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Steven D Buckingham

Publications and source records attributed to Steven D Buckingham.

14 recordsLinked to original sources
PubMed2006

Neonicotinoid insecticides display partial and super agonist actions on native insect nicotinic acetylcholine receptors.

Nicotinic acetylcholine receptors (nAChRs) are present in high density in insect nervous tissue and are targeted by neonicotinoid insecticides. Improved understanding of the actions of these insecticides will assist in the development of new compounds. Here, we have used whole-cell patch-clamp recording of cholinergic neurons cultured from the central nervous system of 3rd instar Drosophila larvae to examine the actions of acetylcholine (ACh) and nicotine, as well as the neonicotinoids imidacloprid, clothianidin and P-CH-clothianidin on native nAChRs of these neurons. Dose-response data yield an EC(50) value for ACh of 19 microm. Both nicotine and imidacloprid act as low efficacy agonists at native nAChRs, evoking maximal current amplitudes 10-14% of those observed for ACh. Conversely, clothianidin and P-CH-clothianidin evoke maximal current amplitudes up to 56% greater than those evoked by 100 microm ACh in the same neurons. This is the first demonstration of 'super' agonist actions of an insecticide on native insect nAChRs. Cell-attached recordings indicate that super agonism results from more frequent openings at the largest (63.5 pS) conductance state observed.

Acetylcholine↗
PubMed2006

Replacement of asparagine with arginine at the extracellular end of the second transmembrane (M2) region of insect GABA receptors increases sensitivity to penicillin G.

The actions of penicillin-G (PCG) on wild-type and mutant Drosophila GABA receptor (RDL) subunits expressed in Xenopus oocytes were studied under two-electrode voltage-clamp. PCG was found to be a non-competitive antagonist of homomeric Drosophila RDL receptors with an IC(50) of 20.41 +/- 1.66 mM at EC(50) GABA. Substitution of a single amino acid (N318R) at the extracellular end of the channel lining region of the RDL subunit increased the potency of GABA approximately four fold, and increased the IC(50) of PCG to 5.09 +/- 0.38 mM. Although the antagonism by PCG on wild-type RDL receptors was independent of membrane potential, PCG action on the N318R mutant showed pronounced voltage-dependency, being much more effective at positive membrane potentials. Thus, in RDL homomers, the replacement of N318 by R318, a residue present at the equivalent position in vertebrate GABA(A) receptors, confers a vertebrate-like PCG pharmacology to the N318R mutant receptor. The A301S mutation that confers resistance to dieldrin did not significantly affect the antagonism by PCG.

Animals↗
PubMed2006

Atypical phenotypes from flatworm Kv3 channels.

Divergence of the Shaker superfamily of voltage-gated (Kv) ion channels early in metazoan evolution created numerous electrical phenotypes that were presumably selected to produce a wide range of excitability characteristics in neurons, myocytes, and other cells. A comparative approach that emphasizes this early radiation provides a comprehensive sampling of sequence space that is necessary to develop generally applicable models of the structure-function relationship in the Kv potassium channel family. We have cloned and characterized two Shaw-type potassium channels from a flatworm (Notoplana atomata) that is arguably a representative of early diverging bilaterians. When expressed in Xenopus oocytes, one of these cloned channels, N.at-Kv3.1, exhibits a noninactivating, outward current with slow opening kinetics that are dependent on both the holding potential and the activating potential. A second Shaw-type channel, N.at-Kv3.2, has very different properties, showing weak inward rectification. These results demonstrate that broad phylogenetic sampling of proteins of a single family will reveal unexpected properties that lead to new interpretations of structure-function relationships.

Animals↗
PubMed2006

Invertebrate studies and their ongoing contributions to neuroscience.

Invertebrates have been deployed very successfully in experimental studies of the nervous system and neuromuscular junctions. Many important discoveries on axonal conduction, synaptic transmission, integrative neurobiology and behaviour have been made by investigations of these remarkable animals. Their advantages as model organisms for investigations of nervous systems include (a) the large diameter of neurons, glia and muscle cells of some invertebrates, thereby facilitating microelectrode recordings; (b) simple nervous systems with few neurons, enhancing the tractability of neuronal circuitry; and (c) well-defined behaviours, which lend themselves to physiological and genetic dissection. Genetic model organisms such as Drosophila melanogaster and Caenorhabditis elegans have provided powerful genetic approaches to central questions concerning nervous system development, learning and memory and the cellular and molecular basis of behaviour. The process of attributing function to particular gene products has been greatly accelerated in recent years with access to entire genome sequences and the application of reverse genetic (e.g. RNA interference, RNAi) and other post-genome technologies (e.g. microarrays). Studies of many other invertebrates, notably the honeybee (Apis mellifera), a nudibranch mollusc (Aplysia californica), locusts, lobsters, crabs, annelids and jellyfish have all assisted in the development of major concepts in neuroscience. The future is equally bright with ease of access to genome-wide reverse genetic technologies, and the development of optical recordings using voltage and intracellular calcium sensors genetically targeted to selected individual and groups of neurons.

Animals↗
PubMed2005

Data mining for protein-protein interactions in invertebrate model organisms.

Well-annotated genome databases are available for many invertebrate species, notably the fruitfly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans. An adequate interpretation of this information at the biological level requires the exploration of the interactions between the gene products. Knowledge of protein interactions and the components of cell signalling pathways in the fly and worm are particularly valuable as hypotheses can be rapidly tested using the powerful genetic toolkits available. Invertebrates offer additional experimental advantages when attempting to characterise protein-protein interactions (PPIs). Their relatively small genome size compared to mammals helps to reduce missed interactions due to redundancy, and their function can be addressed using forward (mutants) and reverse (RNA interference) genetics. However, the researcher looking for evidence of PPIs for a protein of interest is faced with the challenge of extracting interaction data from sources that are highly varied, such as the results of microarray experiments in the unstructured text of research papers. This challenge is greatly reduced by a range of public databases of curated information, as well as publicly available, enhanced search engines, which can provide either direct experimental evidence for a PPI, or valuable clues for generating new hypotheses.

Animals↗
PubMed2005

A7DB: a relational database for mutational, physiological and pharmacological data related to the alpha7 nicotinic acetylcholine receptor.

BACKGROUND: Nicotinic acetylcholine receptors (nAChRs) are pentameric proteins that are important drug targets for a variety of diseases including Alzheimer's, schizophrenia and various forms of epilepsy. One of the most intensively studied nAChR subunits in recent years has been alpha7. This subunit can form functional homomeric pentamers (alpha7)5, which can make interpretation of physiological and structural data much simpler. The growing amount of structural, pharmacological and physiological data for these receptors indicates the need for a dedicated and accurate database to provide a means to access this information in a coherent manner. DESCRIPTION: A7DB http://www.lgics.org/a7db/ is a new relational database of manually curated experimental physiological data associated with the alpha7 nAChR. It aims to store as much of the pharmacology, physiology and structural data pertaining to the alpha7 nAChR. The data is accessed via web interface that allows a user to search the data in multiple ways: 1) a simple text query 2) an incremental query builder 3) an interactive query builder and 4) a file-based uploadable query. It currently holds more than 460 separately reported experiments on over 85 mutations. CONCLUSIONS: A7DB will be a useful tool to molecular biologists and bioinformaticians not only working on the alpha7 receptor family of proteins but also in the more general context of nicotinic receptor modelling. Furthermore it sets a precedent for expansion with the inclusion of all nicotinic receptor families and eventually all cys-loop receptor families.

Cholinergic Agents↗
PubMed2005

Structure and function of two-pore-domain K+ channels: contributions from genetic model organisms.

K+ channels that possess two pore domains in each channel subunit are common in many animal tissues. Such channels are generated from large families of subunits and are implicated in several functions, including temperature sensation, responses to ischaemia, K+ homeostasis and setting the resting potential of the cell. Their activity can be modulated by polyunsaturated fatty acids, pH and oxygen, and some are candidate targets of volatile anaesthetics. However, despite their potential as targets for novel drugs for human health, comparatively little is known about the molecular basis of their diverse physiological and pharmacological properties. Genetic model organisms have considerable potential for improving our understanding of these channels. In this article, we review the contributions of some of these genetic model organisms to recent advances in our knowledge of two-pore-domain K+ channels.

Animals↗
PubMed2005

Chemistry-to-gene screens in Caenorhabditis elegans.

The nematode worm Caenorhabditis elegans is a genetic model organism linked to an impressive portfolio of fundamental discoveries in biology. This free-living nematode, which can be easily and inexpensively grown in the laboratory, is also a natural vehicle for screening for drugs that are active against nematode parasites. Here, we show that chemistry-to-gene screens using this animal model can define targets of antiparasitic drugs, identify novel candidate drug targets and contribute to the discovery of new drugs for treating human diseases.

Animals↗
PubMed2005

Computer simulations of high-pass filtering in zebrafish larval muscle fibres.

Larval somatic muscle of the zebrafish, Danio rerio, like that of some other organisms, responds to a sustained depolarization with one, and only one, action potential. Here, we report computer simulations, using the NEURON simulation programme, of sodium and potassium currents of somatic muscle of larval Danio rerio to investigate their possible contribution to once-only firing. Our computer model incorporated simulated sodium and potassium ion channels based on steady-state and kinetic parameters derived from a recent electrophysiological study. The model responded to sustained depolarizations with a single action potential at all levels of depolarization above threshold. By varying several parameters of the sodium and potassium currents systematically, the minimum changes necessary to produce repetitive firing were found to be a positive shift in the half-inactivation and a negative shift in the half-activation potentials for the sodium current, accompanied by a slowing of the rate of inactivation to half of the experimentally observed values. This suggests that once-only spiking can be attributed to the steady-state values of activation and inactivation of the sodium current, along with a slower rate of inactivation. Mapping of the resultant firing properties against steady-state and kinetic ion channel parameters revealed a high safety factor for once-only firing and showed that the time constant of inactivation of the sodium current was the key determinant of once-only or repetitive firing. The rapidly inactivating potassium current does not influence once-only firing or the maximum rate of firing in response to periodic excitation in these simulations. Although a contribution of other currents to produce once-only firing has not been excluded, this model suggests that the properties of the sodium current are sufficient to account for once-only firing.

Action Potentials↗
PubMed2004

RNA interference: from model organisms towards therapy for neural and neuromuscular disorders.

Experimental RNA interference (RNAi) leading to the selective knockdown of gene function is induced by introducing into cells either double stranded RNA (dsRNA), or short interfering RNA (siRNA) fragments into which dsRNA is cut. The siRNA triggers degradation of homologous messenger RNA (mRNA). Widely used as a research tool in the genetic model organisms Caenorhabditis elegans, Drosophila melanogaster and mouse to investigate the function of individual genes, RNAi has also been deployed in genome-wide, specific gene-knockdown screens. Recent rapid progress in the application of RNAi to mammalian cells, including neurons and muscle cells, offers new approaches to drug target identification and validation. Advances in targeted delivery of RNAi-inducing molecules has raised the possibility of using RNAi directly as a therapy for a variety of human genetic and other neural and neuromuscular disorders. Here, we review examples of the application of RNAi to worm, fly and mouse models of such diseases aimed at understanding their pathophysiology and we address problems to be solved in developing RNAi-based therapies.

Animals↗
PubMed2004

Sodium and potassium currents of larval zebrafish muscle fibres.

The steady-state and kinetic properties of Na(+) and K(+) currents of inner (white) and outer (red) muscles of zebrafish larvae 4-6 days post-fertilization (d.p.f.) are described. In inner muscle, the outward currents were half-activated at -1.0 mV and half-inactivated at -30.4 mV, and completely inactivated within 100 ms of depolarization. The inward currents of inner fibres were half-activated at -7.3 mV and half-inactivated at -74.5 mV and completely inactivated within 5 ms of depolarization. Inner muscle fibres were found to support action potentials, while no action potentials could be evoked in outer muscles. In inner muscle fibres, all tested levels of depolarizing current above a threshold value evoked only one action potential. However, spiking at frequencies of up to 200 cycles s(-1) was evoked by the injection of depolarizing pulses separated by short hyperpolarizing currents. We suggest that the properties of the inward sodium and outward potassium currents permit high frequency firing in response to a pulsatile depolarizing input of the kind expected in fast swimming, whilst safeguarding against tetany during a strong depolarization.

Action Potentials↗
PubMed2003

Bicuculline-insensitive GABA-gated Cl- channels in the larval nervous system of the moth Manduca sexta.

GABA-gated Cl(-) channels were studied in the nervous system of the larval tobacco hawk moth, Manduca sexta, using electrophysiology, (36)Cl(-) uptake into membrane microsacs and immunocytochemistry. A GABA-induced increase in Cl(-) conductance was recorded from a visually identifiable neurone ( fg1) in the desheathed frontal ganglion. The response was insensitive to the vertebrate GABA(A) receptor antagonist, bicuculline, but was blocked by picrotoxinin. Bicuculline-insensitive, picrotoxinin-sensitive, GABA-stimulated (36)Cl(-) uptake was also detected in membrane microsacs prepared from the isolated larval M. sexta nervous system. Such receptors appear to be the major type of GABA receptor in larval nervous system membrane microsac preparations. An antibody raised against a 17 amino acid peptide, based on the predicted C-terminus of the Drosophila GABA receptor subunit (RDL), stained not only cell bodies, including that of fg1, but also the neuropile in the frontal ganglion, indicating the existence of RDL-like GABA receptor subunits in neurones of this ganglion. Thus, bicuculline-insensitive GABA-gated Cl(-) channels are present in the larval nervous system of M. sexta.

Animals↗
PubMed2002

Role of high-voltage activated potassium currents in high-frequency neuronal firing: evidence from a basal metazoan.

Certain neurons of vertebrates are specialized for high-frequency firing. Interestingly, high-frequency firing is also seen in central neurons in basal bilateral metazoans. Recently, the role of potassium currents with rightward-shifted activation curves in producing high-frequency firing has come under scrutiny. We apply intracellular recording, patch-clamp techniques, and compartmental modeling to examine the roles of rightward-shifted potassium currents in repetitive firing and shaping of action potentials in central neurons of the flatworm, Notoplana atomata (Phylum Platyhelminthes). The kinetic properties of potassium and sodium currents were determined from patch-clamp experiments on dissociated brain cells. To predict the effects of changing the steady-state and kinetic properties of these potassium currents, these data were incorporated into a computer model of a 30-microm spherical cell with the levels of current adjusted to approximate the values recorded in voltage-clamp experiments. The model was able to support regenerative spikes at high frequencies in response to injected current. Current-clamp recordings of cultured cells and of neurons in situ also showed evidence of very-high-frequency firing. Adjusting the ratio of inactivating to non-inactivating potassium currents had little effect upon the firing pattern of the cell or its ability to fire at high frequencies, whereas the presence of the non-inactivating current was necessary for repetitive firing. Computer simulations suggested that the rightward shift in voltage sensitivity confers a raised firing threshold, while rapid channel kinetics underlie high frequency firing, and the large activation range enhances the coding range of the cell.

Action Potentials↗