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PubMed1983

Effects of divalent and trivalent cations on Na+-Ca2+ exchange in cardiac sarcolemmal vesicles.

Inhibition of Na+-Ca2+ exchange processes in canine cardiac sarcolemmal vesicles by several divalent and trivalent cations has been investigated. The order of cation effectiveness in inhibiting initial rates of Nai+-induced Ca2+ uptake in the presence of 140 mM Nai+ and 20 microM Cao2+ is La3+ greater than Nd3+ greater than Tm3+ approximately Y3+ greater than Cd2+ much greater than Sr2+ greater than Ba2+ approximately Mn2+ much greater than Mg2+. The effectiveness of the divalent ions is related to their ionic crystal radius as compared with that of Ca2+. No such relationship was observed for the trivalent ions, which appeared instead to be more effective the larger their radius. Very low concentrations of trivalent ions ((1-6).10(-7)M) caused slight stimulation of Ca2+-exchange uptake. The trivalent ions also inhibited passive and Nao+-induced Ca2+ efflux from sarcolemmal vesicles, in the same concentration range as that for inhibiting uptake. The divalent ions, however, stimulated Ca2+ efflux, possibly via divalent cation-Ca2+ exchange. These various results suggest that the divalent and trivalent cations interact differently with the exchange apparatus in the sarcolemma.

Animals↗
PubMed1988

Lanthanide-stimulated glucose and proline transport across rabbit intestinal brush-border membranes.

Trivalent cations of the lanthanide series (La3+----Yb3+) stimulated uptake of proline or glucose in rabbit small intestinal brush-border membrane vesicles. The lanthanides stimulated uptake to an extent greater than Al3+, choline, and in many cases, Na+. A time-course of Er3+-stimulated glucose uptake gave initial rates and overshoots greater than Na+ stimulation. The best activators were Sm3+, Eu3+ and Tm3+, which stimulated proline initial uptakes by 400-600%, and stimulated glucose uptake by 120-150%, compared to Na+. The best lanthanide cotransport activators possessed high third ionization potentials.

Animals↗
PubMed1980

Crystalline actin tubes. II. The effect of various lanthanide ions on actin tube formation.

The ability of skeletal muscle actin to aggregate in the form of crystalline tubular structures was examined using all 14 of the available trivalent lanthanide cations. Under conditions which normally cause F-actin formation, (0.1 M KCl, pH 6.9) only those lanthanide ions which interact with actin with a molar ratio of 5:1 (Ce3+, Pr3+, Nd3+, Sm3+ and Eu3+) or 6:1 (Gd3+, Tb3+, Dy3+ and Ho3+) can promote the formation of ordered tubular structures. Under the same conditions, the remaining ions (Er3+, Tm3+, Yb3+, Lu3+ as well as La3+) interact with a 7:1 molar ratio of lanthanide to actin, but these were unable to form actin tubes. Actin tube dimensions undergo systematic changes depending on which lanthanide ion binds. The dimensions of the actin monomers and their packing arrangement (i.e. number of rows of subunits per helical repeat and the pitch angle) determines the actin tube diameter. It is suggested that the failure of actin tube formation when 7 lanthanide ions bind is due to additional charge on the monomer, possibly conferring on it a net positive charge.

Actins↗
PubMed1993

Structure/function relationships of muscarinic acetylcholine receptors.

The regions of muscarinic receptors that specify G-protein-coupling and ligand-binding have been defined in several recent studies. Overall, these studies have shown that amino acids within the third cytoplasmic loop of the receptors define their selectivity for different G-proteins, and that multiple, discontinuous epitopes contribute to their selectivities for different ligands. In fact, several competitive and allosteric antagonists can be classified into groups based on which of these epitopes contribute to their subtype selectivity. Site-directed mutagenesis, combined with covalent-labeling studies have suggested that ligands bind to a hydrophobic core of the receptors that is formed by multiple transmembrane (TM) domains. An aspartic acid located in TM3 is likely to bind to the ammonium headgroup of muscarinic ligands, and multiple hydroxyl-containing amino acids contribute to agonist but not antagonist binding. These data are discussed in the context of a computational model of a muscarinic receptor. Our model is based on a sequence alignment with bacteriorhodopsin, a seven TM protein for which a higher resolution structure is available. Most of the mutagenic data can be rationalized in the context of this model, and predict testable hypotheses concerning the mechanism by which ligands control the activity of muscarinic receptors.

Animals↗
PubMed1995

Characterisation of the divalent cation channels of the hepatocyte plasma membrane receptor-activated Ca2+ inflow system using lanthanide ions.

The ability of Gd3+ to inhibit vasopressin-stimulated Ca2+ inflow to hepatocytes was compared with its effect on Mn2+ inflow. In the absence of Gd3+, the stimulation of Mn2+ inflow by vasopressin increased with increasing pH of the extracellular medium. Maximal inhibition of vasopressin-stimulated Ca2+ and Mn2+ inflow by saturating concentrations of Gd3+ was 70 and 30%, respectively. Gd3+ also inhibited thapsigargin-stimulated Ca2+ and Mn2+ inflow with maximal inhibition of 70 and 40%, respectively. It is concluded that vasopressin and thapsigargin each activate two types of Ca2+ inflow processes, one which is sensitive and one which is insensitive to lanthanides. The nature of the pore of the lanthanide-sensitive Ca2+ channel was investigated further using different lanthanides as inhibitors. Tm3+, Gd3+, Eu3+, Nd3+ and La3+ each inhibited vasopressin-stimulated Ca2+ and Mn2+ inflow but had no effect on Ca2+ inflow in the absence of an agonist, or on vasopressin-stimulated release of Ca2+ from intracellular stores. Maximal inhibition of vasopressin-stimulated Ca2+ inflow in the presence of a saturating concentration of each lanthanide ranged from 70-90%. An equation which describes a 1:1 interaction of the lanthanide with a putative binding site in the Ca2+ channel gave a good fit to dose-response curves for the inhibition of vasopressin-stimulated Ca2+ inflow by each lanthanide. Lanthanides in the middle of the series exhibited the lowest dissociation constant (Kd) values. The Kd for Gd3+ increased with increasing extracellular Ca2+ concentration, suggesting competitive inhibition of Ca2+ binding by Gd3+. In the absence of lanthanide, vasopressin-stimulated Mn2+ inflow was substantially reduced when the plasma membrane was depolarised by increasing the extracellular K+ concentration. Changing the membrane potential had little effect on the maximum inhibition by Gd3+ of vasopressin-stimulated Mn2+ inflow. The Kd for inhibition of vasopressin-stimulated Ca2+ inflow by Gd3+, measured at the lowest attainable membrane potential, was about 6-fold lower than the Kd measured at the highest attainable membrane potential. The idea that there is a site in the vasopressin-stimulated lanthanide-sensitive Ca2+ channel composed of carboxylic acid groups which bind Ca2+, Mn2+ or a lanthanide ion is consistent with the data obtained using the different lanthanides.

Animals↗
PubMed1995

Antibody specific for phosphorylated AMPA-type glutamate receptors at GluR2 Ser-696.

Possible phosphorylation sites on the Purkinje cell alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate receptor subunits were identified using in vitro kinase assays of 17 synthetic peptides derived from the transmembrane-3 (TM3) domain to the end of C-terminal of a rat glutamate receptor 2 (GluR2). Only two peptides containing Ser-662 and Ser-696 were found to be efficiently phosphorylated by protein kinase C (PKC). The peptide including Ser-696 was also phosphorylated by protein kinase G (PKG). Another peptide containing Thr-692 of a rat GluRA, clone almost identical to GluR1, was phosphorylated by PKC but not by PKG. Antisera recognizing phosphorylated AMPA receptor subunits at GluR2 Ser-696 or the homologous sites of GluR1/3/4 were produced, and the specificity of one of them, named 12P3, was established by enzyme-linked immunosorbent assay (ELISA), immunoblot and immunoprecipitation analyses. 12P3-immunocytochemistry on cerebellar slices demonstrated an AMPA-induced transient AMPA receptor phosphorylation, which appeared in Purkinje cell dendrites as well as somata immediately after AMPA treatment and disappeared after 20 min. This antibody may be a useful tool to study the role of AMPA receptor phosphorylation in producing synaptic plasticity.

Amino Acid Sequence↗
PubMed1993

Differential effects of prepubertal rat Sertoli cell secreted proteins on somatic testicular and nontesticular cells.

There is little information on the mitogenic and immunoregulatory activities of proteins, secreted by prepubertal Sertoli cells during the stage of meiosis initiation and before creation of the blood-testis barrier. We have previously demonstrated dose-dependent and age-related stimulation of BALB/c 3T3 fibroblasts and quiescent rat prespermatogonia (Kancheva et al., 1990) as well as inhibition of natural killer cell activity of mice, guinea pigs and human lymphocytes (Nikolova et al., 1992) by Sertoli cell-conditioned medium derived from 12-day-old rats. In the current study, using splenic lymphocytes stimulated by PHA, LPS and Con A, we have shown a dose-dependent inhibition of T and B lymphocyte proliferation by prepubertal Sertoli cell-secreted proteins (pSCSP). These results suggest that by the time the blood-testis barrier had been formed, Sertoli cell in rat testis had already synthesized immunoregulatory proteins. In addition we have found that pSCSP stimulate the proliferation of TM3 Leydig but not TM4 Sertoli cells. The differential effect of pSCSP is an expression of the different balance between growth factors secreted by Sertoli cells, which in turn is dependent on the requirements of the cell types at each stage of testicular development.

Animals↗
PubMed1994

Agonist selectivity of glutamate receptors is specified by two domains structurally related to bacterial amino acid-binding proteins.

By exchanging portions of the AMPA receptor subunit GluR3 and the kainate receptor subunit GluR6, we have identified two discontinuous segments of approximately 150 amino acid residues each that control the agonist pharmacology of these glutamate receptors. The first segment (S1) is adjacent and N-terminal to the putative transmembrane domain 1 (TM1), whereas the second segment (S2) is located between the putative TM3 and TM4. Only the simultaneous exchange of S1 and S2 converts the pharmacological profile of the recipient to that of the donor subunit. The two segments identified in this study share sequence similarities with the ligand-binding site of several bacterial periplasmic amino acid-binding proteins. Based on the X-ray structure of these proteins, we propose a model for the glutamate-binding site of ionotropic glutamate receptors.

Amino Acid Sequence↗
PubMed1992

Site-directed mutagenesis of the human dopamine D2 receptor.

Based on amino acid sequence and computer modeling, two conflicting three-dimensional models of the dopamine D2 receptor have been proposed. One model (Dahl et al., 1991, Proc. Natl. Acad. Sci. USA 88, 8111) suggests that dopamine interacts with aspartate 80 of transmembrane (TM) 2 and asparagine 390 of TM6 with the transmembranes arranged in a clockwise manner, while a second model (Hibert et al., 1991, Mol. Pharmacol. 40, 8) suggests that dopamine interacts with aspartate 114 of TM3 and the serines of TM5 (194 and 197) with the transmembranes arranged in a counterclockwise manner when viewed from the extracellular space. The present study tests the latter model by selectively mutating aspartate 114 and serines 194 and 197 of the human dopamine D2 receptor by site-directed mutagenesis. In addition, two methionines (116 and 117) were mutated to evaluate whether residues near aspartate (114) of the dopamine D2 receptor are critical in differentiating dopamine receptor agonists from adrenoceptor agonists. Removal of the negative charge with the mutation of aspartate (114) to either asparagine or glycine led to a total loss of both agonist and antagonist binding. Individual or dual methionine mutations in positions 116 and 117, to make the dopamine D2 binding pocket more closely resemble the beta 2-adrenoceptor, did not result in a change in selectivity toward noradrenergic agonists or antagonists. The serine mutations revealed interesting differences between the dopamine D2 receptor and the adrenoceptors. In particular, serine 197 appeared more important than serine 194 for agonist binding. In addition, the binding of one agonist (N-0437) was unaffected by individual serine mutations, while the binding of some antagonists, such as raclopride and spiperone, was significantly altered. These findings are discussed in relation to ligand structure and their interactions with the putative binding pocket.

Amino Acid Sequence↗
PubMed2005

Site-directed mutagenesis of CCR2 identified amino acid residues in transmembrane helices 1, 2, and 7 important for MCP-1 binding and biological functions.

Monocyte chemotactic protein-1 (MCP-1) binds its G-protein-coupled seven transmembrane (TM) receptor, CCR2B, and causes infiltration of monocytes/macrophages into areas of injury, infection or inflammation. To identify functionally important amino acid residues in CCR2B, we made specific mutations of nine residues selected on the basis of conservation in chemokine receptors and located TM1 (Tyr(49)), TM2 (Leu(95)), TM3 (Thr(117) and Tyr(120)), and TM7 (Ala(286), Thr(290), Glu(291), and His(297)) and in the extracellular loop 3 (Glu(278)). MCP-1 binding was drastically affected only by mutations in TM7. Reversing the charge at Glu(291) (E291K) and at His(297) (H297D) prevented MCP binding although substitution with Ala at either site had little effect, suggesting that Glu(291) and His(297) probably stabilize TM7 by their ionic interaction. E291A elicited normal Ca(2+) influx. H297A, Y49F in TM1 and L95A in TM2 that showed normal MCP-1 binding did not elicit Ca(2+) influx and elicited no adenylate cyclase inhibition at any MCP-1 concentration. MCP-1 treatment of HEK293 cells caused lamellipodia formation only when they expressed CCR2B. The mutants that showed no Ca(2+) influx and adenylate cyclase inhibition by MCP-1 treatment showed lamellipodia formation and chemotaxis. Our results show that induction of lamellipodia formation, but not Ca(2+) influx and adenylate cyclase inhibition, is necessary for chemotaxis.

Adenylyl Cyclase Inhibitors↗
PubMed2005

Binding modes of dihydroquinoxalinones in a homology model of bradykinin receptor 1.

We report the first homology model of human bradykinin receptor B1 generated from the crystal structure of bovine rhodopsin as a template. Using an automated docking procedure, two B1 receptor antagonists of the dihydroquinoxalinone structural class were docked into the receptor model. Site-directed mutagenesis data of the amino acid residues in TM1, TM3, TM6, and TM7 were incorporated to place the compounds in the binding site of the homology model of the human B1 bradykinin receptor. The best pose in agreement with the mutation data was selected for detailed study of the receptor-antagonist interaction. To test the model, the calculated antagonist-receptor binding energy was correlated with the experimentally measured binding affinity (K(i)) for nine dihydroquinoxalinone analogs. The model was used to gain insight into the molecular mechanism for receptor function and to optimize the dihydroquinoxalinone analogs.

Amino Acid Sequence↗
PubMed2005

Transmembrane residues define the action of isoflurane at the GABAA receptor alpha-3 subunit.

The gamma-aminobutyric acid type A (GABA(A)) receptor is the target of a structurally diverse group of sedative, hypnotic, and anesthetic drugs, including the volatile anesthetic isoflurane. Previous studies on the GABA(A) receptor have suggested the existence of a cavity located between transmembrane (TM) segments 2 and 3 in both alpha-1 and alpha-2 subunits, within which volatile anesthetics might bind. In this study, we have used site-directed mutagenesis to investigate the involvement of homologous residues of the GABA(A) alpha-3 subunit in allosteric modulation by isoflurane. Mutation of serine residue 294 within the TM2 to histidine or tyrosine increased the potency of GABA and decreased positive modulation by isoflurane. Mutation of alanine residue 315 within the TM3 to tryptophan increased the potency of GABA and abolished isoflurane modulation. The activity of the intravenous anesthetic propofol was unaltered from wild-type at these mutant receptors. These findings are consistent with the action of isoflurane on a critical site within the transmembrane domains of the receptor and suggest a degree of functional homology between the GABA(A) alpha-1, -2, and -3 subunits.

Anesthetics, Inhalation↗
PubMed2006

A cannabinoid receptor 1 mutation proximal to the DRY motif results in constitutive activity and reveals intramolecular interactions involved in receptor activation.

Activation of a G-protein-coupled receptor involves changes in specific microdomain interactions within the transmembrane region of the receptor. Here, we have focused on the role of L207, proximal to the DRY motif of the human cannabinoid receptor 1 in the interconversion of the receptor resting and active states. Ligand binding analysis of the mutant receptor L207A revealed an enhanced affinity for agonists (three- to six-fold) and a diminished affinity for inverse agonists (19- to 35-fold) compared to the wild-type receptor, properties characteristic of constitutive activity. To further examine whether this mutant adopts a ligand-independent, active form, treatment with GTPgammaS was used to inhibit G protein coupling. Under these conditions, the L207A receptor exhibited a 10-fold increase in affinity for the inverse agonist SR141716A, consistent with a shift away from an enhanced precoupled state. Analysis of the cellular activity of the L207A receptor showed elevated basal cyclic AMP accumulation relative to the wild type that is inhibited by SR141716A, consistent with receptor-mediated Gs precoupling. Using toxins to selectively abrogate Gs or Gi coupling, we found that CP55940 nonetheless induced only a Gi response suggesting a strong preference of this ligand-bound form for Gi in this system. Molecular dynamics simulations reveal that the single residue change of L207A impacts the association of TM3 and TM6 in the receptor by altering hydrophobic interactions involving L207, the salt bridge involving the Arg of the DRY motif, and the helical structure of TM6, consistent with events leading to activation. The structural alterations parallel those observed in models of a mutant CB(1) receptor T210I, with established constitutive activity (D'Antona, A.M., Ahn, K.H. and Kendall, D.A., 2006. Mutations of CB1 T210 produce active and inactive receptor forms: correlations with ligand affinity, receptor stability, and cellular localization. Biochemistry, 45, 5606-5617).

Amino Acid Motifs↗
PubMed2004

A 2H solid-state NMR spectroscopic investigation of biomimetic bicelles containing cholesterol and polyunsaturated phosphatidylcholine.

Deuterium solid-state NMR spectroscopy was used to qualitatively study the effects of both 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphatidylcholine (PLiPC) and cholesterol on magnetically aligned phospholipid bilayers (bicelles) as a function of temperature utilizing the chain-perdeuterated probe 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC-d54) in DMPC/dihexanoylPC (DHPC) phospholipid bilayers. The results demonstrate that polyunsaturated PC and cholesterol were successfully incorporated into DMPC/DHPC phospholipid bilayers, leading to a bicelle that will be useful for investigations of eukaryotic membrane protein-lipid interactions. The data indicate that polyunsaturated PC increases membrane fluidity and decreases the minimum magnetic alignment temperature for DMPC/DHPC bicelles. Conversely, the introduction of cholesterol into aligned DMPC/DHPC bilayers decreases fluidity in the membrane and increases the minimum temperature necessary to magnetically align the phospholipid bilayers. Finally, the addition of Tm3+ to magnetically aligned DMPC/DMPC-d54/PLiPC/DHPC bilayers doubles the quadrupolar splittings, indicating that this unique bicelle system can be aligned with the bilayer normal parallel to the static magnetic field.

Biomimetic Materials↗
PubMed2004

Investigating the structural and dynamic properties of n-doxylstearic acid in magnetically-aligned phospholipid bilayers by X-band EPR spectroscopy.

X-band electron paramagnetic resonance (EPR) spectroscopy has been employed to investigate the dynamic properties of magnetically-aligned phospholipid bilayers (bicelles) based on the molecular order parameters (S(mol)), the hyperfine splitting values and the line shapes of the EPR spectra. For the first time, a series of EPR spectra of n-doxylstearic acid spin-labels (n = 5, 7, 12, and 16) incorporated into Tm3+-doped parallel-aligned, Dy3+-doped perpendicular-aligned, and randomly dispersed 1,2-dimyristoyl-sn-glycero-3-phosphocholine/1,2-dihexanoyl-sn-glycero-3-phosphocholine (DMPC/DHPC) bicelles with respect to the direction of the static magnetic field have been investigated as a function of cholesterol content and temperature variation to characterize the orientational aspects along the hydrocarbon acyl chains. Important general observations are that under conditions for which the bicelle is poised in the liquid crystalline phase, the degree of ordering decreases as the nitroxide moiety is transferred toward the end of the stearic acid acyl chains. The addition of cholesterol increases the phase transition temperature and alignment temperature of the DMPC/DHPC phospholipid bilayers and increases the chain order. However, increasing the temperature of the bicelle system decreases the chain order. This report reveals that the dynamic properties of DMPC/DHPC bicelles agree well with other biological and model membrane systems. The results indicate that magnetically-aligned phospholipid bilayers are an excellent model membrane system.

Cyclic N-Oxides↗
PubMed2004

Effects of amiodarone on mutant Na+/Ca2+ exchangers expressed in CCL 39 cells.

Using the whole cell voltage clamp, we reported previously that amiodarone acutely inhibits Na+/Ca2+ exchange current (INCX) in guinea pig cardiac ventricular myocytes. Intracellular application of trypsin via the patch pipette attenuated the blocking effect of amiodarone, suggesting that amiodarone affects the Na+/Ca2+ exchanger (NCX) from the cytoplasmic side. Here, we attempted to detect the site of amiodarone inhibition using wild type NCX1, mutants, and NCX3 expressed in CCL39 fibroblasts. INCX was recorded by ramp pulses. Amiodarone at 30 microM inhibited INCX by 80% in cells expressing wild type NCX1. However, 30 microM amiodarone inhibited INCX by about 55% in cells expressing mutant NCX1 with amino acids 217-671 (DeltaXIP) or 247-671 (Delta247-671) deleted in the long intracellular loop between the transmembrane segments (TM) 5 and 6. INCXs from NCX mutants deleted of cytoplasmic TM1-2, TM3-4 or the C-terminus were inhibited by amiodarone to a similar extent as the wild type. Amiodarone also inhibited INCX of NCX3 by 76%. These results suggest that a long intracellular loop may be involved in the inhibition of NCX1 by amiodarone, but that other intracellular loops, XIP region or C terminus are not involved in the amiodarone inhibition of NCX1.

Amiodarone↗
PubMed2005

Individual changes in lamotrigine plasma concentrations during pregnancy.

Eleven pregnant women on lamotrigine (LTG) monotherapy were retrospectively reviewed. A significant decrease in the ratio of plasma LTG concentration-to-dose by 65.1% was observed during the second trimester (TM2) (p=0.0058) and by 65.8% during TM3 (p=0.0045) compared to pre-pregnancy values. Five patients experienced seizure deterioration during pregnancy. The pharmacokinetic changes display marked inter-patient variation, which stresses the importance of evaluating each woman individually by closely monitoring LTG concentrations until term.

Adult↗
PubMed2006

Genome-wide analysis of the MADS-box gene family in Populus trichocarpa.

Populus trichocarpa (poplar) is distinguished from its herbaceous counterparts Arabidopsis and rice by its woody structure, perennial life cycle, and dioecious, two-whorled flowers. MADS-box genes are known to be involved in many important processes during plant development. Investigation of the poplar genome revealed 105 putative functional MADS-box genes and 12 pseudogenes. These numbers are comparable to those in Arabidopsis. However, poplar has 64 type II MADS-box genes, implying a higher birth rate when compared with Arabidopsis (64 vs.47). In addition to duplications for floral organ identity genes, TM3-like, StMADS11, ANR1 and Bs clades were significantly increased. As indicated by the phylogenetic analysis, there exist at least two MIKC* MADS-box genes in the poplar genome. In contrast, only 41 putative functional type I genes and 9 pseudogenes were found, suggesting that the poplar type I MADS-box genes have experienced a lower rate in both birth and death. Poplar MADS-box genes were distributed on all linkage groups (LGs), except LG XIX. Like other gene families, poplar MADS-box gene family has expanded through tandem gene duplication and segmental duplication events as demonstrated by two genomic regions where clustered MADS-box genes exhibited high similarities in the MADS-box domains. A survey of poplar EST sequences showed that MADS-box genes were expressed in wood and cambium tissues, which are specific to woody plants. The expression of common MADS-box gene in tree-specific tissues suggests that the novel function combinations of a set of genes similar to those in herbaceous plants may account for the development of woody characteristics in poplar.

Arabidopsis↗