Stanley C. Froehner

Molecular Neurobiology of Synaptogenesis

Synaptic transmission in the nervous system depends on highly specialized distributions of io n channels for speed and specificity. At the neuromuscular junction, nicotinic acetylcholine recepto rs (AChR) are localized to postsynaptic sites in extraordinarily high concentrations - approximately 10,000 receptors per square micron of membrane surface - where they are poised to bind acetylcholine released from the presynaptic terminal. Since these receptors are transmembrane multisubunit protein s inherently capable of diffusion within the plane of the membrane, they must interact with other pro teins which anchor them at the synapse. Using biochemical and molecular biological approaches, we ha ve identified one protein that participates in the process. A protein of 43,000 molecular weight (43 K protein) is associated peripherally with cytoplasmic domains of AChR. We have demonstrated a role for the 43K protein in AChR clustering by coexpression with the receptor. In Xenopus oocytes injecte d with in vitro-synthesized RNA encoding each of the receptor subunits, AChR are uniformly distribute d on the surface. Coexpression with RNA encoding the 43K protein causes clustering of AChR. The abi lity to cluster seems to reside entirely in the 43K protein since it will form clusters even in the a bsence of AChR. We are investigating by site directed mutagenesis the molecular mechanisms by which AChR and 43K protein interact with the goal of understanding how this interaction is regulated. We a re also applying a molecular approach to study the role of other postsynaptic membrane proteins, such as the dystrophin family of proteins and dystrophin-associated proteins, in neuromuscular synaptogen esis.

Calcium channels, which are integral to the process of transmitter release, are localized in the presynaptic terminal directly across the synaptic gap from AChR clusters. Little is known about the mechanisms that anchor calcium channels at sites of transmitter release. We are developing monoc lonal antibodies to these calcium channels with the goal of studying the mechanisms that regulate the coordinated clustering of presynaptic calcium channels with postsynaptic neurotransmitter receptors during synaptogenesis.