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David J Adams

Publications and source records attributed to David J Adams.

At least 55 records · Page 3Linked to original sources

Mutagenic insertion and chromosome engineering resource (MICER).

Embryonic stem cell technology revolutionized biology by providing a means to assess mammalian gene function in vivo. Although it is now routine to generate mice from embryonic stem cells, one of the principal methods used to create mutations, gene targeting, is a cumbersome process. Here we describe the indexing of 93,960 ready-made insertional targeting vectors from two libraries. 5,925 of these vectors can be used directly to inactivate genes with an average targeting efficiency of 28%. Combinations of vectors from the two libraries can be used to disrupt both alleles of a gene or engineer larger genomic changes such as deletions, duplications, translocations or inversions. These indexed vectors constitute a public resource (Mutagenic Insertion and Chromosome Engineering Resource; MICER) for high-throughput, targeted manipulation of the mouse genome.

Animals↗

The alpha2delta auxiliary subunit reduces affinity of omega-conotoxins for recombinant N-type (Cav2.2) calcium channels.

The omega-conotoxins from fish-hunting cone snails are potent inhibitors of voltage-gated calcium channels. The omega-conotoxins MVIIA and CVID are selective N-type calcium channel inhibitors with potential in the treatment of chronic pain. The beta and alpha(2)delta-1 auxiliary subunits influence the expression and characteristics of the alpha(1B) subunit of N-type channels and are differentially regulated in disease states, including pain. In this study, we examined the influence of these auxiliary subunits on the ability of the omega-conotoxins GVIA, MVIIA, CVID and analogues to inhibit peripheral and central forms of the rat N-type channels. Although the beta3 subunit had little influence on the on- and off-rates of omega-conotoxins, coexpression of alpha(2)delta with alpha(1B) significantly reduced on-rates and equilibrium inhibition at both the central and peripheral isoforms of the N-type channels. The alpha(2)delta also enhanced the selectivity of MVIIA, but not CVID, for the central isoform. Similar but less pronounced trends were also observed for N-type channels expressed in human embryonic kidney cells. The influence of alpha(2)delta was not affected by oocyte deglycosylation. The extent of recovery from the omega-conotoxin block was least for GVIA, intermediate for MVIIA, and almost complete for CVID. Application of a hyperpolarizing holding potential (-120 mV) did not significantly enhance the extent of CVID recovery. Interestingly, [R10K]MVIIA and [O10K]GVIA had greater recovery from the block, whereas [K10R]CVID had reduced recovery from the block, indicating that position 10 had an important influence on the extent of omega-conotoxin reversibility. Recovery from CVID block was reduced in the presence of alpha(2)delta in human embryonic kidney cells and in oocytes expressing alpha(1B-b). These results may have implications for the antinociceptive properties of omega-conotoxins, given that the alpha(2)delta subunit is up-regulated in certain pain states.

Amino Acid Sequence↗

Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2.

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na(+) channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na(+) (Na(v)) channels contain typical PY motifs (PPXY), and a further Na(v) contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na(v) channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na(+) channel (Na(v)1.2 and Na(v)1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2, Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed the activity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na(v) channels in vivo.

Amino Acid Sequence↗

Structures of muO-conotoxins from Conus marmoreus. I nhibitors of tetrodotoxin (TTX)-sensitive and TTX-resistant sodium channels in mammalian sensory neurons.

The microO-conotoxins are an intriguing class of conotoxins targeting various voltage-dependent sodium channels and molluscan calcium channels. In the current study, we have shown MrVIA and MrVIB to be the first known peptidic inhibitors of the transient tetrodotoxin-resistant (TTX-R) Na(+) current in rat dorsal root ganglion neurons, in addition to inhibiting tetrodotoxin-sensitive Na(+) currents. Human TTX-R sodium channels are a therapeutic target for indications such as pain, highlighting the importance of the microO-conotoxins as potential leads for drug development. Furthermore, we have used NMR spectroscopy to provide the first structural information on this class of conotoxins. MrVIA and MrVIB are hydrophobic peptides that aggregate in aqueous solution but were solubilized in 50% acetonitrile/water. The three-dimensional structure of MrVIB consists of a small beta-sheet and a cystine knot arrangement of the three-disulfide bonds. It contains four backbone "loops" between successive cysteine residues that are exposed to the solvent to varying degrees. The largest of these, loop 2, is the most disordered part of the molecule, most likely due to flexibility in solution. This disorder is the most striking difference between the structures of MrVIB and the known delta- and omega-conotoxins, which along with the microO-conotoxins are members of the O superfamily. Loop 2 of omega-conotoxins has previously been shown to contain residues critical for binding to voltage-gated calcium channels, and it is interesting to speculate that the flexibility observed in MrVIB may accommodate binding to both sodium and molluscan calcium channels.

Amino Acid Sequence↗

Overexpressed Ca(v)beta3 inhibits N-type (Cav2.2) calcium channel currents through a hyperpolarizing shift of ultra-slow and closed-state inactivation.

It has been shown that beta auxiliary subunits increase current amplitude in voltage-dependent calcium channels. In this study, however, we found a novel inhibitory effect of beta3 subunit on macroscopic Ba(2+) currents through recombinant N- and R-type calcium channels expressed in Xenopus oocytes. Overexpressed beta3 (12.5 ng/cell cRNA) significantly suppressed N- and R-type, but not L-type, calcium channel currents at "physiological" holding potentials (HPs) of -60 and -80 mV. At a HP of -80 mV, coinjection of various concentrations (0-12.5 ng) of the beta3 with Ca(v)2.2alpha(1) and alpha(2)delta enhanced the maximum conductance of expressed channels at lower beta3 concentrations but at higher concentrations (>2.5 ng/cell) caused a marked inhibition. The beta3-induced current suppression was reversed at a HP of -120 mV, suggesting that the inhibition was voltage dependent. A high concentration of Ba(2+) (40 mM) as a charge carrier also largely diminished the effect of beta3 at -80 mV. Therefore, experimental conditions (HP, divalent cation concentration, and beta3 subunit concentration) approaching normal physiological conditions were critical to elucidate the full extent of this novel beta3 effect. Steady-state inactivation curves revealed that N-type channels exhibited "closed-state" inactivation without beta3, and that beta3 caused an approximately 40-mV negative shift of the inactivation, producing a second component with an inactivation midpoint of approximately -85 mV. The inactivation of N-type channels in the presence of a high concentration (12.5 ng/cell) of beta3 developed slowly and the time-dependent inactivation curve was best fit by the sum of two exponential functions with time constants of 14 s and 8.8 min at -80 mV. Similar "ultra-slow" inactivation was observed for N-type channels without beta3. Thus, beta3 can have a profound negative regulatory effect on N-type (and also R-type) calcium channels by causing a hyperpolarizing shift of the inactivation without affecting "ultra-slow" and "closed-state" inactivation properties.

Animals↗

Chemical and functional identification and characterization of novel sulfated alpha-conotoxins from the cone snail Conus anemone.

An LC/MS analysis with diagnostic screening for the detection of peptides with posttranslational modifications revealed the presence of novel sulfated peptides within the alpha-conotoxin molecular mass range in Conus anemone crude venom. A functional assay of the extract showed activity at several neuronal nicotinic acetylcholine receptors (nAChRs). Three sulfated alpha-conotoxins (AnIA, AnIB, and AnIC) were identified by LC/MS and assay-directed fractionation and sequenced after purification. The most active of these, alpha-AnIB, was further characterized and used to investigate the influence of posttranslational modifications on affinity. Synthetic AnIB exhibited subnanomolar potency at the rat alpha3beta2 nAChR (IC50 0.3 nM) and was 200-fold less active on the rat alpha7 nAChR (IC50 76 nM). The unsulfated peptide [Tyr16]AnIB showed a 2-fold and 10-fold decrease in activities at alpha3beta2 (IC50 0.6 nM) and alpha7 (IC50 836 nM) nAChR, respectively. Likewise, removal of the C-terminal amide had a greater influence on potency at the alpha7 (IC50 367 nM) than at the alpha3beta2 nAChR (IC50 0.5 nM). Stepwise removal of two N-terminal glycine residues revealed that these residues affect the binding kinetics of the peptide. Comparison with similar 4/7-alpha-conotoxin sequences suggests that residue 11 (alanine or glycine) and residue 14 (glutamine) constitute important determinants for alpha3beta2 selectivity, whereas the C-terminal amidation and sulfation at tyrosine-16 favor alpha7 affinity.

Animals↗

Potassium channels and membrane potential in the modulation of intracellular calcium in vascular endothelial cells.

The endothelium plays a vital role in the control of vascular functions, including modulation of tone; permeability and barrier properties; platelet adhesion and aggregation; and secretion of paracrine factors. Critical signaling events in many of these functions involve an increase in intracellular free Ca(2+) concentration ([Ca(2+)](i)). This rise in [Ca(2+)](i) occurs via an interplay between several mechanisms, including release from intracellular stores, entry from the extracellular space through store depletion and second messenger-mediated processes, and the establishment of a favorable electrochemical gradient. The focus of this review centers on the role of potassium channels and membrane potential in the creation of a favorable electrochemical gradient for Ca(2+) entry. In addition, evidence is examined for the existence of various classes of potassium channels and the possible influence of regional variation in expression and experimental conditions.

Animals↗

Fungal cell wall chitinases and glucanases.

The fungal cell wall is a complex structure composed of chitin, glucans and other polymers, and there is evidence of extensive cross-linking between these components. The wall structure is highly dynamic, changing constantly during cell division, growth and morphogenesis. Hydrolytic enzymes, closely associated with the cell wall, have been implicated in the maintenance of wall plasticity and may have roles during branching and cross-linking of polymers. Most fungal cell wall hydrolases identified to date have chitinase or glucanase activity and this short article reviews the apparent functions of these enzymes in unicellular and filamentous fungi, and the mechanisms that regulate enzyme activity in yeasts.

Cell Wall↗

Functional maturation of isolated neural progenitor cells from the adult rat hippocampus.

Although neural progenitor cells (NPCs) may provide a source of new neurons to alleviate neural trauma, little is known about their electrical properties as they differentiate. We have previously shown that single NPCs from the adult rat hippocampus can be cloned in the presence of heparan sulphate chains purified from the hippocampus, and that these cells can be pushed into a proliferative phenotype with the mitogen FGF2 [Chipperfield, H., Bedi, K.S., Cool, S.M. & Nurcombe, V. (2002) Int. J. Dev. Biol., 46, 661-670]. In this study, the active and passive electrical properties of both undifferentiated and differentiated adult hippocampal NPCs, from 0 to 12 days in vitro as single-cell preparations, were investigated. Sparsely plated, undifferentiated NPCs had a resting membrane potential of approximately -90 mV and were electrically inexcitable. In > 70%, ATP and benzoylbenzoyl-ATP evoked an inward current and membrane depolarization, whereas acetylcholine, noradrenaline, glutamate and GABA had no detectable effect. In Fura-2-loaded undifferentiated NPCs, ATP and benzoylbenzoyl-ATP evoked a transient increase in the intracellular free Ca(2+) concentration, which was dependent on extracellular Ca(2+) and was inhibited reversibly by pyridoxalphosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS), a P2 receptor antagonist. After differentiation, NPC-derived neurons became electrically excitable, expressing voltage-dependent TTX-sensitive Na(+) channels, low- and high-voltage-activated Ca(2+) channels and delayed-rectifier K(+) channels. Differentiated cells also possessed functional glutamate, GABA, glycine and purinergic (P2X) receptors. Appearance of voltage-dependent and ligand-gated ion channels appears to be an important early step in the differentiation of NPCs.

Animals↗

Auxiliary subunit regulation of high-voltage activated calcium channels expressed in mammalian cells.

The effects of auxiliary calcium channel subunits on the expression and functional properties of high-voltage activated (HVA) calcium channels have been studied extensively in the Xenopus oocyte expression system, but are less completely characterized in a mammalian cellular environment. Here, we provide the first systematic analysis of the effects of calcium channel beta and alpha(2)-delta subunits on expression levels and biophysical properties of three different types (Ca(v)1.2, Ca(v)2.1 and Ca(v)2.3) of HVA calcium channels expressed in tsA-201 cells. Our data show that Ca(v)1.2 and Ca(v)2.3 channels yield significant barium current in the absence of any auxiliary subunits. Although calcium channel beta subunits were in principle capable of increasing whole cell conductance, this effect was dependent on the type of calcium channel alpha(1) subunit, and beta(3) subunits altogether failed to enhance current amplitude irrespective of channel subtype. Moreover, the alpha(2)-delta subunit alone is capable of increasing current amplitude of each channel type examined, and at least for members of the Ca(v)2 channel family, appears to act synergistically with beta subunits. In general agreement with previous studies, channel activation and inactivation gating was regulated both by beta and by alpha(2)-delta subunits. However, whereas pronounced regulation of inactivation characteristics was seen with the majority of the auxiliary subunits, effects on voltage dependence of activation were only small (< 5 mV). Overall, through a systematic approach, we have elucidated a previously underestimated role of the alpha(2)-delta(1) subunit with regard to current enhancement and kinetics. Moreover, the effects of each auxiliary subunit on whole cell conductance and channel gating appear to be specifically tailored to subsets of calcium channel subtypes.

Animals↗

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors.

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Adenosine Triphosphate↗

HADHB, HuR, and CP1 bind to the distal 3'-untranslated region of human renin mRNA and differentially modulate renin expression.

Production of renin is critically dependent on modulation of REN mRNA stability. Here we sought to elucidate the molecular mechanisms involved. Transfections of renin-expressing Calu-6 cells with reporter constructs showed that a cis-acting 34-nucleotide AU-rich "renin stability regulatory element" in the REN 3'-untranslated region (3'-UTR) contributes to basal REN mRNA instability. Yeast three-hybrid screening with the REN 3'-UTR as bait isolated HADHB (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein) beta-subunit) as a novel REN mRNA-binding protein. Recombinant HADHB bound specifically to the 3'-UTR of REN mRNA, as did the known mRNA stabilizers HuR and CP1 (poly(C)-binding protein-1). This required the renin stability regulatory element. Forskolin, which augments REN mRNA stability in Calu-6 cells, increased binding of several proteins, including HuR and CP1, to the REN 3'-UTR, whereas 4-bromocrotonic acid, a specific thiolase inhibitor, decreased binding and elevated renin protein levels. Upon decreasing HADHB mRNA with RNA interference, renin protein and mRNA stability increased, whereas RNA interference against HuR caused these to decrease. Immunoprecipitation and reverse transcription-PCR of Calu-6 extracts confirmed that HADHB, HuR, and CP1 each associate with REN mRNA in vivo. Intracellular imaging revealed distinct localization of HADHB to mitochondria, HuR to nuclei, and CP1 throughout the cell. Immunohistochemistry demonstrated enrichment of HADHB in renin-producing renal juxtaglomerular cells. In conclusion, HADHB, HuR, and CP1 are novel REN mRNA-binding proteins that target a cis-element in the 3'-UTR of REN mRNA and regulate renin production. cAMP-mediated increased REN mRNA stability may involve stimulation of HuR and CP1, whereas REN mRNA decay may involve thiolase-dependent pathways.

3' Untranslated Regions↗

Inhibition of the norepinephrine transporter by the venom peptide chi-MrIA. Site of action, Na+ dependence, and structure-activity relationship.

chi-Conopeptide MrIA (chi-MrIA) is a 13-residue peptide contained in the venom of the predatory marine snail Conus marmoreus that has been found to inhibit the norepinephrine transporter (NET). We investigated whether chi-MrIA targeted the other members of the monoamine transporter family and found no effect of the peptide (100 microM) on the activity of the dopamine transporter and the serotonin transporter, indicating a high specificity of action. The binding of the NET inhibitors, [3H]nisoxetine and [3H]mazindol, to the expressed rat and human NET was inhibited by chi-MrIA with the conopeptide displaying a slight preference toward the rat isoform. For both radioligands, saturation binding studies showed that the inhibition by chi-MrIA was competitive in nature. It has previously been demonstrated that chi-MrIA does not compete with norepinephrine, unlike classically described NET inhibitors such as nisoxetine and mazindol that do. This pattern of behavior implies that the binding site for chi-MrIA on the NET overlaps the antidepressant binding site and is wholly distinct from the substrate binding site. The inhibitory effect of chi-MrIA was found to be dependent on Na+ with the conopeptide becoming a less effective blocker of [3H]norepinephrine by the NET under the conditions of reduced extracellular Na+. In this respect, chi-MrIA is similar to the antidepressant inhibitors of the NET. The structure-activity relationship of chi-MrIA was investigated by alanine scanning. Four residues in the first cysteine-bracketed loop of chi-MrIA and a His in loop 2 played a dominant role in the interaction between chi-MrIA and the NET. H alpha chemical shift comparisons indicated that side-chain interactions at these key positions were structurally perturbed by the replacement of Gly-6. From these data, we present a model of the structure of chi-MrIA that shows the relative orientation of the key binding residues. This model provides a new molecular caliper for probing the structure of the NET.

Amino Acid Sequence↗

Allosteric alpha 1-adrenoreceptor antagonism by the conopeptide rho-TIA.

A peptide contained in the venom of the predatory marine snail Conus tulipa, rho-TIA, has previously been shown to possess alpha1-adrenoreceptor antagonist activity. Here, we further characterize its pharmacological activity as well as its structure-activity relationships. In the isolated rat vas deferens, rho-TIA inhibited alpha1-adrenoreceptor-mediated increases in cytosolic Ca2+ concentration that were triggered by norepinephrine, but did not affect presynaptic alpha2-adrenoreceptor-mediated responses. In radioligand binding assays using [125I]HEAT, rho-TIA displayed slightly greater potency at the alpha 1B than at the alpha 1A or alpha 1D subtypes. Moreover, although it did not affect the rate of association for [3H]prazosin binding to the alpha 1B-adrenoreceptor, the dissociation rate was increased, indicating non-competitive antagonism by rho-TIA. N-terminally truncated analogs of rho-TIA were less active than the full-length peptide, with a large decline in activity observed upon removal of the fourth residue of rho-TIA (Arg4). An alanine walk of rho-TIA confirmed the importance of Arg4 for activity and revealed a number of other residues clustered around Arg4 that contribute to the potency of rho-TIA. The unique allosteric antagonism of rho-TIA resulting from its interaction with receptor residues that constitute a binding site that is distinct from that of the classical competitive alpha1-adrenoreceptor antagonists may allow the development of inhibitors that are highly subtype selective.

Adrenergic alpha-1 Receptor Antagonists↗

The activity of camptothecin analogues is enhanced in histocultures of human tumors and human tumor xenografts by modulation of extracellular pH.

BACKGROUND: Most solid human tumors exist in an acidic microenvironment, due in part to inefficient vasculature and a higher intrinsic rate of glycolysis. This leads to a tumor-selective pH gradient, which can be exploited therapeutically with antitumor agents such as the camptothecins (CPTs). Previous work in this laboratory has shown that camptothecin activity is enhanced 40- to 60-fold in monolayer cell culture by reducing the extracellular pH to 6.8. Three-dimensional histoculture has been shown to be a technique that allows human tumor tissue to grow in an in vivo-like way with maintenance of tissue histology and function and drug sensitivity for long periods of time. PURPOSE: In the current study, we utilized these features of histoculture to study new analogues of camptothecin that have superior pharmacological properties. METHODS: We evaluated six CPT analogues in histocultures of human brain, neuroblastoma, breast, colon, and prostate tumors. Fragments were exposed to 10,11-methylenedioxy-CPT (MDC), 7-chloromethyl-MDC, SN-38, topotecan (TPT), 9-amino-CPT, 10-amino-CPT, paclitaxel, 5-fluorouracil, 4-hydroperoxycyclophosphamide and doxorubicin, and antitumor activity was assessed. For in vivo tumor outgrowth studies, fragments were treated in parallel, implanted into nude mice, and monitored for development of tumors. RESULTS. Against 15 of 16 tumor xenografts and all primary tumor samples tested, all compounds were cytotoxic at pH 7.4 (IC(50) range 13-921 microM). MDC, SN-38, TPT, and 9-amino-CPT achieved an average 5-fold increase in activity (range 3-14) at pH 6.8, while 7-chloromethyl-MDC was enhanced 8-fold (range 6-14). The most potentiated analogue was 10-amino-CPT at 27-fold (range 17-49). In contrast, the other agents were active against one or more tumor types but were not enhanced by acidic pH. Importantly, the toxicity of MDC in histoculture of D54 glioma xenografts strongly correlated with the outgrowth of treated fragments subsequently implanted in vivo. CONCLUSION: Evaluation of anticancer drug activity in native-state histoculture supports the concept that pH modulation may be an important approach to improve the selectivity and antitumor effectiveness of camptothecin-based chemotherapy.

Animals↗

Muscarinic and nicotinic ACh receptor activation differentially mobilize Ca2+ in rat intracardiac ganglion neurons.

The origin of intracellular Ca2+ concentration ([Ca2+]i) transients stimulated by nicotinic (nAChR) and muscarinic (mAChR) receptor activation was investigated in fura-2-loaded neonatal rat intracardiac neurons. ACh evoked [Ca2+]i increases that were reduced to approximately 60% of control in the presence of either atropine (1 microM) or mecamylamine (3 microM) and to <20% in the presence of both antagonists. Removal of external Ca2+ reduced ACh-induced responses to 58% of control, which was unchanged in the presence of mecamylamine but reduced to 5% of control by atropine. The nAChR-induced [Ca2+]i response was reduced to 50% by 10 microM ryanodine, whereas the mAChR-induced response was unaffected by ryanodine, suggesting that Ca2+ release from ryanodine-sensitive Ca2+ stores may only contribute to the nAChR-induced [Ca2+]i responses. Perforated-patch whole cell recording at -60 mV shows that the rise in [Ca2+]i is concomitant with slow outward currents on mAChR activation and with rapid inward currents after nAChR activation. In conclusion, different signaling pathways mediate the rise in [Ca2+]i and membrane currents evoked by ACh binding to nicotinic and muscarinic receptors in rat intracardiac neurons.

Animals↗

Alpha-conotoxins PnIA and [A10L]PnIA stabilize different states of the alpha7-L247T nicotinic acetylcholine receptor.

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [A10L]PnIA and alanine scan derivatives of [A10L]PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L]PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nm, respectively. Rates of onset of inhibition were similar for PnIA and [A10L]PnIA; however, the rate of recovery was slower for [A10L]PnIA, indicating that the increased potency of [A10L]PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [A10L]PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [A10L]PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nm. In contrast, [A10L]PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [A10L]PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [A10L]PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

Amino Acid Sequence↗

Macrocyclic inhibitors of the bacterial cell wall biosynthesis enzyme MurD.

Computer-based molecular design has been used to produce a series of new macrocyclic systems targeted against the bacterial cell wall biosynthetic enzyme MurD. Following their preparation, which involved a novel metathesis-based cyclisation as the key step, these systems were found to show good inhibition when assayed against the MurD enzyme.

Binding Sites↗