Effects of the Arg9Cys and Arg25Cys mutations on phospholamban's conformational equilibrium in membrane bilayers

Approximately, 70% of the Ca²⁺ ion transport into the sarcoplasmic reticulum is catalyzed by the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), whose activity is endogenously regulated by phospholamban (PLN). PLN comprises a TM inhibitory region and a cytoplasmic regulatory region that harbors a consensus sequence for cAMP-dependent protein kinase (PKA). The inhibitory region binds the ATPase, reducing its apparent Ca²⁺ binding affinity. β-adrenergic stimulation activates PKA, which phosphorylates PLN at Ser 16, reversing its inhibitory function. Mutations and post-translational modifications of PLN may lead to dilated cardiomyopathy (DCM) and heart failure. PLN's cytoplasmic region interconverts between a membrane-associated T state and a membrane-detached R state. The importance of these structural transitions on SERCA regulation is emerging, but the effects of natural occurring mutations and their relevance to the progression of heart disease are unclear. Here we use solid-state NMR spectroscopy to investigate the structural dynamics of two lethal PLN mutations, R9C and R25C, which lead to DCM. We found that the R25C mutant enhances the dynamics of PLN and shifts the conformational equilibrium toward the R state confirmation, whereas the R9C mutant drives the amphipathic cytoplasmic domain toward the membrane-associate state, enriching the T state population. The changes in membrane interactions caused by these mutations may explain the aberrant regulation of SERCA.     

Interaction of a Sarcolipin Pentamer and Monomer with the Sarcoplasmic Reticulum Calcium Pump,SERCA

The sequential rise and fall of cytosolic calcium underlies the contraction-relaxation cycle of muscle cells. Whereas contraction is initiated by the release of calcium from the sarcoplasmic reticulum, muscle relaxation involves the active transport of calcium back into the sarcoplasmic reticulum. This reuptake of calcium is catalyzed by the sarcoendoplasmic reticulum Ca²⁺-ATPase (SERCA), which plays a lead role in muscle contractility. The activity of SERCA is regulated by small membrane protein subunits, the most well-known being phospholamban (PLN) and sarcolipin (SLN). SLN physically interacts with SERCA and differentially regulates contractility in skeletal and atrial muscle. SLN has also been implicated in skeletal muscle thermogenesis. Despite these important roles, the structural mechanisms by which SLN modulates SERCA-dependent contractility and thermogenesis remain unclear. Here, we functionally characterized wild-type SLN and a pair of mutants, Asn⁴-Ala and Thr⁵-Ala, which yielded gain-of-function behavior comparable to what has been found for PLN. Next, we analyzed two-dimensional crystals of SERCA in the presence of wild-type SLN by electron cryomicroscopy. The fundamental units of the crystals are antiparallel dimer ribbons of SERCA, known for decades as an assembly of calcium-free SERCA molecules induced by the addition of decavanadate. A projection map of the SERCA-SLN complex was determined to a resolution of 8.5 Å, which allowed the direct visualization of an SLN pentamer. The SLN pentamer was found to interact with transmembrane segment M3 of SERCA, although the interaction appeared to be indirect and mediated by an additional density consistent with an SLN monomer. This SERCA-SLN complex correlated with the ability of SLN to decrease the maximal activity of SERCA, which is distinct from the ability of PLN to increase the maximal activity of SLN. Protein-protein docking and molecular dynamics simulations provided models for the SLN pentamer and the novel interaction between SERCA and an SLN monomer.     

Lack of effects of calcium · calmodulin-dependent phosphorylation on Ca2+ release from cardiac sarcoplasmic reticulum

Canine cardiac sarcoplasmic reticulum is phosphorylated by an endogenous calcium · calmodulin-dependent protein kinase and phosphorylation occurs mainly on a 27 kDa proteolipid, called phospholamban. To determine whether this phosphorylation has any effect on Ca²⁺ release, sarcoplasmic reticulum vesicles were phosphorylated by the calcium · calmodulin-dependent protein kinase, while non-phosphorylated vesicles were preincubated under identical conditions but in the absence of ATP to avoid phosphorylation. Both non-phosphorylated and phosphorylated vesicles were centrifuged to remove calmodulin, and subsequently used for Ca²⁺ release studies. Calcium loading was carried out either by the active calcium pump or by incubation with high (5 mM) calcium for longer periods. Phosphorylation of sarcoplasmic reticulum by calcium · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca²⁺ released from cardiac sarcoplasmic reticulum vesicles loaded under passive conditions and on the apparent ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange from cardiac sarcoplasmic reticulum vesicles loaded under active conditions. Thus, it appears that calcium · calmodulin-dependent protein kinase mediated phosphorylation of cardiac sarcoplasmic reticulum is not involved in the regulation of Ca²⁺ release and ⁴⁵Ca²⁺ − ⁴⁰Ca²⁺ exchange.     

The Phospholamban Pentamer Alters Function of the Sarcoplasmic Reticulum Calcium Pump SERCA

The interaction of phospholamban (PLN) with the sarco-endoplasmic reticulum Ca²⁺-ATPase (SERCA) pump is a major regulatory axis in cardiac muscle contractility. The prevailing model involves reversible inhibition of SERCA by monomeric PLN and storage of PLN as an inactive pentamer. However, this paradigm has been challenged by studies demonstrating that PLN remains associated with SERCA and that the PLN pentamer is required for the regulation of cardiac contractility. We have previously used two-dimensional (2D) crystallization and electron microscopy to study the interaction between SERCA and PLN. To further understand this interaction, we compared small helical crystals and large 2D crystals of SERCA in the absence and presence of PLN. In both crystal forms, SERCA molecules are organized into identical antiparallel dimer ribbons. The dimer ribbons pack together with distinct crystal contacts in the helical versus large 2D crystals, which allow PLN differential access to potential sites of interaction with SERCA. Nonetheless, we show that a PLN oligomer interacts with SERCA in a similar manner in both crystal forms. In the 2D crystals, a PLN pentamer interacts with transmembrane segments M3 of SERCA and participates in a crystal contact that bridges neighboring SERCA dimer ribbons. In the helical crystals, an oligomeric form of PLN also interacts with M3 of SERCA, though the PLN oligomer straddles a SERCA-SERCA crystal contact. We conclude that the pentameric form of PLN interacts with M3 of SERCA and that it plays a distinct structural and functional role in SERCA regulation. The interaction of the pentamer places the cytoplasmic domains of PLN at the membrane surface proximal to the calcium entry funnel of SERCA. This interaction may cause localized perturbation of the membrane bilayer as a mechanism for increasing the turnover rate of SERCA.     

Comparing the structure and dynamics of phospholamban pentamer in its unphosphorylated and pseudo-phosphorylated states

Human phospholamban (PLN), a 30 kDa homopentamer in the sarcoplasmic reticulum (SR) membrane, controls the magnitude of heart muscle contraction and relaxation by regulating the calcium pumping activity of the SR Ca(2+)-ATPase (SERCA). When PLN is not phosphorylated, it binds and inhibits SERCA. Phosphorylation of PLN at S16 or T17 releases such inhibitory effect. It remains a matter of debate whether phosphorylation perturbs the structure of PLN, which in turn affects its interaction with SERCA. Here we examine by NMR spectroscopy the structure and dynamics of PLN pentamer with a physiologically relevant, phosphorylation-mimicking mutation, S16E. Based on extensive NMR data, including NOEs, dipolar couplings, and solvent exchange of backbone amides, we conclude that the phosphorylation-mimicking mutation does not perturb the pentamer structure. However, (15)N R(1) and R(2) relaxation rates and (15)N((1)H) NOEs suggest subtle differences in the dynamics of the extramembrane portion of the protein.     

Structure, Dynamics, and Ion Conductance of the Phospholamban Pentamer

A 52-residue membrane protein, phospholamban (PLN) is an inhibitor of an adenosine-5′-triphosphate-driven calcium pump, the Ca²⁺-ATPase. Although the inhibition of Ca²⁺-ATPase involves PLN monomers, in a lipid bilayer membrane, PLN monomers form stable pentamers of unknown biological function. The recent NMR structure of a PLN pentamer depicts cytoplasmic helices extending normal to the bilayer in what is known as the bellflower conformation. The structure shows transmembrane helices forming a hydrophobic pore 4 Å in diameter, which is reminiscent of earlier reports of possible ion conductance through PLN pentamers. However, recent FRET measurements suggested an alternative structure for the PLN pentamer, known as the pinwheel model, which features a narrower transmembrane pore and cytoplasmic helices that lie against the bilayer. Here, we report on structural dynamics and conductance properties of the PLN pentamers from all-atom (AA) and coarse-grained (CG) molecular dynamics simulations. Our AA simulations of the bellflower model demonstrate that in a lipid bilayer membrane or a detergent micelle, the cytoplasmic helices undergo large structural fluctuations, whereas the transmembrane pore shrinks and becomes asymmetric. Similar asymmetry of the transmembrane region was observed in the AA simulations of the pinwheel model; the cytoplasmic helices remained in contact with the bilayer. Using the CG approach, structural dynamics of both models were investigated on a microsecond timescale. The cytoplasmic helices of the CG bellflower model were observed to fall against the bilayer, whereas in the CG pinwheel model the conformation of the cytoplasmic helices remained stable. Using steered molecular dynamics simulations, we investigated the feasibility of ion conductance through the pore of the bellflower model. The resulting approximate potentials of mean force indicate that the PLN pentamer is unlikely to function as an ion channel.     

Mechanism of Inhibition of Calcium Uptake into Sarcoplasmic Reticulum by Notexin,a Neurotoxic and Myotoxic Polypeptide

Notexin belongs to a class of snake venom neurotoxins and myotoxins that have phospholipase A₂ activity. Previous studies have shown that these toxins affect target cells differently from phospholipases that are not neurotoxic or myotoxic. Notexin inhibited the Ca²⁺ uptake into fragmented sarcoplasmic reticulum from rabbit skeletal muscle, but it did not cause an efflux of previously accumulated Ca²⁺ or inhibit the Ca²⁺–ATPase activity. It is suggested that notexin specifically binds to and decreases the conductance for Ca²⁺ of the Ca²⁺ pump and/or the conductance of a channel for an ion that facilitates Ca²⁺ transport. The K⁺ ionophore valinomycin reversed the notexin-induced inhibition of Ca²⁺ uptake into sarcoplasmic reticulum, suggesting that the molecular target of notexin could be a K⁺ channel. Two types of reconstitution experiments make it unlikely that notexin acts by degrading a minor lipid that is resistant to hydrolysis by nontoxic phospholipases A₂. Notexininactivated sarcoplasmic reticulum vesicles were reactivated (with respect to Ca²⁺ uptake) by simple solubilization with detergent and subsequent reconstitution by detergent removal. Second, notexin was still active on sarcoplasmic reticulum vesicles after >94% of the lipids were replaced by soybean phosphoglycerides during the reconstitution procedure.     

Can proton pumping by SERCA enhance the regulatory role of phospholamban and sarcolipin?

The effect of the incorporation of phosphorylated phospholamban (pPLN) and sarcolipin (SLN) in mercury-supported self-assembled lipid monolayers and in lipid bilayers tethered to mercury via a hydrophilic spacer was investigated by voltammetric techniques and electrochemical impedance spectroscopy. It was shown that pPLN and SLN do not permeabilize lipid bilayers toward ions at physiological pH. However, they exert a permeabilizing action toward inorganic monovalent cations such as K⁺ and Tl⁺, but not toward divalent cations such as Ca²⁺ and Cd^2 +, following a small decrease in pH. This behavior can be associated with their regulatory action on the Ca-ATPase of the sarcoplasmic reticulum (SERCA). SERCA pumps two Ca²⁺ ions from the cytosol to the lumen of the sarcoplasmic reticulum (SR) and two protons in the opposite direction, causing a transient decrease of pH in the immediate vicinity of its cytoplasmic domain. This decrease is expected to activate the liberated pPLN molecules and SLN to make the SR membrane leakier toward K⁺ and Na⁺ and the SLN ion channel to translocate small inorganic anions, such as Cl⁻. The effect of pPLN and SLN, which becomes synergic when they are both present in the SR membrane, is expected to favor a rapid equilibration of ions on both sides of the membrane.     

Structural constraints on the transmembrane and juxtamembrane regions of the phospholamban pentamer in membrane bilayers: Gln29 and Leu52

The Ca²⁺-ATPase of cardiac muscle cells transports Ca²⁺ ions against a concentration gradient into the sarcoplasmic reticulum and is regulated by phospholamban, a 52-residue integral membrane protein. It is known that phospholamban inhibits the Ca²⁺ pump during muscle contraction and that inhibition is removed by phosphorylation of the protein during muscle relaxation. Phospholamban forms a pentameric complex with a central pore. The solid-state magic angle spinning (MAS) NMR measurements presented here address the structure of the phospholamban pentamer in the region of Gln22-Gln29. Rotational echo double resonance (REDOR) NMR measurements show that the side chain amide groups of Gln29 are in close proximity, consistent with a hydrogen-bonded network within the central pore. ¹³C MAS NMR measurements are also presented on phospholamban that is 1-¹³C-labeled at Leu52, the last residue of the protein. pH titration of the C-terminal carboxyl group suggests that it forms a ring of negative charge on the lumenal side of the sarcoplasmic reticulum membrane. The structural constraints on the phospholamban pentamer described in this study are discussed in the context of a multifaceted mechanism for Ca²⁺ regulation that may involve phospholamban as both an inhibitor of the Ca²⁺ ATPase and as an ion channel.     

Co-Expression of SERCA Isoforms,Phospholamban and Sarcolipin in Human Skeletal Muscle Fibers

Sarcolipin (SLN) and phospholamban (PLN) inhibit the activity of sarco(endo)plasmic reticulum Ca²⁺-ATPases (SERCAs) by reducing their apparent affinity for Ca²⁺. A ternary complex between SLN, PLN, and SERCAs results in super-inhibition of SERCA activity. Analysis of skeletal muscle homogenate has limited our current understanding of whether SLN and PLN regulate SERCA1a, SERCA2a, or both in skeletal muscle and whether SLN and PLN are co-expressed in skeletal muscle fibers. Biopsies from human vastus lateralis were analyzed through single fiber Western blotting and immunohisto/fluorescence staining to circumvent this limitation. With a newly generated SLN antibody, we report for the first time that SLN protein is present in human skeletal muscle. Addition of the SLN antibody (50 µg) to vastus lateralis homogenates increased the apparent Ca²⁺ affinity of SERCA (K _Ca, pCa units) (-Ab, 5.85 ± 0.02 vs. +Ab, 5.95 ± 0.02) and maximal SERCA activity (μmol/g protein/min) (-Ab, 122 ± 6.4 vs. +Ab, 159 ± 11) demonstrating a functional interaction between SLN and SERCAs in human vastus lateralis. Specifically, our results suggest that although SLN and PLN may preferentially regulate SERCA1a, and SERCA2a, respectively, physiologically they both may regulate either SERCA isoform. Furthermore, we show that SLN and PLN co-immunoprecipitate in human vastus lateralis homogenate and are simultaneously expressed in 81% of the fibers analyzed with Western blotting which implies that super-inhibition of SERCA may exist in human skeletal muscle. Finally, we demonstrate unequivocally that mouse soleus contains PLN protein suggesting that super-inhibition of SERCA may also be important physiologically in rodent skeletal muscle.     

cAMP-dependent protein kinase A selects the excited state of the membrane substrate phospholamban

Phosphorylation of membrane proteins is a central regulatory and signaling mechanism across cell compartments. However, the recognition process and phosphorylation mechanism of membrane-bound substrates by kinases are virtually unknown. cAMP-dependent protein kinase A (PKA) is a ubiquitous enzyme that phosphorylates several soluble and membrane-bound substrates. In cardiomyocytes, PKA targets phospholamban (PLN), a membrane protein that inhibits the sarcoplasmic reticulum Ca²⁺-ATPase (SERCA). In the unphosphorylated state, PLN binds SERCA, reducing the calcium uptake and generating muscle contraction. PKA phosphorylation of PLN at S16 in the cytoplasmic helix relieves SERCA inhibition, initiating muscle relaxation. Using steady-state kinetic assays, NMR spectroscopy, and molecular modeling, we show that PKA recognizes and phosphorylates the excited, membrane-detached R-state of PLN. By promoting PLN from a ground state to an excited state, we obtained a linear relationship between rate of phosphorylation and population of the excited state of PLN. The conformational equilibrium of PLN is crucial to regulate the extent of PLN phosphorylation and SERCA inhibition.     

Early calcium handling imbalance in pressure overload-induced heart failure with nearly normal left ventricular ejection fraction

Heart failure with preserved ejection fraction (HFpEF) is a common clinical syndrome associated with high morbidity and mortality. Therapeutic options are limited due to a lack of knowledge of the pathology and its evolution. We investigated the cellular phenotype and Ca²⁺ handling in hearts recapitulating HFpEF criteria. HFpEF was induced in a portion of male Wistar rats four weeks after abdominal aortic banding. These animals had nearly normal ejection fraction and presented elevated blood pressure, lung congestion, concentric hypertrophy, increased LV mass, wall stiffness, impaired active relaxation and passive filling of the left ventricle, enlarged left atrium, and cardiomyocyte hypertrophy. Left ventricular cell contraction was stronger and the Ca²⁺ transient larger. Ca²⁺ cycling was modified with a RyR2 mediated Ca²⁺ leak from the sarcoplasmic reticulum and impaired Ca²⁺ extrusion through the Sodium/Calcium exchanger (NCX), which promoted an increase in diastolic Ca²⁺. The Sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA2a) and NCX protein levels were unchanged. The phospholamban (PLN) to SERCA2a ratio was augmented in favor of an inhibitory effect on the SERCA2a activity. Conversely, PLN phosphorylation at the calmodulin-dependent kinase II (CaMKII)-specific site (PLN-Thr17), which promotes SERCA2A activity, was increased as well, suggesting an adaptive compensation of Ca²⁺ cycling. Altogether our findings show that cardiac remodeling in hearts with a HFpEF status differs from that known for heart failure with reduced ejection fraction. These data also underscore the interdependence between systolic and diastolic “adaptations” of Ca²⁺ cycling with complex compensative interactions between Ca²⁺ handling partner and regulatory proteins.     

The potassium channel opener NS1619 modulates calcium homeostasis in muscle cells by inhibiting SERCA

NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) is widely used as a large-conductance Ca²⁺-activated K⁺ (BK_Ca) channel opener. It was previously reported that activation of BK_Ca channels by NS1619 could protect the cardiac muscle against ischaemia and reperfusion injury. This study reports the effects of NS1619 on intracellular Ca²⁺ homeostasis in H9C2 and C2C12 cells as well as its molecular mechanism of action. The effects of NS1619 on Ca²⁺ homeostasis in C2C12 and H9C2 cells were assessed using the Fura-2 fluorescence method. Ca²⁺ uptake by sarcoplasmic reticulum (SR) vesicles isolated from rat skeletal muscles and sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA) activity were measured. The effect of NS1619 on the isometric force of papillary muscle contraction in the guinea pig heart was also examined. H9C2 and C2C12 cells treated with NS1619 released Ca²⁺ from internal stores in a concentration-dependent manner. Ca²⁺ accumulation by the SR vesicles was inhibited by NS1619 treatment. NS1619 also decreased the activity of SERCA derived from rat skeletal muscle. The calcium release from cell internal stores and inhibition of SERCA by NS1619 are pH dependent. Finally, NS1619 had a profound effect on the isometric force of papillary muscle contraction in the guinea pig heart. These results indicate that NS1619 is a potent modulator of the intracellular Ca²⁺ concentration in H9C2 and C1C12 cells due to its interaction with SRs. The primary target of NS1619 is SERCA, which is located in SR vesicles. The effect of NS1619-mediated SERCA inhibition on cytoprotective processes should be considered.     

Interaction sites among phospholamban, sarcolipin, and the sarco(endo)plasmic reticulum Ca(2+)-ATPase

A robust cross-link between Gln²³ in phospholamban (PLN) and Lys³²⁸ in the sarco(endo)plasmic reticulum Ca²⁺ ATPase (SERCA1a) is formed in the presence or absence of oxidant and is susceptible to both PLN phosphorylation and SERCA1a Ca²⁺ binding. This cross-link provides precisely the evidence needed to support our earlier proposal that collision of the PLN transmembrane helix at Asn²⁷ with the cytosolic extension of M4 at Leu³²¹ leads to unwinding of the helix. In a study of site-specific interactions among PLN, sarcolipin (SLN), and SERCA1a, we determined that mutations of some specific amino acids in PLN or SLN diminish either the super-inhibition imposed on SERCA1a function by the PLN-SLN binary complex or the physical interactions between PLN and SLN or both. These results have led to a revision of our earlier model for the PLN-SLN-SERCA1a complex.     

Thermogenic activity of Ca-ATPase from skeletal muscle heavy sarcoplasmic reticulum: The role of ryanodine Ca channel

The sarcoplasmic reticulum Ca²⁺ ATPase 1 (SERCA 1) is able to handle the energy derived from ATP hydrolysis in such a way as to determine the parcel of energy that is used for Ca²⁺ transport and the fraction that is converted into heat. In this work we measured the heat production by SERCA 1 in the two sarcoplasmic reticulum (SR) fractions: the light fraction (LSR), which is enriched in SERCA and the heavy fraction (HSR), which contains both the SERCA and the ryanodine Ca²⁺ channel. We verified that although HSR cleaved ATP at faster rate than LSR, the amount of heat released during ATP hydrolysis by HSR was smaller than that measured by LSR. Consequently, the amount of heat released per mol of ATP cleaved (ΔH^cal) by HSR was lower compared to LSR. In HSR, the addition of 5 mM Mg²⁺ or ruthenium red, conditions that close the ryanodine Ca²⁺ channel, promoted a decrease in the ATPase activity, but the amount of heat released during ATP hydrolysis remained practically the same. In this condition, the ΔH^cal values of ATP hydrolysis increased significantly. Neither Mg²⁺ nor ruthenium red had effect on LSR. Thus, we conclude that heat production by SERCA 1 depends on the region of SR in which the enzyme is inserted and that in HSR, the ΔH^cal of ATP hydrolysis by SERCA 1 depends on whether the ryanodine Ca²⁺ channel is opened or closed.     

On the Function of Pentameric Phospholamban: Ion Channel or Storage Form?

Phospholamban (PLN) is an integral membrane protein that inhibits the sarcoplasmic reticulum Ca²⁺-ATPase, thereby regulating muscle contractility. We report a combined electrochemical and theoretical study demonstrating that the pentameric PLN does not possess channel activity for conducting chloride or calcium ions across the lipid membrane. This suggests that the pentameric configuration of PLN primarily serves as a storage form for the regulatory function of muscle relaxation by the PLN monomer.     

The anti-apoptotic protein HAX-1 interacts with SERCA2 and regulates its protein levels to promote cell survival

Cardiac contractility is regulated through the activity of various key Ca²⁺-handling proteins. The sarco(endo)plasmic reticulum (SR) Ca²⁺ transport ATPase (SERCA2a) and its inhibitor phospholamban (PLN) control the uptake of Ca²⁺ by SR membranes during relaxation. Recently, the antiapoptotic HS-1–associated protein X-1 (HAX-1) was identified as a binding partner of PLN, and this interaction was postulated to regulate cell apoptosis. In the current study, we determined that HAX-1 can also bind to SERCA2. Deletion mapping analysis demonstrated that amino acid residues 575–594 of SERCA2's nucleotide binding domain are required for its interaction with the C-terminal domain of HAX-1, containing amino acids 203-245. In transiently cotransfected human embryonic kidney 293 cells, recombinant SERCA2 was specifically targeted to the ER, whereas HAX-1 selectively concentrated at mitochondria. On triple transfections with PLN, however, HAX-1 massively translocated to the ER membranes, where it codistributed with PLN and SERCA2. Overexpression of SERCA2 abrogated the protective effects of HAX-1 on cell survival, after hypoxia/reoxygenation or thapsigargin treatment. Importantly, HAX-1 overexpression was associated with down-regulation of SERCA2 expression levels, resulting in significant reduction of apparent ER Ca²⁺ levels. These findings suggest that HAX-1 may promote cell survival through modulation of SERCA2 protein levels and thus ER Ca²⁺ stores.     

An Energy-Barrier Model for the Permeation of Monovalent and Divalent Cations Through the Maxi Cation Channel in the Plasma Membrane of Rye Roots

The depolarization-activated, high-conductance ``maxi' cation channel in the plasma membrane of rye (Secale cereale L.) roots is permeable to a wide variety of monovalent and divalent cations. The permeation of K⁺, Na⁺, Ca²⁺ and Ba²⁺ through the pore could be simulated using a model composed of three energy barriers and two ion binding sites (a 3B2S model), which assumed single-file permeation and the possibility of double cation occupancy. The model had an asymmetrical free energy profile. Differences in permeation between cations were attributed primarily to differences in their free energy profiles in the regions of the pore adjacent to the extracellular solution. In particular, the height of the central free energy peak differed between cations, and cations differed in their affinities for ion binding sites. Significant ion repulsion occurred within the pore, and the mouths of the pore had considerable surface charge. The model adequately described the diverse current vs. voltage (I/V) relationships obtained over a wide variety of experimental conditions. It described the phenomena of non-Michaelian unitary conductance vs. activity relationships for K⁺, Na⁺ and Ca²⁺, differences in selectivity sequences obtained from measurements of conductance and permeability ratios, changes in relative cation permeabilities with solution composition, and the complex effects of Ba²⁺ and Ca²⁺ on K⁺ currents through the channel. The model enabled the prediction of unitary currents and ion fluxes through the maxi cation channel under physiological conditions. It could be used, in combination with data on the kinetics of the channel, as input to electrocoupling models allowing the relationships between membrane voltage, Ca²⁺ influx and Ca²⁺ signaling to be studied theoretically. Received: 29 April 1998/Revised: 20 November 1998     

Characterization of calmodulin-mediated phosphorylation of cardiac muscle sarcoplasmic reticulum

Sarcoplasmic reticulum, isolated from canine cardiac muscle, was phosphorylated in the presence of exogenous cAMP-dependent protein kinase or calmodulin. This phosphorylation has been shown previously to activate sarcoplasmic reticulum calcium uptake (LePeuch et al. (1979) Biochemistry18, 5150–5157). Calmodulin appeared to activate an endogenous protein kinase present in sarcoplasmic reticulum membranes. The incorporation of phosphate increased with time. However, once all the ATP was consumed, the level of phosphorylated protein started to decrease due to the action of an endogenous protein phosphatase. Dephosphorylation occurred even when the level of phosphorylated sarcoplasmic reticulum remained constant at high ATP concentrations. The phosphorylation of sarcoplasmic reticulum in the presence of calmodulin, increased as the pH was increased from pH 5.5 to 8.5. This phosphorylation was only inhibited by KCl concentrations greater than 100 mm. The apparent K_m of cAMP-dependent protein kinase for ATP was 5.2 ± 0.2 × 10^?5m, and of the calmodulin-dependent protein kinase for ATP was 3.67 ± 0.29 × 10^?5m. Phosphorylation was maximally activated by 5–10 mm MgCl₂; higher MgCl₂ concentrations inhibited this phosphorylation. Thus the calmodulin-dependent phosphorylation of cardiac sarcoplasmic reticulum could be maximally activated at sarcoplasmic concentrations of K⁺, Mg²⁺, and ATP. The calmodulindependent phosphorylation was half-maximally activated at Ca²⁺ concentrations that were significantly greater than those required to promote the formation of the sarcoplasmic reticulum Ca-activated ATPase phosphoprotein intermediate. Thus at sarcoplasmic Ca²⁺ concentrations that might be expected during systole, the sarcoplasmic reticulum calcium pump would be fully activated before any significant calmodul-independent sarcoplasmic reticulum phosphorylation occurred. However, under certain pathological conditions when the sarcoplasmic Ca²⁺ becomes elevated (e.g., in ischemia) the kinase could be activated so that the sarcoplasmic reticulum would be phosphorylated and calcium uptake augmented. Thus, the calmodulin-dependent protein kinase may only function when the heart needs to rescue itself from a possibly fatal calcium overload.     

Effects of monovalent cations on Ca uptake by skeletal and cardiac muscle sarcoplasmic reticulum

Ca²⁺ transport by the sarcoplasmic/endoplasmic reticulum Ca²⁺ ATPase (SERCA) is sensitive to monovalent cations. Possible K⁺ binding sites have been identified in both the cytoplasmic P-domain and the transmembrane transport-domain of the protein. We measured Ca²⁺ transport into SR vesicles and SERCA ATPase activity in the presence of different monovalent cations. We found that the effects of monovalent cations on Ca²⁺ transport correlated in most cases with their direct effects on SERCA. Choline⁺, however, inhibited uptake to a greater extent than could be accounted for by its direct effect on SERCA suggesting a possible effect of choline on compensatory charge movement during Ca²⁺ transport. Of the monovalent cations tested, only Cs⁺ significantly affected the Hill coefficient of Ca²⁺ transport (n_H). An increase in n_H from ∼2 in K⁺ to ∼3 in Cs⁺ was seen in all of the forms of SERCA examined. The effects of Cs⁺ on the maximum velocity of Ca²⁺ uptake were also different for different forms of SERCA but these differences could not be attributed to differences in the putative K⁺ binding sites of the different forms of the protein.