Neurotransmitter receptor assembly and expression

The general focus of the research in our laboratory is determination of the neurotransmitter receptors responsible for the rapid postsynaptic response in nerve and muscle. These receptors are members of a large molecular family that includes nicotinic acetylcholine receptors (AChRs), GABAA receptors, glycine receptors and ionotropic glutamate receptors. All of these receptors are oligomeric membrane proteins with subunits surrounding an ion channel that opens when neurotransmitters bind to the receptor.

Subunit folding and assembly of the "muscle-type" nicotinic AChR

One of our projects is to understand how nerve and muscle cells fold and assemble the subunits into a functioning receptor and express it in the postsynaptic membrane. The AChR was chosen because its composition and stoichiometry is known. The AChR is composed of four subunits, a, b, g and d, that assemble into a2bgd pentamers. In order to make precise measurements of the AChR subunits in vivo, the AChR subunit cDNAs cloned from the ray Torpedo californica and from mouse have been stably integrated into the genome of mammalian tissue culture cell lines. Expression of the AChRs in these foreign cells allows large amounts of the receptors to be grown up for biochemical and functional assays and allows the AChRs to be studied in the absence of other synaptic proteins, which might otherwise complicate our studies. Using a variety of recombinant DNA, biochemical and pharmacological techniques, we are presently dissecting the events involved in the AChR subunit folding and assembly.

Neuronal nicotinic AChRs

Nicotinic AChR receptors are found in the central and peripheral nervous systems of almost all animals. As the site where nicotine binds in the brain, these receptors are responsible for nicotine addiction and may also play a role in Alzheimer's disease. Neuronal AChRs differ from the muscle-type AChRs in that they are subdivided into distinct pharmacological subtypes. Each subtype appears to be composed of a different set of subunit isoforms. These subunits are distinct from, yet homologous to the muscle subunits. We are searching for the subunit composition of these neuronal AChR subtypes. Different combinations of the subunit cDNAs will be expressed in mammalian cell lines system to see if specific pharmacological properties of a subtype can be reproduced in this reconstituted system. Attempts will also be made to identify subunit composition by purifying the receptor subtypes from different neuronal sources.

 Rangwala, F., R. C. Drisdel, S. Rakhilin, E. Ko, P. Atluri, A. B. Harkins, A. P. Fox, S. B. Salman, W. N.Green. (1997) Neuronal a-Bungarotoxin receptors differ structurally from other nicotinic acetylcholine receptors. J Neurosci, 17: 8201-8212.

 Eertmoed, A. L., Y. Vallejo and W. N. Green. (1997) Transient expression of heteromeric ion channels. Methods in Enzymology (in press).