• Development of acetylcholine-binding protein (AChBP) as a biosensor for serotonin ligands

      Ataian, Yeganeh; Schulte, Marvin; Duffy, Lawrence; Kuhn, Thomas; Bult-Ito, Abel (2018-05)
      Acetylcholine-binding protein (AChBP) is a water-soluble novel protein with a high sequence similarity (15-30% identity) to ligand-gated ion channel (LGIC) receptors. The crystal structure of AChBP is used to study the extracellular domain of the pentameric LGICs such as nicotinic acetylcholine receptors (nAChRs) and 5-hydroxytryptamine type 3 receptors (5-HT₃Rs); and homology models have been developed to study receptor-ligand interactions. The 5-HT₃ serotonin receptors are potential therapeutic targets for multiple nervous system disorders such as alcohol and drug dependence, anxiety, depression, schizophrenia, sleep, cognition, memory, and chemotherapy-induced and post-operative nausea and vomiting. Therefore, the ligands that target the 5-HT₃Rs are considered powerful therapeutic agents. As such, 5-HT₃ serotonin receptors have been the targets of drug discovery efforts. The main objective of the current protein engineering project was to develop a soluble serotonin-binding protein using AChBP, which would mimic the specificity of the native 5-HT₃ serotonin receptor. Once developed, this soluble protein would be used as a model to design an array of receptors, which could be placed on biosensors for high-throughput drug screening (HTDS). The results of site-directed mutagenesis of AChBP demonstrated that mutation of certain AChBP residues to its equivalent in serotonin resulted in an increased affinity of AChBP for serotonin ligands, and that each individual mutation increased the affinity of AChBP to a certain degree. It indicates that this approach is going in the right direction but multiple mutations will probably be needed to get to an AChBP whose affinity is equivalent to wild-type serotonin. In addition, the most significant changes appeared to be in the C-loop as it produced the largest increase in affinity of AChBP for serotonin agonists. The results also support the proposed C-loop closure model for the receptor, and based on data presented here, a new alignment of the C-loop is suggested.
    • Methods of temperature & metabolism reduction in rats and possible influence on human health

      Bailey, Isaac R.; Drew, Kelly L.; Kuhn, Thomas B.; Rasley, Brian T. (2017-10)
      Spaceflight poses unique and significant hazards; the maintenance of human health remains a large part of the National Aeronautic and Space Administration (NASA) strategic goals and work remains to be done if we wish to maintain a long-term presence in space. The effects of ionizing radiation and bone density loss are some of the primary health related problems which need to be addressed. One of the main purposes of this research is to translate aspects of thermoregulation and metabolism reduction in hibernating species to a non-hibernating species in- order to devise alternative methods of preventing DNA damage and loss of bone density in astronauts. A second purpose for this research applies the same approach in emergency medicine, having potential as conjunctive therapy for cardiac arrest victims. Targeted temperature management (TTM; formerly known as therapeutic hypothermia) is the standard of care for these patients and is applied to increase survival rates and reduces neurological deficit. Stimulating Central Nervous System (CNS) A1 adenosine receptors inhibits shivering and non-shivering thermogenesis, inducing a hibernation-like response in hibernating species. A similar phenomenon occurs when using this technique in non-hibernating species such as rats. The adenosine A1 agonist, N6-cyclohexyladenosine (CHA) was utilized in all 3 of the experiments to determine how dose, diet, ambient temperature, and finally surface temperature affects the thermoregulatory response in Sprague-dawley rats. In addition to CHA, the partial agonist capadenoson was also tested for thermolytic efficacy (that is, the efficacy to abolish thermogenesis). Surface temperature control using a temperature controlled cage designed and built by myself in combination with IV CHA was found to be most effective in maintaining a target temperature of 32°C without risk of over-cooling. Results from these experiments suggests that the new standard technique in studying TTM using small animals should be similar to what is currently used in clinics; surface temperature modulation.