Identifying the Interactions between Animal Toxins and Human nAChRs: The Role of Snake PLA2 in Interacting with nAChR alpha Subunits

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PROJECT SUMMARY/ABSTRACT Nicotinic acetylcholine receptors (nAChRs) are involved in a variety of fundamental physiological processes, and dysfunction in these receptors is associated with many human disorders such as Alzheimer’s and Parkinson’s diseases. The structure and function of human nAChRs are extensively studied; however, the combinations of different nAChR subunits making various nAChR subtypes and their extracellular regions binding a structurally diverse range of ligands, pose challenges in high-throughput (HT) and systematic investigation of nAChRs. Fortunately, animal toxins provide a unique tool to study the structure and function of nAChR because they have been fine-tuned through millions of years of natural selection to present high specificity and selectivity towards specific proteins such as nAChRs. Our long-term goal is to identify universal or selective animal toxins that interact with human nAChRs, and elucidate the mechanistic underpinnings underlying their interactions. To achieve this goal, the overall objective of this application is to establish HT approaches for identifying the interactions between snake toxins and nAChRs subunits, as well as deciphering the role of toxins in interacting with human nAChRs by proposing the following three specific aims: 1) Generate a snake toxin ORFeome library. The unique snake toxin transcript open-reading frames (ORFs) will be identified from the existing venom gland cDNA library of a medically important snake (Crotalus atrox), and individually ligated into a “donor” vector to create a snake toxin ORFeome library that can be directly used for the interaction detections with any human proteins including nAChRs. 2) Determine the interactions of snake toxins with human nAChR subunits. The interactions between snake toxins and human nAChR subunits, with focus on the detection between venom phospholipase A2 (PLA2) and nAChR α subunits, will be identified by utilizing a HT yeast two-hybrid (HT-Y2H) system, followed by co-affinity purification/mass spectrometry (AP/MS). 3) Decipher the role of venom toxins in interacting with nAChR subunits. We will develop a HT pharmacological assay to determine if snake toxins such as PLA2 act as agonist or antagonist against human nAChRs. The nAChRs such as α1β1γδ, α3β4, α4β2, and α7, will be individually transfected into human cells such as HEK293 for measuring the calcium dynamics and membrane potential elicited by PLA2-nAChR interactions. The research results are expected to be impactful because: 1) it will provide valuable resources for the scientific community, university faculty and students for subsequent biological research and biomedical applications; and 2) it will provide a unique tool for HT and systematic investigation of the structure and function of nAChRs. In addition, the project will create numerous opportunities for undergraduate students to improve their research skills in biomedical science.