Atomic-Level Mechanisms for Phospholamban Regulation of the Calcium Pump

We performed protein pKa calculations and molecular dynamics (MD) simulations of the calcium pump (sarcoplasmic reticulum Ca²⁺-ATPase (SERCA)) in complex with phospholamban (PLB). X-ray crystallography studies have suggested that PLB locks SERCA in a low-Ca²⁺-affinity E2 state that is incompatible with metal-ion binding, thereby blocking the conversion toward a high-Ca²⁺-affinity E1 state. Estimation of pKa values of the acidic residues in the transport sites indicates that at normal intracellular pH (7.1–7.2), PLB-bound SERCA populates an E1 state that is deprotonated at residues E309 and D800 yet protonated at residue E771. We performed three independent microsecond-long MD simulations to evaluate the structural dynamics of SERCA-PLB in a solution containing 100 mM K⁺ and 3 mM Mg²⁺. Principal component analysis showed that PLB-bound SERCA lies exclusively along the structural ensemble of the E1 state. We found that the transport sites of PLB-bound SERCA are completely exposed to the cytosol and that K⁺ ions bind transiently (≤5 ns) and nonspecifically (nine different positions) to the two transport sites, with a total occupancy time of K⁺ in the transport sites of 80%. We propose that PLB binding to SERCA populates a novel (to our knowledge) E1 intermediate, E1⋅H⁺₇₇₁. This intermediate serves as a kinetic trap that controls headpiece dynamics and depresses the structural transitions necessary for Ca²⁺-dependent activation of SERCA. We conclude that PLB-mediated regulation of SERCA activity in the heart results from biochemical and structural transitions that occur primarily in the E1 state of the pump.     

Distinctive Features of Catalytic and Transport Mechanisms in Mammalian Sarco-endoplasmic Reticulum Ca2+ ATPase (SERCA) and Cu+ (ATP7A/B) ATPases

Ca²⁺ (sarco-endoplasmic reticulum Ca²⁺ ATPase (SERCA)) and Cu⁺ (ATP7A/B) ATPases utilize ATP through formation of a phosphoenzyme intermediate (E-P) whereby phosphorylation potential affects affinity and orientation of bound cation. SERCA E-P formation is rate-limited by enzyme activation by Ca²⁺, demonstrated by the addition of ATP and Ca²⁺ to SERCA deprived of Ca²⁺ (E2) as compared with ATP to Ca²⁺-activated enzyme (E1·2Ca²⁺). Activation by Ca²⁺ is slower at low pH (2H⁺·E2 to E1·2Ca²⁺) and little sensitive to temperature-dependent activation energy. On the other hand, subsequent (forward or reverse) phosphoenzyme processing is sensitive to activation energy, which relieves conformational constraints limiting Ca²⁺ translocation. A “H⁺-gated pathway,” demonstrated by experiments on pH variations, charge transfer, and Glu-309 mutation allows luminal Ca²⁺ release by H⁺/Ca²⁺ exchange. As compared with SERCA, initial utilization of ATP by ATP7A/B is much slower and highly sensitive to temperature-dependent activation energy, suggesting conformational constraints of the headpiece domains. Contrary to SERCA, ATP7B phosphoenzyme cleavage shows much lower temperature dependence than EP formation. ATP-dependent charge transfer in ATP7A and -B is observed, with no variation of net charge upon pH changes and no evidence of Cu⁺/H⁺ exchange. As opposed to SERCA after Ca²⁺ chelation, ATP7A/B does not undergo reverse phosphorylation with P_i after copper chelation unless a large N-metal binding extension segment is deleted. This is attributed to the inactivating interaction of the copper-deprived N-metal binding extension with the headpiece domains. We conclude that in addition to common (P-type) phosphoenzyme intermediate formation, SERCA and ATP7A/B possess distinctive features of catalytic and transport mechanisms.     

Synergism between polyamines and ROS in the induction of Ca2+ and K+ fluxes in roots

Stress conditions cause increases in ROS and polyamines levels, which are not merely collateral. There is increasing evidence for the ROS participation in signaling as well as for polyamine protective roles under stress. Polyamines and ROS, respectively, inhibit cation channels and induce novel cation conductance in the plasma membrane. Our new results indicate that polyamines and OH^• also stimulate Ca²⁺ pumping across the root plasma membrane. Besides, polyamines potentiate the OH^•-induced non-selective current and respective passive K⁺ and Ca²⁺ fluxes. In roots this synergism, however, is restricted to the mature zone, whereas in the distal elongation zone only the Ca²⁺ pump activation is observed. Remodeling the plasma membrane ion conductance by OH^• and polyamines would impact K⁺ homeostasis and Ca²⁺ signaling under stress.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATpase of cardiac sarcoplasmic reticulum

ATP and the divalent cations Mg²⁺ and Ca²⁺ regulated K⁺ stimulation of the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K⁺-stimulated ATPase activity of the Ca²⁺ pump by two mechanisms. First, ATP chelated free Mg²⁺ and, at low ionized Mg²⁺ concentrations, K⁺ was shown to be a potent activator of ATP hydrolysis. In the absence of K⁺ ionized Mg²⁺ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K⁺ the concentration of ionized Mg²⁺ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K⁺-stimulation. With a saturating concentration of ionized Mg²⁺, stimulation by K⁺ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K⁺ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold.Activation of K⁺-stimulated ATPase activity by Ca²⁺ was maximal at anionized Ca²⁺ concentration of approx. 1 μM. At very high concentrations of either Ca²⁺ or Mg²⁺, basal Ca²⁺-dependent ATPase activity persisted, but the enzymic response to K⁺ was completely inhibited. The results provide further evidence that the Ca²⁺-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Anomalous DNA binding by E2 regulatory protein driven by spacer sequence TATA

We have investigated the anomalously weak binding of human papillomavirus (HPV) regulatory protein E2 to a DNA target containing the spacer sequence TATA. Experiments in magnesium (Mg²⁺) and calcium (Ca²⁺) ion buffers revealed a marked reduction in cutting by DNase I at the CpG sequence in the protein-binding site 3′ to the TATA spacer sequence, Studies of the cation dependence of DNA-E2 affinities showed that upon E2 binding the TATA sequence releases approximately twice as many Mg²⁺ ions as the average of the other spacer sequences. Binding experiments for TATA spacer relative to ATAT showed that in potassium ion (K⁺) the E2 affinity of the two sequences is nearly equal, but the relative dissociation constant (K_d) for TATA increases in the order K⁺ < Na⁺ < Ca²⁺ < Mg²⁺. Except for Mg²⁺, K_d for TATA relative to ATAT is independent of ion concentration, whereas for Mg²⁺ the affinity for TATA drops sharply as ion concentration increases. Thus, ions of increasing positive charge density increasingly distort the E2 binding site, weakening the affinity for protein. In the case of Mg²⁺, additional ions are bound to TATA that require displacement for protein binding. We suggest that the TATA sequence may bias the DNA structure towards a conformation that binds the protein relatively weakly.     

ATP,pH and Mg2+ modulate a cation current in Beta vulgaris vacuoles: A possible shunt conductance for the vacuolar H+-ATPase

Macroscopic instantaneous and time-dependent currents have been measured in the vacuolar membrane of Beta vulgaris using a patch clamp configuration analogous to whole cell mode. At low cytosolic Ca²⁺ and in the absence of Mg²⁺, only an instantaneous current was observed. This current is carried predominantly by cations (P_KP_Cl 71, p_nap_cl 41 and arginine is also conducted). The instantaneous current can be activated by ATP^4– (e.g., ATP-activated mean K⁺ current density was –20 mA.m^–2 at a membrane voltage of –20 mV) and by increasing cytosolic pH and Mg²⁺ (raising Mg²⁺ from 0 to 0.4 mm induced a mean current density increase of –7 mA.m^–2 at –20 mV). Such current can be activated by simultaneous addition of putative in vivo concentrations of ATP^4–/MgATP/Mg _free ²⁺ (in the presence of bafilomycin to inhibit the vacuolar ATPase) and further modulated by cytosolic pH. With vacuolar K⁺ concentration greater than that of the cytosol, activation of the instantaneous current would mediate vacuolar K⁺ release over the range of physiological membrane voltage. It is argued that the ATP^4–-activated current, in addition to acting as a K⁺ mobilization pathway, could provide a counter-ion (shunt) conductance, allowing the two electrogenic H⁺ pumps which reside in the vacuolar membrane to acidify the vacuolar lumen.A separate time-dependent current, which was not observed at low Ca²⁺ concentrations (less than 500 nm) could also be elicited by addition of Mg²⁺ at the cytoplasmic membrane face. This current was stimulated by increasing cytoplasmic pH.The authors are grateful to the BBSRC for financial support (Grant PG87/529) and to the Royal Society (University Research Fellowship to J.M.D.). We thank C. Abbott, K. Partridge and J. Robinson for plant cultivation; A. Amtmann, A. Bertl, D. Gradmann and G. Thiel for helpful discussion.     

Activation by Ca2+ and block by divalent ions of the K+ channel in the membrane of cytoplasmic drops fromChara australis

Summary Patch-clamp studies of cytoplasmic drops from the charophyteChara australis have previously revealed K⁺ channels combining high conductance (170 pS) with high selectivity for K⁺, which are voltage activated. The cation-selectivity sequence of the channel is shown here to be: K⁺>Rb⁺>NH ₄ ⁺ Na⁺ and Cl^–. Divalent cytosolic ions reduce the K⁺ conductance of this channel and alter its K⁺ gating in a voltage-dependent manner. The order of blocking potency is Ba²⁺>Sr²⁺>Ca²⁺>Mg²⁺. The channel is activated by micromolar cytosolic Ca²⁺, an activation that is found to be only weakly voltage dependent. However, the concentration dependence of calcium activation is quite pronounced, having a Hill coefficient of three, equivalent to three bound Ca²⁺ needed to open the channel. The possible role of the Ca²⁺-activated K⁺ channel in the tonoplast ofChara is discussed.     

Differential effects of heavy metal ions on Ca2+-dependent K+ channels

Summary 1. The ability of various divalent metal ions to substitute for Ca²⁺ in activating distinct types of Ca²⁺-dependent K⁺ [K⁺(Ca²⁺] channels has been investigated in excised, inside-out membrane patches of human erthrocytes and of clonal N1E-115 mouse neuroblastoma cells using the patch clamp technique. The effects of the various metal ions have been compared and related to the effects of Ca²⁺.2. At concentrations between 1 and 100 µM Pb²⁺, Cd²⁺ and Co²⁺ activate intermediate conductance K⁺(Ca²⁺) channels in erythrocytes and large conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Pb²⁺ and Co²⁺, but not Cd²⁺, activate small conductance K⁺(Ca²⁺) channels in neuroblastoma cells. Mg²⁺ and Fe²⁺ do not activate any of the K⁺(Ca²⁺) channels.3. Rank orders of the potencies for K⁺(Ca²⁺) activation are Pb²⁺, Cd²⁺>Ca²⁺, Co²⁺>>Mg²⁺, Fe²⁺ for the intermediate erythrocyte K⁺(Ca²⁺) channel, and Pb²⁺, Cd²⁺>Ca²⁺>Co²⁺>>Mg²⁺, Fe²⁺ for the small, and Pb²⁺>Ca²⁺>Co²⁺>>Cd²⁺, Mg²⁺, Fe²⁺ for the large K⁺(Ca²⁺) channel in neuroblastoma cells.4. At high concentrations Pb²⁺, Cd²⁺, and Co²⁺ block K⁺(Ca²⁺) channels in erythrocytes by reducing the opening frequency of the channels and by reducing the single channel amplitude. The potency orders of the two blocking effects are Pb²⁺>Cd²⁺, Co²⁺>>Ca²⁺, and Cd²⁺>Pb²⁺, Co²⁺>>Ca²⁺, respectively, and are distinct from the potency orders for activation.5. It is concluded that the different subtypes of K⁺(Ca²⁺) channels contain distinct regulatory sites involved in metal ion binding and channel opening. The K⁺(Ca²⁺) channel in erythrocytes appears to contain additional metal ion interaction sites involved in channel block.     

Termination of translation in eukaryotes is mediated by the quaternary eRF1*eRF3*GTP*Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg²⁺. We show that (i) eRF3 binds GDP (K_d = 1.9 μM) and this interaction depends only minimally on the Mg²⁺ concentration; (ii) GTP binds to eRF3 (K_d = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg²⁺ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg²⁺, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.     

Reduced Model Captures Mg2+-RNA Interaction Free Energy of Riboswitches

The stability of RNA tertiary structures depends heavily on Mg²⁺. The Mg²⁺-RNA interaction free energy that stabilizes an RNA structure can be computed experimentally through fluorescence-based assays that measure Γ₂₊, the number of excess Mg²⁺ associated with an RNA molecule. Previous explicit-solvent simulations predict that the majority of excess Mg²⁺ ions interact closely and strongly with the RNA, unlike monovalent ions such as K⁺, suggesting that an explicit treatment of Mg²⁺ is important for capturing RNA dynamics. Here we present a reduced model that accurately reproduces the thermodynamics of Mg²⁺-RNA interactions. This model is able to characterize long-timescale RNA dynamics coupled to Mg²⁺ through the explicit representation of Mg²⁺ ions. KCl is described by Debye-Hückel screening and a Manning condensation parameter, which represents condensed K⁺ and models its competition with condensed Mg²⁺. The model contains one fitted parameter, the number of condensed K⁺ ions in the absence of Mg²⁺. Values of Γ₂₊ computed from molecular dynamics simulations using the model show excellent agreement with both experimental data on the adenine riboswitch and previous explicit-solvent simulations of the SAM-I riboswitch. This agreement confirms the thermodynamic accuracy of the model via the direct relation of Γ₂₊ to the Mg²⁺-RNA interaction free energy, and provides further support for the predictions from explicit-solvent calculations. This reduced model will be useful for future studies of the interplay between Mg²⁺ and RNA dynamics.     

Functional reconstitution of the mitochondrial Ca2+/H+ antiporter Letm1

The leucine zipper, EF hand–containing transmembrane protein 1 (Letm1) gene encodes a mitochondrial inner membrane protein, whose depletion severely perturbs mitochondrial Ca²⁺ and K⁺ homeostasis. Here we expressed, purified, and reconstituted human Letm1 protein in liposomes. Using Ca²⁺ fluorophore and ⁴⁵Ca²⁺-based assays, we demonstrate directly that Letm1 is a Ca²⁺ transporter, with apparent affinities of cations in the sequence of Ca²⁺ ≈ Mn²⁺ > Gd³⁺ ≈ La³⁺ > Sr²⁺ >> Ba²⁺, Mg²⁺, K⁺, Na⁺. Kinetic analysis yields a Letm1 turnover rate of 2 Ca²⁺/s and a K_m of ∼25 µM. Further experiments show that Letm1 mediates electroneutral 1 Ca²⁺/2 H⁺ antiport. Letm1 is insensitive to ruthenium red, an inhibitor of the mitochondrial calcium uniporter, and CGP-37157, an inhibitor of the mitochondrial Na⁺/Ca²⁺ exchanger. Functional properties of Letm1 described here are remarkably similar to those of the H⁺-dependent Ca²⁺ transport mechanism identified in intact mitochondria.     

Interaction of Ca2+ with (Na+ + K+)-ATPase: Properties of the Ca2+-stimulated phosphatase activity

Ca²⁺ inhibited the Mg²⁺-dependent and K⁺-stimulated p-nitrophenylphosphatase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase. In the absence of K⁺, however, a Mg²⁺-dependent and Ca²⁺-stimulated phosphatase was observed, the maximal velocity of which, at pH 7.2, was about 20% of that of the K⁺-stimulated phosphatase. The Ca²⁺-stimulated phosphatase, like the K⁺-stimulated activity, was inhibited by either ouabain or Na⁺ or ATP. Ouabain sensitivity was decreased with increase in Ca²⁺, but the K_0.5 values of the inhibitory effects of Na⁺ and ATP were independent of Ca²⁺ concentration. Optimal pH was 7.0 for Ca²⁺-stimulated activity, and 7.8–8.2 for the K⁺-stimulated activity. The ratio of the two activities was the same in several enzyme preparations in different states of purity. The data indicate that (a) Ca²⁺-stimulated phosphatase is catalyzed by (Na⁺ + K⁺)-ATPase; (b) there is a site of Ca²⁺ action different from the site at which Ca²⁺ inhibits in competition with Mg²⁺; and (c) Ca²⁺ stimulation can not be explained easily by the action of Ca²⁺ at either the Na⁺ site or the K⁺ site.     

Ca2+-Dependent activities of (Na+ + K+)-ATPase

Experiments on the effects of varying concentrations of Ca²⁺ on the Mg²⁺ + Na⁺-dependent ATPase activity of a highly purified preparation of dog kidney (Na⁺ + K⁺)-ATPase showed that Ca²⁺ was a partial inhibitor of this activity. When Ca²⁺ was added to the reaction mixture instead of Mg²⁺, there was a ouabain-sensitive Ca²⁺ + Na⁺-dependent ATPase activity the maximal velocity of which was 30 to 50% of that of Mg²⁺ + Na⁺-dependent activity. The apparent affinities of the enzyme for Ca²⁺ and CaATP seemed to be higher than those for Mg²⁺ and MgATP. Addition of K⁺, along with Ca²⁺ and Na⁺, increased the maximal velocity and the concentration of ATP required to obtain half-maximal velocity. The maximal velocity of the ouabain-sensitive Ca²⁺ + Na⁺ + K⁺-dependent ATPase was about two orders of magnitude smaller than that of Mg²⁺ + Na⁺ + K⁺-dependent activity. In agreement with previous observations, it was shown that in the presence of Ca²⁺, Na⁺, and ATP, an acid-stable phosphoenzyme was formed that was sensitive to either ADP or K⁺. The enzyme also exhibited a Ca²⁺ + Na⁺-dependent ADP-ATP exchange activity. Neither the inhibitory effects of Ca²⁺ on Mg²⁺-dependent activities, nor the Ca²⁺-dependent activities were influenced by the addition of calmodulin. Because of the presence of small quantities of endogenous Mg²⁺ in all reaction mixtures, it could not be determined whether the apparent Ca²⁺-dependent activities involved enzyme-substrate complexes containing Ca²⁺ as the divalent cation or both Ca²⁺ and Mg²⁺.     

Divalent cations and the phosphatase activity of the (Na + K)-dependent ATPase

Phosphatase activity of a kidney (Na + K)-ATPase preparation was optimally active with Mg²⁺ plus K⁺. Mn²⁺ was less effective and Ca²⁺ could not substitute for Mg²⁺. However, adding Ca²⁺ with Mg²⁺ or substituting Mn²⁺ for Mg²⁺ activated it appreciably in the absence of added K⁺, and all three divalent cations decreased apparent affinity for K⁺. Inhibition by Na⁺ decreased with higher Mg²⁺ concentrations, when Ca²⁺ was added, and when Mn²⁺ was substituted for Mg²⁺. Dimethyl sulfoxide, which favorsE ₂ conformations of the enzyme, increased apparent affinity for K⁺, whereas oligomycin, which favorsE ₁ conformations, decreased it. These observations are interpretable in terms of activation through two classes of cation sites. (i) At divalent cation sites, Mg²⁺ and Mn²⁺, favoring (under these conditions)E ₂ conformations, are effective, whereas Ca²⁺, favoringE ₁, is not, and monovalent cations complete. (ii) At monovalent cation sites divalent cations compete with K⁺, and although Ca²⁺ and Mn²⁺ are fairly effective, Mg²⁺ is a poor substitute for K⁺, while Na⁺ at these sites favorsE ₁ conformations. K⁺ increases theK _m for substrate, but both Ca²⁺ and Mn²⁺ decrease it, perhaps by competing with K⁺. On the other hand, phosphatase activity in the presence of Na⁺ plus K⁺ is stimulated by dimethyl sulfoxide, by higher concentrations of Mg²⁺ and Mn²⁺, but not by adding Ca²⁺; this is consistent with stimulation occurring through facilitation of an E₁ to E₂ transition, perhaps an E₁-P to E₂-P step like that in the (Na + K)-ATPase reaction sequence. However, oligomycin stimulates phosphatase activity with Mg²⁺ plus Na⁺ alone or Mg²⁺ plus Na⁺ plus low K⁺: this effect of oligomycin may reflect acceleration, in the absence of adequate K⁺, of an alternative E₂-P to E₁ pathway bypassing the monovalent cation-activated steps in the hydrolytic sequence.     

Characterization of K+-dependent and K+-independentp-nitrophenylphosphatase activity of synaptosomes

These experiments examined effects of several ligands on the K⁺ p-nitrophenylphosphatase activity of the (Na⁺,K⁺)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K⁺-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K⁺ was approximately 75% less than that observed when K⁺ was included in the incubation medium. The response to the H⁺ concentrations was different: K⁺-independent activity showed a pH optimum around 6.5–7.0, while the K⁺-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K⁺-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K⁺-independent activity. Na⁺ did not affect K⁺-independent activity at all, while the same ligand concentration inhibited sharply the K⁺-dependent activity; this inhibition was not competitive with the substrate,p-nitrophenyl phosphate. K⁺-dependent activity was stimulated by Mg²⁺ with low affinity (millimolar range), and 3 mM Mg²⁺ produced a slight stimulation of the activity in absence of K⁺, which could be interpreted as Mg²⁺ occupying the K⁺ sites. Ca²⁺ had no appreciable effect on the activity in the absence of K⁺. However, in the presence of K⁺ a sharp inhibition was found with all Ca²⁺ concentrations studied. ATP (0.5 mM) did not affect the K⁺-independent activity, but this nucleotide behaved as a competitive inhibitor top-nitrophenylphosphate. Pi inhibited activity in the presence of K⁺, competively to the substrate, so it could be considered as the second product of the reaction sequence.Abbreviations used p-NPP p-nitrophenylphosphate - p-NPPase rho-nitrophenylphosphatase activity     

A missense mutation converts the Na+,K+-ATPase into an ion channel and causes therapy-resistant epilepsy

The ion pump Na⁺,K⁺-ATPase is a critical determinant of neuronal excitability; however, its role in the etiology of diseases of the central nervous system (CNS) is largely unknown. We describe here the molecular phenotype of a Trp931Arg mutation of the Na⁺,K⁺-ATPase catalytic α1 subunit in an infant diagnosed with therapy-resistant lethal epilepsy. In addition to the pathological CNS phenotype, we also detected renal wasting of Mg²⁺. We found that membrane expression of the mutant α1 protein was low, and ion pumping activity was lost. Arginine insertion into membrane proteins can generate water-filled pores in the plasma membrane, and our molecular dynamic (MD) simulations of the principle states of Na⁺,K⁺-ATPase transport demonstrated massive water inflow into mutant α1 and destabilization of the ion-binding sites. MD simulations also indicated that a water pathway was created between the mutant arginine residue and the cytoplasm, and analysis of oocytes expressing mutant α1 detected a nonspecific cation current. Finally, neurons expressing mutant α1 were observed to be depolarized compared with neurons expressing wild-type protein, compatible with a lowered threshold for epileptic seizures. The results imply that Na⁺,K⁺-ATPase should be considered a neuronal locus minoris resistentia in diseases associated with epilepsy and with loss of plasma membrane integrity.     

Signal transduction in red bloodcells of the lizards Ameiva ameiva and Tupinambis merianae (Squamata, Teiidae)

The fluorescent calcium probe, Fluo-3, AM was used to measure the intracellular calcium concentration in red blood cells (RBCs) of the teiid lizards Ameiva ameiva and Tupinambis merianae. The cytosolic [Ca²⁺] is maintained around 20nM and the cells contain membrane-bound Ca²⁺pools. One pool appears to be identifiable with the endoplasmic reticulum (ER) inasmuch as addition of the sarco-endoplasmic reticulum Ca²⁺ATPase, SERCA, inhibitor thapsigargin induces an increase in cytosolic [Ca²⁺both in the presence and in the absence of extracellular Ca²⁺. In addition to the ER, an acidic compartment appears to be involved in Ca²⁺storage, as collapse of intracellular pHgradients by monensin, a Na⁺–H⁺exchanger, and nigericin, a K⁺–H⁺exchanger, induce the release of Ca²⁺from internal pools. A vacuolar H⁺pump, sensitive to NBD-Cl and bafilomycin appears to be necessary to load the acidic Ca²⁺pools. Finally, the purinergic agonist ATP triggers a rapid and transient increase of [Ca²⁺]_cin the cells from both lizard species, mostly by mobilization of the cation from internal stores.     

Regulation of cardiac sarcolemmal Ca2+ channels and Ca2+ transporters by thyroid hormone

In order to examine the regulatory role of thyroid hormone on sarcolemmal Ca²⁺-channels, Na⁺–Ca²⁺ exchange and Ca²⁺-pump as well as heart function, the effects of hypothyroidism and hyperthyroidism on rat heart performance and sarcolemmal Ca²⁺-handling were studied. Hyperthyroid rats showed higher values for heart rate (HR), maximal rates of ventricular pressure development+(dP/dt)max and pressure fall–(dP/dt)max, but shorter time to peak ventricular pressure (TPVP) and contraction time (CT) when compared with euthyroid rats. The left ventricular systolic pressure (LVSP) and left ventricular end-diastolic pressure (LVEDP), as well as aortic systolic and diastolic pressures (ASP and ADP, respectively) were not significantly altered. Hypothyroid rats exhibited decreased values of LVSP, HR, ASP, ADP, +(dP/dt)max and –(dP/dt)max but higher CT when compared with euthyroid rats; the values of LVEDP and TPVP were not changed. Studies with isolated-perfused hearts showed that while hypothyroidism did not modulate the inotropic response to extracellular Ca²⁺ and Ca²⁺ channel blocker verapamil, hyperthyroidism increased sensitivity to Ca²⁺ and decreased sensitivity to verapamil in comparison to euthyroid hearts. Studies of [³H]-nitrendipine binding with purified cardiac sarcolemmal membrane revealed decreased number of high affinity binding sites (B_max) without any change in the dissociation constant for receptor-ligand complex (K_d) in the hyperthyroid group when compared with euthyroid sarcolemma; hypothyroidism had no effect on these parameters. The activities of sarcolemmal Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake and ouabain-sensitive Na⁺–K⁺ ATPase were decreased whereas the Mg²⁺-ATPase activity was increased in hypothyroid hearts. On the other hand, sarcolemmal membranes from hyperthyroid samples exhibited increased ouabain-sensitive Na⁺–K⁺ ATPase activity, whereas Ca²⁺-stimulated ATPase, ATP-dependent Ca²⁺ uptake, and Mg²⁺-ATPase activities were unchanged. The V_max and K_a for Ca²⁺ of cardiac sarcolemmal Na⁺–Ca²⁺ exchange were not altered in both hyperthyroid and hypothyroid states. These results indicate that the status of sarcolemmal Ca²⁺-transport processes is regulated by thyroid hormones and the modification of Ca²⁺-fluxes across the sarcolemmal membrane may play a crucial role in the development of thyroid state-dependent contractile changes in the heart.     

Influence of monovalent ions on the activity of the (Ca2+ + Mg2+)-ATPase and Ca2+-transport of human red blood cells

In reconstituted human red blood cells a difference was found in (Ca²⁺ + Mg²⁺)-ATPase activity and in Ca²⁺ efflux at 37°C, depending on the side of the membrane at which the monovalent cations K⁺ and Na⁺ were placed. Under the conditions used, (Ca²⁺ + Mg²⁺)-ATPase activity and Ca²⁺ efflux was highest when K⁺ ($\text{35 ± 0.5}\text{mM (± S.E.)}$, mean of four experiments) was at the inside and Na⁺ (130 mM) at the outside of the ghost membrane.     

Mechanisms of Extracellular NO and Ca<Superscript>2+</Superscript> Regulating the Growth of Wheat Seedling Roots

Our previous studies suggested the cross talk of nitric oxide (NO) with Ca²⁺ in regulating stomatal movement. However, its mechanism of action is not well defined in plant roots. In this study, sodium nitroprusside (SNP, a NO donor) showed an inhibitory effect on the growth of wheat seedling roots in a dose-dependent manner, which was alleviated through reducing extracellular Ca²⁺ concentration. Analyzing the content of Ca²⁺ and K⁺ in wheat seedling roots showed that SNP significantly promoted Ca²⁺ accumulation and inhibited K⁺ accumulation at a higher concentration of extracellular Ca²⁺, but SNP promoted K⁺ accumulation in the absence of extracellular Ca²⁺. To gain further insights into Ca²⁺ function in the NO-regulated growth of wheat seedling roots, we conducted the patch-clamped protoplasts of wheat seedling roots in a whole cell configuration. In the absence of extracellular Ca²⁺, NO activated inward-rectifying K⁺ channels, but had little effects on outward-rectifying K⁺ channels. In the presence of 2 mmol L⁻¹ CaCl₂ in the bath solution, NO significantly activated outward-rectifying K⁺ channels, which was partially alleviated by LaCl₃ (a Ca²⁺ channel inhibitor). In contrast, 2 mmol L⁻¹ CaCl₂ alone had little effect on inward or outward-rectifying K⁺ channels. Thus, NO inhibits the growth of wheat seedling roots likely by promoting extracellular Ca²⁺ influx excessively. The increase in cytosolic Ca²⁺ appears to inhibit K⁺ influx, promotes K⁺ outflux across the plasma membrane, and finally reduces the content of K⁺ in root cells.