Our long-term goal is to elucidate the molecular mechanisms underlying ligand-neurotransmitter receptor interactions utilizing the muscle nicotinic acetylcholine receptor (AChR) as a model. Although the AChR was first purified and cloned nearly 20 years ago, detailed structural information of this protein is still very limited. This is mainly due to the inherent difficulty of crystallizing membrane proteins. Hence, or main objective is to express large quantities of soluble, N-terminal extracellular domains of the AChR alpha and delta subunits, and to determine their 3-dimensional structure. This domain, in the alpha subunit, comprises almost half the subunit mass and contains the binding site for alpha-bungarotoxin (alpha-BuTx) as well as the so-called major immunogenic region (MIR). This region is the target of autoimmune antibodies in myasthenia gravis. In addition, this domain contains the structural information for assembly with the delta subunit to form alpha- delta heterodimers which can bind cholinergic ligands, such as acetylcholine (ACh) and d-tubocurarine (dTC).
The specific aims of this proposal are: 1) to identify a minimal soluble sequence on the alpha subunit that can fold to form an alpha-BuT binding site and the MIR; 2) to define the minimal domains on the alpha and domain subunits that can form a soluble heterodimer with a high affinity ACh-binding site. Properly folded alpha N-terminal domain and the alphadelta heterodimer will be expressed as secretory proteins in yeast, and purified to homogeneity; 3) to determine the 3D solution structure of the alpha extracellular domain in free form and in complex with alpha-BuTx using multi-dimensional NMR. Also, structural elements on the alphadelta heterodimer interaction with ACh and dTC will be delineated using transferred NOE; 4) to crystalize the alphadelta heterodimer-ACh complex and the MIR-autoimmune antibody complex in order to study their structure using x-ray diffraction methods. Completion of this project will allow us to solve the long-sought structure of the ligand-binding sites of the AChR. It will also provide insight into the common structural elements that determine the function of other ligand-gated ion channels, such as the GABA, glycine and 5-HT/3 receptors. As these proteins play important roles in the pathogenesis of pain, mental illness and other common disorders such as epilepsy and stroke, information on their structure is essential for the rational drug design for more selective therapeutic agents. Finally, techniques to be developed through this work should facilitate future structural studies of these and other ligand-gated ion channels.
