Activation process of calcium-dependent potassium channel in Euhadra neurons: involvement of calcium/calmodulin and subsequent protein phosphorylation.

1. The activation process of Ca(2+)-dependent potassium channel was studied electrophysiologically and pharmacologically using identified neurons of the land snail, Euhadra peliomphala. 2. Ca(2+)-mediated delayed outward K current (IKD) was dose-dependently reduced by the calmodulin inhibitors, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5, week) and N-(6-aminohexyl)-5-chloro-naphthalenesulfonamide (W-7, potent). These antagonists also caused a slight membrane depolarization and increase in impulse discharge frequency with decrease in the amplitude of both action potential and after hyperpolarization. 3. The cAMP-dependent protein kinase inhibitor N-[2-(methylamino) ethyl]-5-isoquinoline-sulfonamide (H-8) did not produce any significant effect on IKD and membrane potential. 4. Calmodulin, when injected into the neuron which had been treated with either W-5 or W-7, transiently restored the suppressed IKD nearly to the pretreatment level, and caused hyperpolarization of the cell. In contrast, calcium chloride, intracellularly injected in the same way, had little effect on both the IKD and the membrane potential shifted by these antagonists. 5. Intracellular injection of kinase II, a Ca2+/calmodulin-dependent protein kinase, caused an increase in the IKD and membrane hyperpolarization. Similar but weak effects were produced when a catalytic subunit (CS) of cAMP-dependent protein kinase was intracellularly injected. However, the neurons pretreated with W-7 no longer had any detectable increase in the IKD and hyperpolarization of the membrane. 6. These results suggest the possibility that Ca2+/camodulin-dependent protein phosphorylation may finally mediate the activation of a certain number of potassium channels.     

Calmodulin/calmodulin-dependent protein kinase II mediates SAAF-induced motility activation of ascidian sperm

Ca2+-influx and membrane hyperpolarization by sperm-activating and -attracting factor (SAAF) released from the unfertilized egg of the ascidians Ciona cause a transient increase in cAMP, which triggers activation of sperm motility. We demonstrated here the presence of Ca2+-binding protein, calmodulin (CaM), and CaM-dependent kinase II (CaMKII) in the sperm. CaM antagonist, W-7, and CaMKII inhibitor, KN-93, suppressed SAAF-induced membrane hyperpolarization, increase in cAMP, and activation of sperm motility, but inactive analogues of W-7 and KN-93, namely W-5 and KN-92, respectively, did not. Subsequent addition of K+ ionophore, valinomycin, hyperpolarized the plasma membrane, increased cAMP, and conferred motility to the immotile sperm even in the presence of W-7 and KN-93. Addition of IBMX activated motility of sperm, which has been immobilized by W-7 and KN-93. These suggest that increased [Ca2+]i through influx of Ca2+ by SAAF binds to CaM to activate CaMKII. The activated CaMKII may cause membrane hyperpolarization to increase cAMP, which triggers the activation of sperm motility in Ciona.     

The newly synthesized selective Ca2+/calmodulin dependent protein kinase II inhibitor KN-93 reduces dopamine contents in PC12h cells.

We reported that one of the isoquinolinesulfonamide derivatives, KN-62, is a potent and specific inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII) (Tokumitsu, H., Chijiwa, T., Hagiwara, M., Mizutani, A., Terasawa, M. and Hidaka, H. (1990) J. Biol. Chem. 265, 4315-4320). We have now investigated the inhibitory property of a newly synthesized methoxybenzenesulfonamide, KN-93, on CaMKII activity in situ and in vitro. KN-93 elicited potent inhibitory effects on CaMKII phosphorylating activity with an inhibition constant of 0.37 microM but this compound had no significant effects on the catalytic activity of cAMP-dependent protein kinase, Ca2+/phospholipid dependent protein kinase, myosin light chain kinase and Ca(2+)-phosphodiesterase. KN-93 also inhibited the autophosphorylation of both the alpha- and beta-subunits of CaMKII. Kinetic analysis indicated that KN-93 inhibits CaMKII, in a competitive fashion against calmodulin. To evaluate the regulatory role of CaMKII on catecholamine metabolism, we examined the effect of KN-93 on dopamine (DA) levels in PC12h cells. The DA levels decreased in the presence of KN-93. Further, the tyrosine hydroxylase (TH) phosphorylation induced by KCl or acetylcholine was significantly suppressed by KN-93 in PC12h cells while events induced by forskolin or 8-Br-cAMP were not affected. These results suggest that KN-93 inhibits DA formation by modulating the reaction rate of TH to reduce the Ca(2+)-mediated phosphorylation levels of the TH molecule.     

Primary structure of the cAMP-dependent phosphorylation site of the plasma membrane calcium pump

The primary structure of a region of the erythrocyte plasma membrane calcium pump which is phosphorylated by the cAMP-dependent protein kinase has been determined. The sequence is A-P-T-K-R-N-S-S(P)-P-P-P-S-P-D. The site is located between the calmodulin binding domain and the C-terminus of the ATPase. The ATPase is phosphorylated only at this site by the cAMP-dependent protein kinase, and the phosphorylation is inhibited by calmodulin. The effect of the phosphorylation is to decrease the Km for Ca2+ of the purified ATPase from about 10 microM to about 1.4 microM and to increase the Vmax of ATP hydrolysis about 2-fold.     

Phosphorylation of smooth muscle myosin light chain kinase by Ca2+-activated, phospholipid-dependent protein kinase

Protein kinase C incorporates phosphate into two sites of myosin light chain kinase (MLC-kinase) in the absence of calmodulin. Phosphorylation is all but abolished in the presence of Ca2+ and calmodulin, suggesting that both sites of phosphorylation are close to the calmodulin binding site. The phosphorylation of MLC-kinase results in an approximately 10-fold increase in the dissociation constant of MLC-kinase for calmodulin. Following phosphorylation (2 mol/mol of enzyme) of MLC-kinase by protein kinase C, an additional 2 mol of phosphate can be incorporated into the MLC-kinase apoenzyme by the cAMP-dependent protein kinase. Different maps of phosphopeptides were obtained by tryptic hydrolysis from MLC-kinase preparations phosphorylated by each kinase. The phosphorylation sites for the cAMP-dependent kinase were located in a fragment of approximately 25,000 daltons. In contrast the phosphorylation sites for protein kinase C are found in a much smaller tryptic peptide. These results suggest that the phosphorylation sites on MLC-kinase are different for protein kinase C and for cAMP-dependent protein kinase. However, phosphorylation in both regions results in a reduced affinity for calmodulin.     

Inhibition of G2/M progression in Schizosaccharomyces pombe by a mutant calmodulin kinase II with constitutive activity.

Intracellular signaling by the second messenger Ca2+ through its receptor calmodulin (CaM) regulates cell function via the activation of CaM-dependent enzymes. Previous studies have shown that cell cycle progression at G1/S and G2/M is sensitive to intracellular CaM levels. However, little is known about the CaM-regulated enzymes involved. Protein phosphorylation has been shown to be important for cell-cycle regulation. Because CaM regulates several protein kinases, and at least one protein phosphatase, our studies are focusing on the roles of these enzymes within the cell cycle. As an initial approach to this problem, cDNAs encoding either normal or mutant calcium/calmodulin kinase II (CaMKII) have been expressed in Schizosaccharomyces pombe. The results show that overexpression of a constitutively active mutant CaMKII caused cell-cycle arrest in G2. Arrest was associated with a failure to activate the p34/cdc2 protein kinase. Expression of the mutant CaMKII in strains of S. pombe with altered timing of mitosis revealed that this effect is not mediated either by cdc25+ or wee1+, suggesting that CaMKII may regulate G2/M progression by another mechanism.     

Regulation of human platelet membrane Ca2+ transport by cAMP- and calmodulin-dependent phosphorylation

We have examined the effects of added cAMP-dependent protein kinase and endogenous calmodulin-dependent kinase on Ca2+ transport in purified internal membranes from human platelets. Both Ca2+ uptake and Ca2+-ATPase activity were maximally stimulated about 2-fold by addition of cAMP-dependent protein kinase. Cyclic AMP-dependent protein kinase inhibitor reduced both Ca2+ uptake and Ca2+-ATPase activities at concentrations which also inhibited cAMP-dependent protein phosphorylation. In addition, concerted stimulation of Ca2+-ATPase by exogenous calmodulin and added catalytic subunit of cAMP-dependent protein kinase was observed. A 22-kDa protein was phosphorylated by both cAMP-dependent and calmodulin-dependent kinases at the same rate as stimulation of the Ca2+-ATPase. Cyclic AMP-dependent phosphorylation of the 22-kDa polypeptide was inhibited by the protein kinase inhibitor and calmodulin-dependent phosphorylation was inhibited by chlorpromazine and EGTA. These results are consistent with the hypothesis that one mode of control of Ca2+ homeostasis in platelets may be similar to the phospholamban system in cardiac muscle.     

Ca2+/calmodulin-dependent protein phosphorylation associated with the cytoskeleton of quiescent rat fibroblast (3Y1) cells.

Endogenous phosphorylation of the crude membrane fraction of cultured 3Y1 fibroblast cells was enhanced by the addition of Ca2+/calmodulin. Both Ca2+/calmodulin-dependent protein kinase activity and its substrate were present in a cytoskeletal fraction, obtained as a pellet after washing of the membrane fraction with 2 mM EGTA, 0.6 M NaCl, and 1% Triton X-100. The phosphorylatable protein in the Triton X-insoluble fraction was identified by immunoblotting as vimentin. This endogenous phosphorylation induced by calmodulin was inhibited by the addition of KN-62, a specific Ca2+/calmodulin-dependent protein kinase II inhibitor, in a dose-dependent manner. However, phosphorylation of the 59 kDa protein (vimentin) in this fraction was not stimulated by adding both phosphatidyl serine and cAMP, thereby suggesting the absence of protein kinase C or of cAMP-dependent protein kinase in this fraction. The protein kinase associated with the Triton X-insoluble fraction phosphorylated the Ca2+/calmodulin-dependent protein kinase II-specific site of synapsin I from the bovine cortex. Two-dimensional phosphopeptide maps of vimentin indicated that a major phosphopeptide phosphorylated by the endogenous calmodulin-dependent kinase also appears to be the same as a major phosphopeptide phosphorylated by the exogenous Ca2+/calmodulin-dependent protein kinase II. Our results suggest that cytoskeleton-associated Ca2+/calmodulin-dependent protein kinase II regulates dynamic cellular functions through the phosphorylation of cytoskeletal elements in non-neural cells.     

The role of Ca2+ and cyclic AMP in the phosphorylation of rat-liver soluble proteins by endogenous protein kinases

Rat liver soluble proteins were phosphorylated by endogenous protein kinase with [gamma-32P]ATP. Proteins were separated in dodecyl sulphate slab gels and detected with the aid of autoradiography. The relative role of cAMP-dependent, cAMP-independent and Ca2+-activated protein kinases in the phosphorylation of soluble proteins was investigated. Heat-stable inhibitor of cAMP-dependent protein kinase inhibits nearly completed the phosphorylation of seven proteins, including L-type pyruvate kinase. The phosphorylation of eight proteins is not influenced by protein kinase inhibitor. The phosphorylation of six proteins, including phosphorylase, is partially inhibited by protein kinase inhibitor. These results indicate that phosphoproteins of rat liver can be subdivided into three groups: phosphoproteins that are phosphorylated by (a) cAMP-dependent protein kinase or (b) cAMP-independent protein kinase; (c) phosphoproteins in which both cAMP-dependent and cAMP-independent protein kinase play a role in the phosphorylation. The relative phosphorylation rate of substrates for cAMP-dependent protein kinase is about 15-fold the phosphorylation rate of substrates for cAMP-independent protein kinase. The Km for ATP of cAMP-dependent protein kinase and phosphorylase kinase is 8 microM and 38 microM, respectively. Ca2+ in the micromolare range stimulates the phosphorylation of (a) phosphorylase, (b) a protein with molecular weight of 130 000 and (c) a protein with molecular weight of 15 000. The phosphate incorporation into a protein with molecular weight of 115 000 is inhibited by Ca2+. Phosphorylation of phosphorylase and the 15 000-Mr protein in the presence of 100 microM Ca2+ could be completely inhibited by trifluoperazine. It can be concluded that calmodulin is involved in the phosphorylation of at least two soluble proteins. No evidence for Ca2+-stimulated phosphorylation of subunits of glycolytic or gluconeogenic enzymes, including pyruvate kinase, was found. This indicates that it is unlikely that direct phosphorylation by Ca2+-dependent protein kinases is involved in the stimulation of gluconeogenesis by hormones that act through a cAMP-independent, Ca2+-dependent mechanism.     

CaM kinase II regulation of CRHSP-28 phosphorylation in cultured mucosal T84 cells

Ca(2+)-regulated heat-stable protein of 28 kDa (CRHSP-28; a member of the tumor protein D52 family) is highly expressed in exocrine glands and was shown to regulate digestive enzyme secretion from pancreatic acinar cells. We found CRHSP-28 highly expressed in cultured mucosal secretory T84 cells, consistent with an important regulatory role in apical membrane trafficking. Stimulation of cells with carbachol (CCh) induced rapid, concentration-dependent phosphorylation of CRHSP-28 on at least two serine residues. Isoelectric focusing and immunoblotting were used to characterize cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation depends on elevated cellular Ca2+, being maximally induced by ionomycin and thapsigargin and fully inhibited by BAPTAAM. In vitro phosphorylation of recombinant CRHSP-28 was 10-fold greater by casein kinase II (CKII) than Ca2+/calmodulin-dependent protein kinase II (CaMKII). However, phosphopeptide mapping studies demonstrated that CaMKII induced an identical phosphopeptide profile to endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated phosphorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated that CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaMKII was also localized to the subapical cytoplasm and was clearly displaced following actin filament disruption. These data suggest that CRHSP-28 phosphorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells.     

Phosphorylation of bovine brain 81-kDa acidic calmodulin binding protein (ACAMP-81) in vitro.

We found a novel 81-kDa acidic protein (ACAMP-81) in the bovine brain membrane fraction, which bound to calmodulin in a Ca(2+)-dependent manner. The present study reveals physicochemical properties and phosphorylation of this protein with various protein kinases in vitro. The Stokes radius and sedimentation coefficient were calculated to be 52 A and 2.05 S, respectively, suggesting that the structure of ACAMP-81 is highly elongated. Purified Ca2+/phospholipid-dependent protein kinase (protein kinase C), cAMP-dependent protein kinase, and Ca2+/calmodulin-dependent protein kinase II (Ca2+/CaM kinase II) catalyzed the incorporation of 1.46, 0.72, and 0.44 mol of phosphate/mol of ACAMP-81, respectively. The amino acid residues of ACAMP-81 phosphorylated by either protein kinase C or cAMP-dependent protein kinase were almost exclusively on serine. Sequential phosphorylation of ACAMP-81 by cAMP-dependent protein kinase and protein kinase C resulted in the additional incorporation of 1.15 mol of [32P]phosphate into ACAMP-81. Comparison of phosphopeptide maps of ACAMP-81 phosphorylated by each kinase revealed that there are two classes of phosphorylatable polypeptide, one is phosphorylatable by both protein kinases which contained two polypeptides and the others are specific sites for protein kinase C.     

Differential modulation of Kv4.2 and Kv4.3 channels by calmodulin-dependent protein kinase II in rat cardiac myocytes

In this work we have combined biochemical and electrophysiological approaches to explore the modulation of rat ventricular transient outward K(+) current (I(to)) by calmodulin kinase II (CaMKII). Intracellular application of CaMKII inhibitors KN93, calmidazolium, and autocamtide-2-related inhibitory peptide II (ARIP-II) accelerated the inactivation of I(to), even at low [Ca(2+)]. In the same conditions, CaMKII coimmunoprecipitated with Kv4.3 channels, suggesting that phosphorylation of Kv4.3 channels modulate inactivation of I(to). Because channels underlying I(to) are heteromultimers of Kv4.2 and Kv4.3, we have explored the effect of CaMKII on human embryonic kidney (HEK) cells transfected with either of those Kvalpha-subunits. Whereas Kv4.3 inactivated faster upon inhibition of CaMKII, Kv4.2 inactivation was insensitive to CaMKII inhibitors. However, Kv4.2 inactivation became slower when high Ca(2+) was used in the pipette or when intracellular [Ca(2+)] ([Ca(2+)](i)) was transiently increased. This effect was inhibited by KN93, and Western blot analysis demonstrated Ca(2+)-dependent phosphorylation of Kv4.2 channels. On the contrary, CaMKII coimmunoprecipitated with Kv4.3 channels without a previous Ca(2+) increase, and the association was inhibited by KN93. These results suggest that both channels underlying I(to) are substrates of CaMKII, although with different sensitivities; Kv4.2 remain unphosphorylated unless [Ca(2+)](i) increases, whereas Kv4.3 are phosphorylated at rest. In addition to the functional impact that phosphorylation of Kv4 channels could cause on the shape of action potential, association of CaMKII with Kv4.3 provides a new role of Kv4.3 subunits as molecular scaffolds for concentrating CaMKII in the membrane, allowing Ca(2+)-dependent modulation by this enzyme of the associated Kv4.2 channels.     

Substrate-selective and calcium-independent activation of CaMKII by α-actinin

Protein-protein interactions are thought to modulate the efficiency and specificity of Ca(2+)/calmodulin (CaM)-dependent protein kinase II (CaMKII) signaling in specific subcellular compartments. Here we show that the F-actin-binding protein α-actinin targets CaMKIIα to F-actin in cells by binding to the CaMKII regulatory domain, mimicking CaM. The interaction with α-actinin is blocked by CaMKII autophosphorylation at Thr-306, but not by autophosphorylation at Thr-305, whereas autophosphorylation at either site blocks Ca(2+)/CaM binding. The binding of α-actinin to CaMKII is Ca(2+)-independent and activates the phosphorylation of a subset of substrates in vitro. In intact cells, α-actinin selectively stabilizes CaMKII association with GluN2B-containing glutamate receptors and enhances phosphorylation of Ser-1303 in GluN2B, but inhibits CaMKII phosphorylation of Ser-831 in glutamate receptor GluA1 subunits by competing for activation by Ca(2+)/CaM. These data show that Ca(2+)-independent binding of α-actinin to CaMKII differentially modulates the phosphorylation of physiological targets that play key roles in long-term synaptic plasticity.     

A specific 70K protein found in epileptic rat cortex: induction of bursting activity and negative resistance by its intracellular application in Euhadra neurons

The effect of 70-kD protein (P70, a specific protein found in cobalt-induced epileptogenic focus of rat cerebral cortex) on membrane properties was examined in identified neurons of the snail, Euhadra peliomphala, using the pressure injection method combined with the voltage-clamp technique. In neurons that normally exhibited spontaneous regular firing, intracellular injection of P70 elicited bursting activity and a negative slope resistance (NSR) region in their current-voltage (I-V) curve in a manner corresponding to the duration of its injection. These responses were suppressed by prior injection of an antibody to P70 into the neurons, and were markedly inhibited by a reduction of extracellular Na+ ions and the anticonvulsant agent phenytoin, but not by Co(2+)-substituted Ca(2+)-free saline. In addition, intracellularly applied P70 potentiated both bursting activity and the NSR induced by a Na channel activator, veratridine. However, prior application of a saturating dose of this activator occluded the effect of P70. These results suggest that P70 elicits a Na(+)-dependent negative resistance, which may contribute to the generation of bursting activity.     

Phosphorylation of ryanodine receptors

Both cardiac and skeletal muscle ryanodine receptors (RyRs) are parts of large complexes that include a number of kinases and phosphatases. These RyRs have several potential phosphorylation sites in their cytoplasmic domains, but the functional consequences of phosphorylation and the identity of the enzymes responsible have been subjects of considerable controversy. Hyperphosphorylation of Ser-2809 in RyR2 (cardiac isoform) and Ser-2843 in RyR1 (skeletal isoform) has been suggested to cause the dissociation of the FK506-binding protein (FKBP) from RyRs, producing "leaky channels," but some laboratories find no relationship between phosphorylation and FKBP binding. Also debated is the identity of the kinases that phosphorylate these serines: cAMP-dependent protein kinase (PKA) versus calmodulin kinase II (CaMKII). Phosphorylation of other targets of these kinases could also alter calcium homeostasis. For example, PKA also phosphorylates phospholamban (PLB), altering the Sarco-endoplasmic reticulum Ca2+ ATPase (SERCA) activity. This review summarizes the major findings and controversies associated with phosphorylation of RyRs.     

Characterization of endogenous protein phosphorylation in isolated rat liver lysosomes

Membranes prepared from highly purified rat liver lysosomes contain endogenous protein-phosphorylation activities. The transfer of phosphate to membrane fractions from [gamma-32P]ATP was analyzed by gel electrophoresis under acidic denaturing conditions. Two phosphopeptides were detected, with molecular weights of 3,000 and 14,000. Phosphorylation of these proteins was unaffected by the addition of cAMP, cGMP, or the heat-stable inhibitor of cAMP-dependent protein kinase. No additional phosphorylation was observed when cAMP-dependent protein kinase was included in the reaction or when exogenous protein kinase substrates were added. The 14,000-dalton 32P-labeled product was formed rapidly in the presence of low concentrations (250 microM) of either Ca2+ or Mg2+. This product was labile under both acidic and alkaline conditions, suggesting that this protein contains an acyl phosphate, present presumably as a catalytic intermediate in a phosphotransferase reaction. The lower molecular weight species required a high concentration (5 mM) of Mg2+ for phosphorylation, and micromolar concentrations of Ca2+ stimulated the Mg2+-dependent activity. The addition of Ca2+ and calmodulin stimulated the phosphorylation reaction to a greater extent than with Ca2+ alone. This activity was strongly inhibited by 0.2 mM LaCl3 and to a lesser extent by 50 microM chlorpromazine or trifluoperazine. These results suggest that the 3000-dalton peptide may be phosphorylated by a Ca2+, calmodulin-dependent kinase associated with the lysosomal membrane.     

Effects of cAMP- and cGMP-dependent protein kinases, and calmodulin on Ca2+ uptake by highly purified sarcolemmal vesicles of vascular smooth muscle

Sarcolemmal fractions of vascular smooth muscles were prepared from porcine thoracic aortae by differential and sucrose density gradient centrifugation. In these fractions, there was a high activity of 5'-nucleotidase, a putative marker enzyme of plasma membrane, and a low activity of rotenone insensitive NADH-cytochrome c reductase a marker of sarcoplasmic reticulum. In these fractions, the Ca2+ uptake was ATP-dependent. A low concentration of saponin which inhibited Ca2+ uptake by the plasma membrane but not by the sarcoplasmic reticulum, inhibited 65% of the Ca2+ uptake of this fraction. The Ca2+ uptake of this fraction was enhanced by cAMP- and cGMP-dependent protein kinases, and by calmodulin. The cAMP-dependent protein kinase enhanced the phosphorylation of 28 and 22 kDa proteins, while the cGMP-dependent protein kinase phosphorylated the 35 kDa protein. The phosphorylation of 100, 75, 65, 41 and 22 kDa proteins was enhanced by Ca2+ and calmodulin. These results indicate that cAMP- and cGMP-dependent protein kinases as well as calmodulin play important roles in Ca2+ transport in the sarcolemma, and that the phosphorylated proteins may be associated with an enhancement of Ca2+ transport in the sarcolemma.     

Calmodulin-mediated regulation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity in isolated cardiac sarcoplasmic reticulum

A severalfold activation of calcium transport and (Ca2+ + Mg2+)-activated ATPase activity by micromolar concentrations of calmodulin was observed in sarcoplasmic reticulum vesicles obtained from canine ventricles. This activation was seen in the presence of 120 mM KCl. The ratio of moles of calcium transported per mol of ATP hydrolyzed remained at about 0.75 when calcium transport and (Ca2+ + Mg2+)-activated ATPase activity were measured in the presence and absence of calmodulin. Thus, the efficiency of the calcium transport process did not change. Stimulation of calcium transport by calmodulin involves the phosphorylation of one or more proteins. The major 32P-labeled protein, as determined by sodium dodecyl sulfate slab gel electrophoresis, was the 22,000-dalton protein called phospholamban. The Ca2+ concentration dependency of calmodulin-stimulated microsomal phosphorylation corresponded to that of calmodulin-stimulated (Ca2+ + Mg2+)-activated ATPase activity. Proteins of 11,000 and 6,000 daltons and other proteins were labeled to a lesser extent. A similar phosphorylation pattern was obtained when microsomes were incubated with cAMP-dependent protein kinase and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. Phosphorylation produced by added cAMP-dependent protein kinase and calmodulin was additive. These studies provided further evidence for Ca2+-dependent regulation of calcium transport by calmodulin in sarcoplasmic reticulum that could play a role in the beat-to-beat regulation of cardiac relaxation in the intact heart.     

Ca2+-dependent K+ permeability of heart sarcolemmal vesicles. Modulation by cAMP-dependent protein kinase activity and by calmodulin

The Ca2+-dependent K+ permeability of heart sarcolemma vesicles was measured by following the transmembrane movement of the charge compensating tetraphenylborate anion. The increase in vesicles permeability induced by Ca2+ is lost when membrane proteins are dephosphorylated by an endogenous protein phosphatase and is restored by a phosphorylation process catalysed by a cAMP-dependent protein kinase. The calmodulin antagonist R 24571 lowers the Ca2+-dependent K+ permeability by decreasing the Ca2+ affinity of the K+ transporting system.