Biomedical subjects

V Gerzanich

Publications and source records attributed to V Gerzanich.

22 records · Page 2Linked to original sources

PubMed1994

Human alpha 7 acetylcholine receptor: cloning of the alpha 7 subunit from the SH-SY5Y cell line and determination of pharmacological properties of native receptors and functional alpha 7 homomers expressed in Xenopus oocytes.

The alpha-bungarotoxin-binding acetylcholine receptors from the human neuroblastoma cell line SH-SY5Y were found to cross-react with some monoclonal antibodies to alpha 7 subunits of nicotinic acetylcholine receptors from chicken brain. The human alpha 7 subunit cDNA from SH-SY5Y was cloned, revealing 94% amino acid sequence identity to rat alpha 7 subunits and 92% identity to chicken alpha 7 subunits. Native human alpha 7 receptors showed affinities for some ligands similar to those previously observed with native chicken alpha 7 receptors, but for other ligands there were large species-specific differences in binding affinity. These results paralleled properties of alpha 7 homomers expressed in Xenopus oocytes. Human alpha 7 homomers exhibited rapidly desensitizing, inwardly rectifying, agonist-induced, cation currents that triggered Ca(2+)-sensitive Cl- channels in the oocytes. A change in efficacy from partial agonist for chicken alpha 7 homomers to full agonist for human alpha 7 homomers was exhibited by 1,1-dimethyl-4-phenylpiperazinium. This result reveals a large species-specific pharmacological difference, despite small differences in alpha 7 sequences. This is important for understanding the effects of these drugs in humans and for identifying amino acids that may contribute to the acetylcholine binding site, for analysis by in vitro mutagenesis. These results also characterize properties of native alpha 7 receptors and alpha 7 homomers that will provide criteria for functional properties expected of structural subunits, when these can be identified, cloned, and coexpressed with alpha 7 subunits.

Amino Acid Sequence↗

PubMed1993

Reporter epitopes: a novel approach to examine transmembrane topology of integral membrane proteins applied to the alpha 1 subunit of the nicotinic acetylcholine receptor.

The development of a novel immunological method called the "reporter epitope" technique to probe the transmembrane topology of integral membrane proteins is described. Using this method, synthetic oligonucleotides encoding epitopes (reporter epitopes) for well characterized monoclonal antibodies (reporter mAbs) were inserted at various locations within the human acetylcholine receptor (AChR) alpha 1 subunit cDNA. The engineered subunits were then expressed along with Torpedo beta 1, gamma, and delta subunits in Xenopus oocytes, and the transmembrane location of the site of insertion was determined by the binding of the 125I-labeled reporter mAbs to whole oocytes. Control reporter epitope insertions at alpha 347 exhibited the expected cytoplasmic location. Reporter epitopes inserted at alpha 429 are located on the extracellular surface. Reporter epitopes that are 16-48 amino acids long do not disrupt assembly or function of hybrid AChRs when inserted near the carboxy terminus (at alpha 429) or in the large cytoplasmic domain (at alpha 347). However, because two reporter epitopes inserted at alpha 157 obliterated subunit assembly and a third reporter epitope when tolerated at this position was inaccessible from the extracellular surface and only marginally accessible after detergent solubilization of the AChRs, a definitive transmembrane location for this region was not possible. Nonetheless, the use of this approach has been successfully demonstrated, and it may be generally applicable to the study of other integral membrane proteins.

Amino Acid Sequence↗

PubMed1993

On the excitatory effects of ATP and its role as a neurotransmitter in coeliac neurons of the guinea-pig.

1. The effects of ATP on neurons from guinea-pig coeliac ganglia were studied to evaluate the possibility that this nucleotide acts as an excitatory neurotransmitter substance. 2. In experiments with intracellular microelectrodes, ATP (> or = 10 nM) depolarized coeliac neurons from the resting potential and produced an increase in the membrane conductance. These excitatory effects of ATP were observed in isolated coeliac ganglia, in acutely dissociated neurons or in cultured neurons. ATP also produced membrane conductance increases in neurons clamped at the resting potential using a single electrode voltage clamp. 3. When studied in the whole-cell configuration of the patch clamp (intracellular Cs+ to block K+ currents; -50 mV holding potential), ATP evoked inward currents in a manner more potent and efficacious than acetylcholine (ACh). 4. Whole-cell currents induced by ATP were inwardly rectifying and reversed at -13 mV in normal Na+ solutions. Changes in extracellular Na+ concentration altered the reversal potential in a manner predicted by the Goldman-Hodgkin-Katz bi-ionic equation with a ratio of Na+ to Cs+ permeability (PNa/PCs) = 0.6. 5. Single channel currents were evoked by ATP in excised (outside-out) patches. Current-voltage relationships for single channel currents exhibited inward rectification. The mean single channel conductance was 22 pS at -50 mV. 6. Antagonists of ATP-gated channels (suramin, Reactive Blue 2) reduced the effects of ATP but not ACh. 7. Antagonists at nicotinic receptors/ion channels (hexamethonium or tubocurarine) reduced the effects of ACh but not ATP. 8. Excitatory synaptic currents were observed in cultures of coeliac neurons. Synaptic currents possessed similar current-voltage relationships to currents produced by ATP, were increased in frequency by K+ depolarization in a Ca(2+)-dependent manner, and were selectively antagonized by ATP antagonists. 9. Local K+ depolarization of the ends of neurites evoked single channel currents characteristic of ATP in outside-out patches when patches were positioned near the region of apparent synaptic contact but not when patches were positioned at remote regions. 10. The results suggest that ATP receptors are linked to ion channels and mediate excitatory synaptic transmission between coeliac neurons.

Acetylcholine↗

PubMed1992

ATP mediates excitatory synaptic transmission in mammalian neurones.

Adenosine 5'-triphosphate (ATP, 0.1-100 microM), produced inward currents in patch-clamped coeliac neurones from guinea-pig when studied in either the whole cell configuration or in excised (outside-out) patches. The P2-purinoceptor antagonists suramin (80-230 microM) or reactive blue 2 (2-20 microM) depressed the ATP-induced currents but not those produced by acetylcholine. Excitatory post-synaptic currents (e.p.s.cs) were observed in cultured neurones. E.p.s.cs had similar current-voltage relationships to currents evoked by ATP in excised patches and were reduced by suramin or reactive blue 2 to a similar extent as ATP currents. The results suggest that ATP is the excitatory neurotransmitter in cultures of these neurones.

Acetylcholine↗