Biomedical subjects

K Dineley-Miller

Publications and source records attributed to K Dineley-Miller.

3 recordsLinked to original sources

PubMed1995

Production of polyclonal antisera that recognize and distinguish between the extracellular domains of neuronal nicotinic acetylcholine receptor subunits.

Ligand-gated ion channels are oligomeric transmembrane proteins that usually contain more than one kind of monomer. The variety of monomers available to participate in oligomer formation and the apparent latitude in acceptable monomer combinations allows considerable diversity. Mechanisms for identifying the monomers comprising specific receptors are needed. We have generated affinity-purified polyclonal antisera that recognize the extracellular domain of nine neuronal nicotinic acetylcholine receptor (nAChR) subunits and distinguish between them. We prepared these antisera by immunizing rabbits with bacterially expressed recombinant protein representing the N-terminal extracellular domain of each neuronal nAChR subunit followed by affinity purification of antibodies against synthetic peptides corresponding to residues 68-81 of the alpha 1 subunit. We demonstrate subunit specificity of each affinity-purified antisera by western blots of the bacterially expressed protein and immunoblot against peptide. We further used these antibodies to demonstrate expression of neuronal nAChR subunits on the surface of transiently transfected simian kidney (COS-7) cells.

Amino Acid Sequence↗

PubMed1993

Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium.

A full-length clone coding for the rat alpha 7 nicotinic receptor subunit was isolated from an adult brain cDNA library and expressed in Xenopus oocytes. A significant proportion of the current through alpha 7-channels is carried by Ca2+. This Ca2+ influx then activates a Ca(2+)-dependent Cl- conductance, which is blocked by the chloride channel blockers niflumic and fluflenamic acid. Increasing the external NaCl concentration caused the reversal potentials for the alpha 7-channels and the Ca(2+)-dependent Cl- channels to be shifted in opposite directions. Under these conditions, agonist application activates a biphasic current with an initial inward current through alpha 7-channels followed by a niflumic acid- and fluflenamic acid-blockable outward current through Ca(2+)-dependent Cl- channels. A relative measure of the Ca2+ permeability was made by measuring the shift in the reversal potential caused by adding 10 mM Ca2+ to the external solution. Measurements made in the absence of Cl-, to avoid artifactual current through Ca(2+)-activated Cl- channels, indicate that alpha 7-homooligomeric channels have a greater relative Ca2+ permeability than the other nicotinic ACh receptors. Furthermore, alpha 7-channels have an even greater relative Ca2+ permeability than the NMDA subtype of glutamate receptors. High levels of alpha 7-transcripts were localized by in situ hybridization in the olfactory areas, the hippocampus, the hypothalamus, the amygdala, and the cerebral cortex. These results imply that alpha 7-containing receptors may play a role in activating calcium-dependent mechanisms in specific neuronal populations of the adult rat limbic system.

Amino Acid Sequence↗

PubMed1992

Gene transcripts for the nicotinic acetylcholine receptor subunit, beta4, are distributed in multiple areas of the rat central nervous system.

Previous in situ hybridization experiments reported that beta4 (beta 4) neuronal nicotinic acetylcholine receptor (nAChR) transcripts were found only in the medial habenula (MHB). Co-expression in Xenopus oocytes of the beta 4 subunit and any one of three ligand-binding or alpha subunits results in the formation of functional nAChRs. Comparisons between the pharmacology of nAChRs expressed in oocytes and the pharmacology of nAChRs found in the rat CNS prompted a further investigation of the localization of transcripts encoding the beta 4 nAChR subunit. Using two beta 4-specific cRNA probes, in situ hybridization was performed in rat brain. beta 4 mRNA was detected at high levels in the presubiculum, parasubiculum, subiculum and dentate gyrus of the hippocampal formation, in layer IV of the isocortex, in the medial habenula, in the interpeduncular nucleus, and in the trigeminal motor nerve nucleus. Moderate hybridization signals were seen in the isocortex (layers I-III), in olfactory regions, in fields CA1 through CA4 of Ammon's horn and the entorhinal cortex of the hippocampal formation, in the supramammillary nucleus, in the pontine nucleus, in the cerebellum, and in the locus coeruleus. No hybridization above background was detected in the septum, basal ganglia, sensory portions of the brainstem, or spinal cord.

Animals↗