22). For example, oxidative modifications of methionines in CaM have been implicated in a decrease in its binding to the pump and in its maximal activating effect (54, 55), and this could be circumvented by activation of a PKA pathway. The results presented in this study indicate that, in the electrocyte, CaM kinase II appears to play a pivotal role in an integrated CaM- and PKA-mediated activation of the plasma membrane Ca2+ pump.



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  		FIG. 5.
Stimulation of the Ca2+-ATPase by PKA requires a functional CaM kinase II. A, Ca2+-ATPase activity in CCM measured as described under "Experimental Procedures" (0.5 µM free Ca2+) in the presence of the cAMP concentrations shown. B, Ca2+-ATPase activity under control conditions (-; no additions), with 10 nM forskolin (FSK), or with 100 nM CTx. C, Ca2+-ATPase activity measured in the presence of cAMP and KN-93 in the combinations shown on the abscissa. Error bars correspond to S.E. values calculated from the absolute activity values and converted to percent values. *, statistically different from the control (no additions).

 
The view that CaM kinase II is an intermediate in the activation of the electrocyte Ca2+-ATPase by CaM and PKA was first supported by the experiments presented in Figs. 5, 6, 7. The stimulatory signal of the cAMP/PKA pathway upon Ca2+-ATPase in CCM (Fig. 5, A and B) was stopped when KN-93, an inhibitor of CaM kinase II, was simultaneously added (Fig. 5C). Moreover, the compensatory effect of CTx when CaM was inactivated by TFP (Fig. 6A) or removed from the membranes (Fig. 7A) was no longer observed in the presence of KN-93 (Fig. 6B). Taken together, these data are compatible with a model of a signaling cascade in which an upstream CTx-sensitive pathway converges to a CaM kinase II-mediated activating step of Ca2+-ATPase. In the literature concerning Ca2+/CaM-activated proteins, there are a small number of significant reports on the cross-talk between PKA and CaM kinase II. For example, in the cardiac sarcoplasmic reticulum, PKA and CaM kinase II stimulate active Ca2+ uptake, an effect that is mediated by phosphorylation of both the Ca2+ pump and its regulatory protein phospholamban (56). Complex interactions have also been shown in parotid acinar cells, where cAMP enhances Ca2+ clearance probably involving CaM kinase II (53). Therefore, it is clear that the latter enzyme could play a central role in the modulatory network of the cellular Ca2+ signals by tightly coupling PKA, CaM, and the Ca2+ pump.



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  		FIG. 6.
The PKA inhibitor PKI{alpha} (A) and the CaM kinase II inhibitor KN-93 (B) block reactivation by CTx of Ca2+-ATPase activity impaired by TFP. CTx, TFP, PKI{alpha}, and KN-93 were combined as indicated on the abscissas in assays using CCM. Errors bars correspond to S.E. values calculated from the absolute activity values and converted to percent values. *, statistical difference with respect to the control.

 
An inactivating effect of KN-93 on a G protein has been shown in a recent study (57), and this could be an alternative explanation as to why CTx failed to modify the inhibition caused by KN-93. Nevertheless, because the addition of cAMP (which acts downstream of a G protein in a signaling cascade activated by CTx) did not result in recovery of Ca2+-ATPase activity when KN-93 was present (Fig. 5C), it is clear that CaM kinase II is the key target in the activation of the electrocyte Ca2+ pump by cAMP/PKA and its inhibition by KN-93.



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  		FIG. 7.
Effects of PKA and CaM kinase II inhibitors on Ca2+-ATPase from CDM in the absence or presence of exogenous CaM. Ca2+-ATPase activity was assayed in CDM as indicated on the abscissas. CTx, KN-93, and PKI{alpha} were combined as shown to investigate their effects on Ca2+-ATPase in CDM in the absence (A) or presence (B) of exogenous CaM. CCM served as the control. Both types of membrane preparations were obtained as described under "Experimental Procedures" and assayed in parallel for Ca2+-ATPase activity. Error bars correspond to S.E. values calculated from the absolute activity values and converted to percent values.

 
Figs. 8 and 9 give direct biochemical evidence of the mechanism proposed here for stimulation of Ca2+-ATPase activity via CaM kinase II-mediated phosphorylation of the Ca2+ pump molecule. Fig. 8 shows that phosphorylation of CaM kinase II was strongly increased upon the addition of either cAMP or CTx and was suppressed by specific blocking of the {alpha}-catalytic subunit of PKA by its specific inhibitor. Three amino acids (Ser50, Thr286, and Ser314; NCBI Protein Database and www.expasy.org/prosite) are found in consensus domains for PKA recognition in CaM kinase II, and one of them appears to be particularly interesting from a regulatory perspective. Thr286 is the key amino acid to be phosphorylated in the autoinhibitory domain Arg283-Gly-Glu-Thr-Val287 (33). Therefore, it may be hypothesized that PKA phosphorylates this residue as an efficient alternative to the autophosphorylation mechanism of CaM kinase II activation (31-33) and/or when CaM binding to its targets is impaired (54, 55). Subsequently, a direct demonstration that phosphorylation of the Ca2+ pump molecule is the culmination of the route that sequentially involves activation of PKA and phosphorylation of CaM kinase II is given in Fig. 9. The increase in hydroxylamine-resistant phosphorylation of the 140-kDa band recognized by antibody 5f10 upon the addition of CTx and the blocking of this effect by KN-93 or PKI{alpha} clearly show that activation of the electrocyte Ca2+-ATPase by PKA has CaM kinase II as an intermediate.



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  		FIG. 8.
Activation of the adenylyl cyclase/PKA route phosphorylates CaM kinase II. A, immunodetection of a specific phospho-Ser/Thr PKA substrate in CaM kinase II using rabbit phospho-Ser/Thr PKA substrate polyclonal antibody as described under "Experimental Procedures." Membranes (CCM) were previously exposed for 30 min to the conditions indicated on the abscissa. Lane 1, control with no additions; lane 2, 5 nM cAMP; lane 3, 10 nM CTx; lane 4, 25 units/ml PKI{alpha} (peptide 5-22). B, densitometric representation of the phosphorylation intensity shown in A. C, immunodetection of CaM kinase II in the same preparation (see also Fig. 4A, inset).

 
The observations presented in this work raise the question of whether CaM can also directly activate the electrocyte Ca2+ pump. Because merely depleting endogenous CaM caused a strong inhibition of Ca2+-ATPase activity (Figs. 3 and 7), it can be proposed that the mechanism described here is an additional and efficient way to regulate plasma membrane Ca2+ pumping activity together with the well known direct activation by CaM (3, 8, 9, 24). Perhaps, the basal phosphorylation of the pump seen in the absence of activators (Fig. 9) may help CaM by reducing the binding of the autoinhibitory domain to the catalytic domain of the Ca2+-ATPase molecule, as proposed previously (58).



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  		FIG. 9.
Hydroxylamine-resistant phosphorylation of Ca2+-ATPase after activation of the PKA pathway is suppressed by blocking CaM kinase II. Phosphorylation of Ca2+-ATPase in the presence of hydroxylamine was carried out as described under "Experimental Procedures" with the combinations of CTx, KN-93, and PKI{alpha} shown on the abscissa. A, 32P signals detected using a phosphor screen. B, PhosphorImager densitometric analysis of the intensity of the 140-kDa 32P-phosphorylated bands (means of two experiments that agreed within 15%). C, immunodetection of plasma membrane Ca2+-ATPase using monoclonal antibody 5f10.

 

   FOOTNOTES
 
This work is dedicated to Dr. Darcy Fontoura de Almeida on his 75th birthday.

* This work was supported in part by grants from the Conselho Nacional de Desenvolvimento Científico e Tecnológico, the Programa de Apoio aos Núcleos de Excelência (Conselho Nacional de Desenvolvimento Científico e Tecnológico/Ministério da Ciência e Tecnologia), the Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (Brazil), and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior/Comité Français d'Evaluation de la Coopération Universitaire avec le Brésil Grant 378 (Brazil/France). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Recipients of a fellowship from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. Back

Recipient of a fellowship from the Conselho Nacional de Desenvolvimento Científico e Tecnológico. Back

** To whom correspondence should be addressed. Tel.: 55-21-2562-6520; Fax: 55-21-2280-8193; E-mail: avieyra{at}biof.ufrj.br.

1 The abbreviations used are: CaM, calmodulin; CaM kinase II, Ca2+/calmodulin-dependent protein kinase II; PKA, protein kinase A; TFP, trifluoperazine dihydrochloride; CTx, cholera toxin; PKI{alpha}, protein kinase A {alpha}-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. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Martha Sorenson for comments.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Schoffeniels, E. (1961) in Bioelectrogenesis: A Comparative Survey of Its Mechanisms with Particular Emphasis on Electric Fishes (Chagas, C., and Paes de Carvalho, A. eds) pp. 147-165, Elsevier Science Publishers B. V., Amsterdam
  2. Esquibel, M. A. (1970) Effect of Calcium and Magnesium on the Neuroelectric Synapses, Ph.D. thesis, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil
  3. Carafoli, E. (1991) Physiol. Rev. 71, 129-153[Free Full Text]
  4. Penniston, J. T., and Enyedi, A. (1998) J. Membr. Biol. 165, 101-109[CrossRef][Medline] [Order article via Infotrieve]
  5. Shull, G. E. (2000) Eur. J. Biochem. 267, 5284-5290[Medline] [Order article via Infotrieve]
  6. Carafoli, E., and Brini, M. (2000) Curr. Opin. Chem. Biol. 4, 152-161[CrossRef][Medline] [Order article via Infotrieve]
  7. Gromadzinska, E., Lachowicz, L., Walkowiak, B., and Zylinska, L. (2001) Biochim. Biophys. Acta 1549, 19-31[CrossRef][Medline] [Order article via Infotrieve]
  8. Strehler, E. E., and Zacharias, D. A. (2001) Physiol. Rev. 81, 21-50[Abstract/Free Full Text]
  9. Carafoli, E., Santella, L., Branca, D., and Brini, M. (2001) Crit. Rev. Biochem. Mol. Biol. 36, 107-260[CrossRef][Medline] [Order article via Infotrieve]
  10. Isshiki, M., Ando, J., Korenaga, R., Kogo, H., Fujimoto, T., Fujita, T., and Kamiya, A. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5009-5014[Abstract/Free Full Text]
  11. Isshiki, M., and Anderson, R. G. W. (1999) Cell Calcium 26, 201-208[CrossRef][Medline] [Order article via Infotrieve]
  12. Tortelote, G. G., Valverde, R. H. F., Lemos, T., Guilherme, A., Einicker-Lamas, M., and Vieyra, A. (2004) FEBS Lett. 576, 31-35[CrossRef][Medline] [Order article via Infotrieve]
  13. Gotter, A. L., Kaetzel, M. A., and Dedman, J. R. (1998) Comp. Biochem. Physiol. A 119, 225-241
  14. Almeida, D. F., Castro, G. O., Miranda, M., and Chagas, C. (1963) Exp. Cell Res. 29, 42-49[CrossRef][Medline] [Order article via Infotrieve]
  15. Keynes, R. D., and Martins-Ferreira, H. (1953) J. Physiol. (Lond.) 119, 315-351
  16. Lowe, J., Araujo, G. M., Pedrenho, A. R., Nunes-Tavares, N., Ribeiro, M. G., and Hassón-Voloch, A. (2004) Biochim. Biophys. Acta 1661, 40-46[Medline] [Order article via Infotrieve]
  17. Oliveira, M. M., Machado, R. D., and Chagas, C. (1978) Comp. Biochem. Physiol. C 61, 17-22[CrossRef]
  18. Amende, L. M., Chock, S. P., and Albers, R. W. (1983) J. Neurochem. 40, 1040-1047[CrossRef][Medline] [Order article via Infotrieve]
  19. Taffarel, M., Coelho-Sampaio, T., Teixeira-Ferreira, A., Somló, C., de Souza, W., Machado, R. D., and Vieyra, A. (1989) J. Histochem. Cytochem. 37, 953-959[Abstract]
  20. Lutsenko, S., and Kaplan, J. H. (1995) Biochemistry 34, 15607-15613[CrossRef][Medline] [Order article via Infotrieve]
  21. Hao, L., Rigaud, J. L., and Inesi, G. (1994) J. Biol. Chem. 269, 14268-14275[Abstract/Free Full Text]
  22. Guerini, D. (1998) Cell Tissue Res. 292, 191-197[CrossRef][Medline] [Order article via Infotrieve]
  23. Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401[Abstract/Free Full Text]
  24. Benaim, G., Zurini, M., and Carafoli, E. (1984) J. Biol. Chem. 259, 8471-8477[Abstract/Free Full Text]
  25. Coelho-Sampaio, T., Ferreira, S. T., Benaim, G., and Vieyra, A. (1991) J. Biol. Chem. 266, 22266-22272[Abstract/Free Full Text]
  26. Filoteo, A. G., Enyedi, A., and Penniston, J. T. (1992) J. Biol. Chem. 267, 11800-11805[Abstract/Free Full Text]
  27. Wang, K. K., Villalobo, A., and Roufogalis, B. D. (1992) Trends Cell Biol. 2, 46-52[CrossRef][Medline] [Order article via Infotrieve]
  28. Hofmann, F., Anagli, J., Carafoli, E., and Vorherr, T. (1994) J. Biol. Chem. 269, 24298-24303[Abstract/Free Full Text]
  29. Munjaal, R. P., Connor, C. G., Turner, R., and Dedman, J. R. (1986) Mol. Cell. Biol. 6, 950-954[Abstract/Free Full Text]
  30. Gotter, A. L., Kaetzel, M. A., and Dedman, J. R. (1997) Comp. Biochem. Physiol. A 118, 81-91[CrossRef]
  31. Means, A. R. (2000) Mol. Endocrinol. 14, 4-13[Free Full Text]
  32. Hook, S. S., and Means, A. R. (2001) Annu. Rev. Pharmacol. Toxicol. 41, 471-505[CrossRef][Medline] [Order article via Infotrieve]
  33. Hudmon, A., and Schulman, H. (2002) Biochem. J. 364, 593-611[CrossRef][Medline] [Order article via Infotrieve]
  34. Chagas, C., Nesralla-Lobão, H., and Rebello, M. A. (1972) C. R. Acad. Sci. (Paris) 274, 1526-1529
  35. Emerick, M. C., and Agnew, W. S. (1989) Biochemistry 28, 8367-8380[CrossRef][Medline] [Order article via Infotrieve]
  36. de Souza, W., Benchimol, M., Somló, C., Machado R. D., and Hassón-Voloch, A. (1979) Cell Tissue Res. 202, 275-281[Medline] [Order article via Infotrieve]
  37. Boumendil-Podevin, E. F., and Podevin, R. A. (1983) Biochim. Biophys. Acta 735, 86-94[Medline] [Order article via Infotrieve]
  38. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275[Free Full Text]
  39. Inesi, G., Kurzmack, M., Coan, C., and Lewis, D. E. (1980) J. Biol. Chem. 255, 3025-3031[Free Full Text]
  40. Sorenson, M. M., Coelho, H. S., and Reuben, J. P. (1986) J. Membr. Biol. 90, 219-230[CrossRef][Medline] [Order article via Infotrieve]
  41. Taussky, H. H., and Shorr, E. (1953) J. Biol. Chem. 202, 675-685[Free Full Text]
  42. Barshop, B. A., Wrenn, R. F., and Frieden, C. (1983) Anal. Biochem. 130, 134-145[CrossRef][Medline] [Order article via Infotrieve]
  43. Caruso-Neves, C., Rangel, L. B., Vives, D., Vieyra, A., Coka-Guevara, S., and Lopes, A. G. (2000) Biochim. Biophys. Acta 1468, 107-114[Medline] [Order article via Infotrieve]
  44. Borke, J. L., Caride, A., Verma, A. K., Penniston, J. T., and Kumar, R. (1989) Am. J. Physiol. 257, R842-R849
  45. Sagara, Y., and Inesi, G. (1991) J. Biol. Chem. 266, 13503-13506[Abstract/Free Full Text]
  46. Yamaotsu, N., Suga, M., and Hirono, S. (2000) Biopolymers 58, 410-421
  47. Sumi, M., Kiuchi, K., Ishikawa, T., Ishii, A., Hagiwara, M., Nagatsu, T., and Hidaka, H. (1991) Biochem. Biophys. Res. Commun. 181, 968-975[CrossRef][Medline] [Order article via Infotrieve]
  48. Graf, E., and Penniston, J. T. (1981) J. Biol. Chem. 256, 1587-1592[Abstract/Free Full Text]
  49. Vieyra, A., Nachbin, L., de Dios-Abad, E., Goldfeld, M., Meyer-Fernandes, J. R., and de Moraes, L. (1986) J. Biol. Chem. 261, 4247-4255[Abstract/Free Full Text]
  50. Erondu, N. E., and Kennedy, M. B. (1985) J. Neurosci. 5, 3270-3277[Abstract]
  51. Kennedy, M. B. (2000) Science 290, 750-754[Abstract/Free Full Text]
  52. Saitoh, T., and Schwartz, J. H. (1985) J. Cell Biol. 100, 835-842[Abstract/Free Full Text]
  53. Bruce, J. I., Yule, D. I., and Shuttleworth, T. J. (2002) J. Biol. Chem. 277, 48172-48181[Abstract/Free Full Text]
  54. Yao, Y., Yin, D., Jas, G. S., Kuczera, K., Williams, T. D., Schoneich, C., and Squier, T. C. (1996) Biochemistry 35, 2767-2787[CrossRef][Medline] [Order article via Infotrieve]
  55. Yin, D., Sun, H., Weaver, R. F., and Squier, T.C. (1999) Biochemistry 38, 13654-13660[CrossRef][Medline] [Order article via Infotrieve]
  56. Xu, A., Hawkins, C., and Narayanan, N. (1997) J. Mol. Cell. Cardiol. 29, 405-418[CrossRef][Medline] [Order article via Infotrieve]
  57. Yue, C., and Sanborn, B. M. (2001) Mol. Cell. Endocrinol. 175, 149-156[CrossRef][Medline] [Order article via Infotrieve]
  58. Carafoli, E. (1994) FASEB J. 8, 993-1002[Abstract]

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