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K M Page

Publications and source records attributed to K M Page.

At least 37 records · Page 2Linked to original sources
PubMed1999

Dissection of the calcium channel domains responsible for modulation of neuronal voltage-dependent calcium channels by G proteins.

The molecular determinants for G-protein regulation of neuronal calcium channels remain controversial. We have generated a series of alpha 1B/alpha 1E chimeric channels, since rat brain alpha 1E (rbEII), unlike human alpha 1E, showed no G-protein modulation. The study, carried out in parallel using D2 receptor modulation of calcium currents in Xenopus oocytes of G beta gamma modulation of calcium currents in COS-7 cells, consistently showed an essential role for domain I (from the N terminus to the end of the I-II loop) of the alpha 1B Ca2+ channel in G-protein regulation, with no additional effect of the C terminal of alpha 1B. The I-II loop alone of alpha 1B, or the I-II loop together with the C-terminal tail, was insufficient to confer G-protein modulation of alpha 1E (rbEII). We have further observed that the alpha 1E clone rbEII is truncated at the N-terminus compared to other alpha 1 subunits, and we isolated a PCR product from rat brain equivalent to a longer N-terminal isoform. The long N-terminal alpha 1E, unlike the short form, showed G-protein modulation. Furthermore, the equivalent truncation of alpha 1B (delta N1-55) abolished G-protein modulation of alpha 1B. Thus, we propose that the N terminus of alpha 1B and alpha 1E calcium channels contains essential molecular determinants for membrane-delimited G-protein inhibition, and that other regions, including the I-II loop and the C terminus, do not play a conclusive role alone.

Amino Acid Sequence↗
PubMed1998

Orally active indole N-oxide PDE4 inhibitors.

This communication describes the synthesis and in vitro and in vivo evaluation of a novel potent series of phosphodiesterase type (IV) (PDE4) inhibitors. Several of the compounds presented possess low nanomolar IC50's for PDE4 inhibition and excellent in vivo activity for inhibition of TNF-alpha levels in LPS challenged mice (mouse endotoxemia model). Emesis studies (dog) and efficacy in a SCW arthritis model for the most potent PDE4 inhibitors are presented.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

The synthesis and biological evaluation of a novel series of indole PDE4 inhibitors I.

This communication describes the synthesis and in vitro evaluation of a novel potent series of phosphodiesterase type (IV) (PDE4) inhibitors. The compounds described contain an indole moiety which replaces the 'rolipram-like' 3-methoxy-4-cyclopentoxy motif. Several of the compounds presented possess low nanomolar IC50's for PDEIV inhibition. In vivo activities determined from measurement of serum TNF-alpha levels in LPS challenged mice (mouse endotoxemia model) are also reported.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1998

The effect of overexpression of auxiliary Ca2+ channel subunits on native Ca2+ channel currents in undifferentiated mammalian NG108-15 cells.

1. High voltage activated (HVA) Ca2+ channels are composed of a pore-forming alpha 1 subunit and the accessory beta and alpha2-delta subunits. However, the subunit composition of low voltage activated (LVA), or T-type, Ca2+ channels has yet to be elucidated. We have examined whether native calcium channels in NG108-15 mouse neuroblastoma x rat glioma hybrid cells, which express predominantly LVA currents when undifferentiated, are modulated by overexpression of accessory calcium channel subunits. 2. Endogenous alpha 1A, B, C, C, and E, and low levels of beta and alpha 2-delta subunit protein were demonstrated in undifferentiated NG108-15 cells. 3. The alpha 2-delta, beta 2a or beta 1b accessory subunits were overexpressed by transfection of the cDNAs into these cells, and the effect examined on the endogenous Ca2+ channel currents. Heterologous expression, particularly of alpha 2-delta but also of beta 2a subunits clearly affected the profile of these currents. Both subunits induced a sustained component in the currents evoked by depolarizing voltages above -30 mV, and alpha 2-delta additionally caused a depolarization in the voltage dependence of current activation, suggesting that it also affected the native T-type currents. In contrast, beta 1b overexpression had no effect on the endogenous Ca2+ currents, despite immunocytochemical evidence for its expression in the transfected cells. 4 These results suggest that in NG108-15 cells, overexpression of the Ca2+ channel accessory subunits alpha 2-delta and beta 2a induce a sustained component of HVA current, and alpha 2-delta also influences the voltage dependence of activation of the LVA current. It is possible that native T-type alpha 1 subunits are not associated with beta subunits.

Animals↗
PubMed1998

Identification of the amino terminus of neuronal Ca2+ channel alpha1 subunits alpha1B and alpha1E as an essential determinant of G-protein modulation.

We have examined the basis for G-protein modulation of the neuronal voltage-dependent calcium channels (VDCCs) alpha1E and alpha1B. A novel PCR product of alpha1E was isolated from rat brain. This contained an extended 5' DNA sequence and was subcloned onto the previously cloned isoform rbEII, giving rise to alpha1Elong whose N terminus was extended by 50 amino acids. VDCC alpha1 subunit constructs were co-expressed with the accessory alpha2-delta and beta2a subunits in Xenopus oocytes and mammalian (COS-7) cells. The alpha1Elong showed biophysical properties similar to those of rbEII; however, when G-protein modulation of expressed alpha1 subunits was induced by activation of co-expressed dopamine (D2) receptors with quinpirole (100 nM) in oocytes, or by co-transfection of Gbeta1gamma2 subunits in COS-7 cells, alpha1Elong, unlike alpha1E(rbEII), was found to be G-protein-modulated, in terms of both a slowing of activation kinetics and a reduction in current amplitude. However, alpha1Elong showed less modulation than alpha1B, and substitution of the alpha1E1-50 with the corresponding region of alpha1B1-55 produced a chimera alpha1bEEEE, with G-protein modulation intermediate between alpha1Elong and alpha1B. Furthermore, deletion of the N-terminal 1-55 sequence from alpha1B produced alpha1BDeltaN1-55, which could not be modulated, thus identifying the N-terminal domain as essential for G-protein modulation. Taken together with previous studies, these results indicate that the intracellular N terminus of alpha1E1-50 and alpha1B1-55 is likely to contribute to a multicomponent site, together with the intracellular I-II loop and/or the C-terminal tail, which are involved in Gbetagamma binding and/or in subsequent modulation of channel gating.

Animals↗
PubMed1998

Role of domain I of neuronal Ca2+ channel alpha1 subunits in G protein modulation.

1. We studied the G protein inhibition of heteromultimeric neuronal Ca2+ channels by constructing a series of chimeric channels between the strongly modulated alpha1B subunit and the alpha1E(rbEII) subunit, which showed no modulation. 2. In parallel studies, alpha1 subunit constructs were co-expressed together with the accessory Ca2+ channel alpha2-delta and beta2a subunits in mammalian (COS-7) cells and Xenopus oocytes. G protein inhibition of expressed Ca2+ channel currents was induced by co-transfection of Gbeta1 and Ggamma2 subunits in COS-7 cells or activation of co-expressed dopamine (D2) receptors by quinpirole (100 nM) in oocytes. 3. The data indicate that transfer of the alpha1B region containing the N-terminal, domain I and the I-II loop (i.e. the alpha1B1-483 sequence), conferred G protein modulation on alpha1E(rbEII), both in terms of a slowing of activation kinetics and a reduction in current amplitude. 4. In contrast, the data are not consistent with the I-II loop and/or the C-terminal forming a unique site for G protein modulation.

Animals↗
PubMed1997

Voltage-dependent binding and calcium channel current inhibition by an anti-alpha 1D subunit antibody in rat dorsal root ganglion neurones and guinea-pig myocytes.

1. The presence of calcium channel alpha 1D subunit mRNA in cultured rat dorsal root ganglion (DRG) neurones and guinea-pig cardiac myocytes was demonstrated using the reverse transcriptase-polymerase chain reaction. 2. An antipeptide antibody targeted at a region of the voltage-dependent calcium channel alpha 1D subunit C-terminal to the pore-forming SS1-SS2 loop in domain IV (amino acids 1417-1434) only bound to this exofacial epitope if the DRG neurones and cardiac myocytes were depolarized with 30 mM K+. 3. Incubation of cells under depolarizing conditions for 2-4 h with the antibody resulted in a maximal inhibition of inward current density of 49% (P < 0.005) for DRGs and 30% (P < 0.05) for cardiac myocytes when compared with controls. 4. S-(-)-Bay K 8644 (1 microM) enhanced calcium channel currents in DRGs by 75 +/- 19% (n = 5) in neurones incubated under depolarizing conditions with antibody that had been preabsorbed with its immunizing peptide (100 micrograms ml-1). This was significantly (P < 0.05) larger than the enhancement by S-(-)-Bay K 8644 that was seen with cells incubated under identical conditions but with antibody alone, which was 15 +/- 4% (n = 5). 5. These results demonstrate the presence of calcium channel alpha 1D subunits in rat DRG neurones and guinea-pig cardiac myocytes. They also show that amino acids 1417-1434 of the alpha 1D subunit are only exposed to the extracellular face of the membrane following depolarization and that the binding of an antibody to these amino acids attenuates calcium channel current and reduces the ability of S-(-)-Bay K 8644 to enhance this current, indicating that it is an L-type current that is attenuated.

Amino Acid Sequence↗
PubMed1997

The intracellular loop between domains I and II of the B-type calcium channel confers aspects of G-protein sensitivity to the E-type calcium channel.

Neuronal voltage-dependent calcium channels undergo inhibitory modulation by G-protein activation, generally involving both kinetic slowing and steady-state inhibition. We have shown previously that the beta-subunit of neuronal calcium channels plays an important role in this process, because when it is absent, greater receptor-mediated inhibition is observed (). We therefore hypothesized that the calcium channel beta-subunits normally may occlude G-protein-mediated inhibition. Calcium channel beta-subunits bind to the cytoplasmic loop between transmembrane domains I and II of the alpha1-subunits (). We have examined the hypothesis that this loop is involved in G-protein-mediated inhibition by making chimeras containing the I-II loop of alpha1B or alpha1A inserted into alpha1E (alpha1EBE and alpha1EAE, respectively). This strategy was adopted because alpha1B (the molecular counterpart of N-type channels) and, to a lesser extent, alpha1A (P/Q-type) are G-protein-modulated, whereas this has not been observed to any great extent for alpha1E. Although alpha1B, coexpressed with alpha2-delta and beta1b transiently expressed in COS-7 cells, showed both kinetic slowing and steady-state inhibition when recorded with GTPgammaS in the patch pipette, both of which were reversed with a depolarizing prepulse, the chimera alpha1EBE (and, to a smaller extent, alpha1EAE) showed only kinetic slowing in the presence of GTPgammaS, and this also was reversed by a depolarizing prepulse. These results indicate that the I-II loop may be the molecular substrate of kinetic slowing but that the steady-state inhibition shown by alpha1B may involve a separate site on this calcium channel.

Animals↗
PubMed1997

Functional expression of rat brain cloned alpha1E calcium channels in COS-7 cells.

The properties of the rat brain alpha1E Ca2+ channel subunit and its modulation by accessory rat brain alpha2-delta and beta1b subunits were studied by transient transfection in a mammalian cell line in order to attempt to reconcile the debate as to whether alpha1E forms a low-voltage-activated (LVA) or high-voltage-activated (HVA) Ca2+ channel and to examine its pharmacology in detail. alpha1E alone was capable of forming an ion-conducting pore in COS-7 cells. The properties of heteromultimeric alpha1E/alpha2-delta/beta1b channels were largely dictated by the presence of the beta1b subunit, which increased current density and tended to produce a hyperpolarizing shift in the voltage dependence of activation and inactivation. alpha1E/alpha2-delta/beta1b channels did not appear to be regulated by Ca2+-induced inactivation. alpha1E was shown to exhibit a unique pharmacological profile. omega-Agatoxin IVA blocked the current in a dose-dependent manner with an IC50 of approximately 50 nM and a maximum inhibition of about 80%, whilst omega-conotoxin MVIIC was without effect. The 1,4-dihydropyridine (DHP) antagonist nicardipine (1 micro;M) produced an inhibition of 51 +/- 7%, whereas the DHP agonist S-(-)BAY K 8644 was without effect. Our findings suggest a re-evaluation of the classification of the alpha1E Ca2+ channel subunit; we propose that rat brain alpha1E forms a novel Ca2+ channel with properties more similar to a subtype of LVA than HVA Ca2+ current.

Animals↗
PubMed1997

Properties of cloned rat alpha1A calcium channels transiently expressed in the COS-7 cell line.

The rat brain alpha1A calcium channel clone has been expressed in COS-7 cells together with the neuronal accessory subunits beta1b and alpha2-delta. From reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry and electrophysiology experiments, we have obtained no evidence that these cells contain any endogenous calcium channels. Transfected cells were identified by co-expression of a cDNA for the reporter Green Fluorescent Protein. From immunocytochemical evidence, a high degree of co-expression was obtained between Green Fluorescent Protein and individual calcium channel subunits. When all three calcium channel subunits (alpha1, alpha2-delta and beta1b) were co-expressed, evidence was obtained that all subunits were present at the cell membrane. Voltage-dependent calcium currents were observed between 24 and 72 h after transfection with the three calcium channel subunits. The current density for the combination alpha1A/alpha2-delta/beta1b was 4.19 +/- 0.69 pA.pF(-1) and the current produced was slowly inactivating. The time constant of inactivation of the maximum I(Ba) was 332 +/- 46 ms (n = 5). The voltage-dependence of activation and steady-state inactivation had voltages of half activation and inactivation of 9.5 +/- 2.5 mV and -30.4 +/- 1.5 mV respectively, and there was little overlap between the two curves. The alpha1A current was completely blocked by 100 microM Cd2+ and was also blocked by omega-conotoxin MVIIC (500 nM). Dose-inhibition curves and analysis of k(on) and k(off) for omega-agatoxin IVA both revealed apparent K(D) values of approximately 11 nM for alpha1A currents, with a k(on) of 7.8 x 10(4) M(-1).s(-1). The results suggest that alpha1A expressed in these cells has some resemblance to the P type component of calcium current observed in native neurons, although it shows a somewhat greater degree of inactivation than native P current, more similar to the Q type current component. It also has an affinity for omega-agatoxin IVA 2-5 fold lower than reported for P current, but approximately 9-fold higher than reported for Q current.

Animals↗
PubMed1997

Importance of the different beta subunits in the membrane expression of the alpha1A and alpha2 calcium channel subunits: studies using a depolarization-sensitive alpha1A antibody.

The plasma membrane expression of the rat brain calcium channel subunits alpha1A, alpha2-delta and the beta subunits beta1b, beta2a, beta3b and beta4 was examined by transient expression in COS-7 cells. Neither alpha1A nor alpha2-delta localized to the plasma membrane, either alone or when coexpressed. However, coexpression of alpha1A or alpha2-delta/alpha1A with any of the beta subunits caused alpha1A and alpha2 to be targetted to the plasma membrane. The alpha1A antibody is directed against an exofacial epitope at the mouth of the pore, which is not exposed unless cells are depolarized, both for native alpha1A channels in dorsal root ganglion neurons and for alpha1A expressed with a beta subunit. This subsidiary result provides evidence that either channel opening or inactivation causes a conformational change at the mouth of the pore of alpha1A. Immunostaining for alpha1A was obtained in depolarized non-permeabilized cells, indicating correct orientation in the membrane only when it was coexpressed with a beta subunit. In contrast, beta1b and beta2a were associated with the plasma membrane when expressed alone. However, this is not a prerequisite to target alpha1A to the membrane since beta3 and beta4 alone showed no differential localization, but did direct the translocation of alpha1A to the plasma membrane, suggesting a chaperone role for the beta subunits.

Animals↗
PubMed1995

The functional role of the binding site aspartate in muscarinic acetylcholine receptors, probed by site-directed mutagenesis.

Mutation of the Asp in transmembrane domain three of the muscarinic receptors to Asn (M1) or Glu (M1 and M2) strongly reduced the high-affinity component of agonist binding, and the M1 phosphoinositide response. Formation of the acetylcholine-receptor binary complex was also strongly inhibited. In contrast, binary complex formation by other agonists, as well as the antagonist (-)-N-methylscopolamine, was less affected. Ionic bonding between the carboxylate oxyanion and the positively-charged headgroup probably anchors acetylcholine when it is bound in its active conformation, but alternative, non-productive, binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands.

Acetylcholine↗
PubMed1995

The role of charge interactions in muscarinic agonist binding, and receptor-response coupling.

Site-directed mutagenesis has been used to evaluate the roles of the key aspartate and arginine residues in transmembrane domain three of the muscarinic receptors. The results suggest that the formation of an ionic bond between the Asp carboxylate group and the onium headgroup is essential to anchor acetylcholine in its active, bound conformation in both binary agonist-receptor and ternary agonist-receptor-G-protein complexes, but that secondary, non-productive binding modes, promoted by non-polar forces, may contribute to binary complex formation by other ligands. The positive charge of the arginyl side-chain is central to the recognition, and subsequent activation of G-proteins by the agonist-M1 mAChR complex.

Amino Acid Substitution↗
PubMed1995

Oxygen control of the Bradyrhizobium japonicum hemA gene.

The hemA gene of Bradyrhizobium japonicum, which encodes the first enzyme in the heme biosynthetic pathway, is regulated by oxygen. Up to ninefold induction of beta-galactosidase activity is seen when cultures of B. japonicum containing either a plasmid-encoded or a chromosomally integrated hemA-lacZ fusion are shifted to restricted aeration. The oxygen effect is mediated via the FixLJ two-component regulatory system, which regulates the expression of a number of genes involved in the nitrogen fixation process in response to low-oxygen conductions; oxygen induction is lost when the hemA-lacZ fusion is expressed in strains of B. japonicum carrying mutations in fixL or fixJ. The B. japonicum hemA promoter region contains a sequence identical to the Escherichia coli Fnr binding site (positions -46 to -33 relative to the hemA transcription start site). Fnr is a regulatory protein necessary for the oxygen-regulated expression of anaerobic respiratory genes. Activity of a hemA-lacZ fusion construct in which the Fnr box-like sequence was replaced with a BglII site is not induced in B. japonicum cultures grown under restricted aeration. The fnr homolog fixK is FixLJ dependent. Collectively, these data suggest a role for the rhizobial Fnr-like protein, FixK, in the regulation of hemA. Furthermore, the coregulation of hemA with symbiotically important genes via FixLJ is consistent with the idea that hemA is required in the nodule as well as under free-living conditions.

Aldehyde Oxidoreductases↗
PubMed1994

Effect of iron availability on expression of the Bradyrhizobium japonicum hemA gene.

Bradyrhizobium japonicum produces delta-aminolevulinic acid, the universal precursor of tetrapyrroles, in a reaction catalyzed by the product of the hemA gene. Expression of the B. japonicum hemA gene is affected by iron availability. Activity of a hemA-lacZ fusion is increased approximately threefold by iron, and RNA analysis indicates that iron regulation is at the level of mRNA accumulation. To our knowledge, this is the first example of an iron-regulated heme biosynthetic gene in prokaryotes.

Aldehyde Oxidoreductases↗
PubMed1992

Determination of the novel hydroxymethyl glutaryl coenzyme A reductase inhibitor (RP 61969) and its dihydroxy acid hydrolysis product in human plasma by reversed-phase high-performance liquid chromatography.

A method is described for the determination of the novel hydroxymethyl glutaryl coenzyme A reductase inhibitor RP 61969 (I) and its hydrolysis product, the dihydroxy acid RP 62420 (II), in human plasma. A structural isomer of I is used as internal standard. Both I and II were extracted from acidified plasma with diethyl ether. The dried residues were reconstituted in the high-performance liquid chromatography mobile phase and chromatographed on a 5 microns ODS2 column. The mobile phase used was aqueous dipotassium phosphate +tetra-n-butyl ammonium bromide (both 10 mM)-acetonitrile-methanol (60:40:5, v/v). At a flow-rate of 1.5 ml min-1 and ambient temperature, the retention time of II is 3.5 min, that of the internal standard is 5 min, and that of I is 8 min. The method has been validated and applied to the assay of plasma samples resulting from a cell-plasma distribution experiment in human whole blood.

Chromatography, High Pressure Liquid↗
PubMed1992

Characterization of the formate (for) locus, which encodes the cytosolic serine hydroxymethyltransferase of Neurospora crassa.

Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon (C1) metabolism, catalyzing the reaction of serine and tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate serves as a donor of C1 units for the synthesis of numerous compounds, including purines, thymidylate, lipids, and methionine. We provide evidence that the formate (for) locus of Neurospora crassa encodes cytosolic SHMT. The for+ gene was localized to a 2.8-kb BglII fragment by complementation (restoration to formate-independent growth) of a strain carrying a recessive for allele, which confers a growth requirement for formate. The for+ gene encodes a polypeptide of 479 amino acids which shows significant similarity to amino acid sequences of SHMT from bacterial and mammalian sources (47 and 60% amino acid identity, respectively). The for+ mRNA has several different start and stop sites. The abundance of for+ mRNA increased in response to amino acid imbalance induced by glycine supplementation, suggesting regulation by the N. crassa cross-pathway control system, which is analogous to general amino acid control in Saccharomyces cerevisiae. This was confirmed by documenting that for+ expression increased in response to histidine limitation (induced by 3-amino-1,2,4-triazole) and that this response was dependent on the presence of a functional cross-pathway control-1 (cpc-1) gene, which encodes CPC1, a positively acting transcription factor. There are at least five potential CPC1 binding sites upstream of the for+ transcriptional start, as well as one that exactly matches the consensus CPC1 binding site in the first intron of the for+ gene.

Amino Acid Sequence↗