Muscarinic acetylcholine receptors (mAChRs) are coupled to G-proteins and activate different second messengers and ion channels. Currently, there are five subtypes of mAChRs (MrM5), and are differentially distributed throughout the body. mAChRs are involved in a variety of disorders such as Schizophrenia, Parkinson's disease (PD), Alzheimer's disease (AD), cardiac arrhythmia, glaucoma, irritable bowel syndrome and asthma. Irrespective of the technological developments, the study of functions and characterization of mAChR subtypes has been lagging behind due to lack of highly selective agonists and antagonists. Several toxins which recognize the mAChRs have been purified and sequenced from the venom of mamba snakes of the genus Dendroaspis. The sequences of the toxins are homologous, but their mode of action and selectivity for mAChR subtypes are different. For example, MT3 toxin shows higher affinity for M4 receptors, and lower affinity for M! receptors, whereas, MT7 is specific for Iv^ receptors. The structure-function relationship of MT3 toxin is essential to establish the critical amino acid residues for the binding to the M4 receptors. Synthetic gene for MT3 toxin and the expression system are available in my laboratory for such studies. The hypothesis of this proposal is that the functional site of MT3 toxin requires multiple amino acid residues which are present in all the three loops of the toxin for its subtype specificity as well as for its action as an antagonist. I, therefore, propose to study the structure-function relationship of MT3 toxin, and the specific aims of my proposal are:
SPECIFIC AIM 1 : Production of MT3/MT7 chimeras and MT3 analogues: This will be achieved by the following experiments: (i) Synthesis MT3 toxin gene, and expression of MT3 toxin in Pichia pastoris using pPICZaA expression vector, and will be purified by the standard methods; (ii) Production of MT3/MT7 chimeras and MT3 analogue; and (iii) The structure-function of MT3/MT7 chimeras and MT3 analogues. The chimeras/analogues will be screened for their binding to Mi-M5 receptors expressed individually in CHO cells using [3H]-NMS binding assay.
SPECIFIC AIM 2 : The mode of interactions of MT3 toxin analogues with its receptors. MT3 analogues with higher selectivity for M4 receptors will be subjected for the agonist/antagonist/allosteric interactions using CHO cells expressing M4 receptors. The completion of this study will establish the specific amino acid residues responsible for MT3 toxin's subtype specificity. The long-term goal of this proposal is to design, and to develop therapeutic agents based on the three-dimensional template on which smaller molecule mimics can be designed for different disorders involving mAChRs. ? ? ?
