The long-term objective of our research is to elucidate the molecular mechanisms underlying the function of nicotinic acetylcholine receptors (AChR). The muscle AChR is a ligand-gated ion channel that mediates fast signal transmission at the neuromuscular junction (NMJ). The receptor protein was first purified nearly two decades ago, but its atomic structure remains poorly defined. With the support of our previous NIH grant, we have identified the minimal ligand-binding domains on the alpha and delta subunits of mouse muscle AChR. In addition, we have established a yeast expression system that allows the production of large quantities of the extracellular domains of x subunit in monomeric form (alpha211) as well as the extracellular domains of both alpha and delta subunits in dimeric form (alphadelta heterodimer). Biochemical and pharmacological studies have demonstrated that the recombinant proteins fold in native-like conformation with high affinity to cholinergic ligands. Furthermore, we have optimized the conditions for uniform isotopic labeling and structural determination of o211 by nuclear magnetic resonance (NMR). Initial screening of conditions for crystallization has led to the production of small crystals of alphadelta heterodimers in complex with acetylcholine (ACh). As the logical extension of our past studies, the specific aims of research proposed in this competing renewal application are: (1) to determine the high-resolution structure of o211 by multidimensional NMR; and (2) to solve the atomic structure of o2i heterodimer in complex with ACh or the autoimmune antibody mAb35 by Xray diffraction. Elucidation of the 3D structure of AChR holds the key to understanding how the receptor interacts with ligands. Such information may also be applicable to studies of other ligand-gated ion channels including GABA, glycine and 5-HT3 receptors. As these proteins play a role in the pathogenesis of pain, dementia, epilepsy, and stroke determination of their structure is essential for the rational design of more selective therapeutic agents. The third specific aim of our research is to study the structure of neural agrin, a protein secreted by motoneurons that induces clustering of AChRs on muscle cell membrane at the NMJ. Multiple forms of agrin that differ in binding properties and bioactivity are generated through alternative splicing of agrin mRNAs in a variety of tissues. How alternative splicing regulates AChR-clustering activity is completely unknown. Here, we propose to solve the solution structure of the C-terminal domains of both neural and muscle agrins by NMR. The structural information will help to recover the molecular mechanisms underlying the important function of agfin during synaptogenesis at the NMJ.
