Receptor Pharmacology & Drug Delivery

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    (2013-04-12) Johnson, Rachel
    Purpose: Acid-sensing ion channels (ASICs) are trimeric, sodium-selective channels located throughout the central and peripheral nervous systems. These ion channels are activated by extracellular protons and involved in ischemia, neurotransmission, and pain nociception. Thus, ASICs are intriguing molecular targets for the development of novel pharmaceutical treatments. However, there is a lack of selective compounds that can differentiate specific ASIC subtypes. One ASIC subtype, the peripherally located ASIC3, is sensitive to extracellular calcium, which acts to stabilize the closed, inactive state. Recently, non-proton ligands, such as 2-guanidine-4-methylquinazoline (GMQ), have been identified to selectively activate ASIC3. Controversy remains over what constitutes the ASIC3 GMQ binding site and how other mediators of ASIC3 activity, such as calcium, influence non-proton ligand activity. We hypothesize that non-proton ligand activation of ASIC3 requires the depletion, or removal, of calcium. Methods: Chimeric receptors combining the extracellular (ETC), transmembrane (TM), and intracellular (ITC) domains of rat ASIC3 and chicken ASIC1 were generated and subcloned into a mammalian expression vector with an amino-terminal GFP tag. Each of these vectors were transfected into Chinese hamster ovarian (CHO) cells where successful transfection was confirmed using fluorescence microscopy. Each chimeric receptor was assessed for function using whole-cell patch clamp electrophysiology. The non-proton ligand GMQ was assessed in each ASIC in the presence and absence of calcium. Results: Our preliminary data confirm that ASIC3 is activated and held open by a lowering of extracellular calcium concentration at high pH (pH 8.0), thus minimizing proton influence. This activation is enhanced by the addition of GMQ to the extracellular solution at the same pH in a concentration dependent manner. Our data suggest that when calcium is removed from the extracellular solution at high pH, GMQ stimulates ASIC3 activity. Conclusions: The non-proton ligand activity, mediated by GMQ, is dependent on the removal of calcium from the ASIC3 calcium block site, which appears to be void in ASIC1. Future studies will focus on elucidating the GMQ activation kinetics and key residues in both the extracellular and transmembrane domains critical for GMQ binding and activity. The understanding of ASIC3 activation via this novel mechanism may allow for the development of novel compounds specifically targeting ASIC3.
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    (2013-04-12) Ashutosh, FNU
    Purpose: Several brain injuries and neurodegenerative diseases implicate excessive production of reactive oxygen species, such as hydrogen peroxide (H2O2) in disease pathogenesis. Catalase, an H2O2 degrading enzyme, is a well-known antioxidant target for therapeutic intervention. However, medical use of catalase is restricted by its labile nature and inadequate delivery to central nervous system. Methods: Here, a nanotechnology approach was evaluated that utilizes catalase-loaded, poly(lactic-co-glycolic acid) nanoparticles (NPs) to deliver catalase in order to protect human neurons from oxidative damage. Results: This study shows highly efficient catalase encapsulation capable of retaining ~ 99% enzymatic activity. NPs released catalase rapidly and antioxidant activity was sustained for over a month. Human neurons took up NPs rapidly and without toxicity. While human neurons are highly sensitive to H2O2, nanoparticle-mediated catalase delivery protected neuronal cultures from H2O2-induced oxidative stress. Catalase-loaded NPs significantly reduced H2O2-induced protein oxidation, DNA damage, mitochondrial membrane transition pore opening, loss of cell membrane integrity and restored cell morphology, neurite network and microtubule-associated protein-2 expression in neuronal cultures. Further, catalase-loaded NPs showed better neuronal recovery from H2O2 pre-exposure than non-encapsulated catalase enzyme, suggesting possible applications in ameliorating stroke relevant oxidative stress. Conclusions: Brain targeting of catalase-loaded NPs may find wide therapeutic applications for oxidative stress-associated acute and chronic neurodegenerative disorders.
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    (2013-04-12) Snell, Heather
    Purpose: Currently we lack a 3-dimensional template GABA interacting with the GABA-rho receptor. We are working towards determining the three-dimensional structure of the GABAA-rho receptor. This will assist in the development of novel therapeutics. Methods: GABA-rho1 was subcloned into a baculovirus expression vector using blunt end PCR and transfected into SF9 insect cells. SF9 cells were harvested, and protein was isolated using metal affinity and size exclusion chromatography. A phosphate buffered saline (PBS) based running buffer was used to stabilize the purified protein. Protein crystallization trials were performed using the lipidic cubic phase technique and observed weekly for crystal formation using a stereomicroscope. Results: GABAA-rho was isolated using metal affinity chromatography. Our size exclusion chromatography studies reveal that using a PBS-based running buffer stabilizes the pentameric arrangement of purified GABAA-rho receptor. Subsequent lipidic cubic phase crystallization trials revealed three conditions what yielded birefringent protein crystals. Conclusions: Previously, we determined the optimum incubation time to yield maximum amount of protein. However, the isolate protein was not stable in the subsequent purification steps. Here, we have identified optimal buffer and detergent conditions to isolate and solubilize the protein. We have also identified crystal conditions that yield crystals. Future experiments will focus on generating additional protein crystals in the presence and absence of ligands, such as GABA and GABAA-rho receptor selective antagonists.
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    (2013-04-12) Agharkar, Amruta S.
    Purpose: The human voltage-gated proton channel (Hv1) is a proton-selective channel implicated in the efflux of protons and is found as a homodimer. Each subunit of the Hv1 proton channel forms the proton-selective pore. Recently, it has been shown that Hv1 is overexpressed in metastaic breast cancer cells and helps in cancer cell proliferation and migration. Although the voltage-sensing domain of the Hv1 proton channel is similar to the voltage sensor of voltage gated channels that have determined three-dimensional structures, the Hv1 proton channel structure remains elusive. Determining the structure of Hv1 channel will serve as a template for designing new therapeutic strategies. Here, we present our progress towards solving the three-dimensional structure of the Hv1 proton channel. Methods: The full length Hv1 proton channel gene was combined with a carboxyl-terminal 6Xhis tag within a baculovirus insect expression vector. Small-scale expression experiments for detergent screening followed by metal affinity chromatography and size exclusion chromatography (SEC) are used to assess protein stability. Our purification and stability assays are coupled with whole cell patch-clamp electrophysiology technique to determine Hv1 construct functionality. Results: The Hv1 channel gene was subcloned into a vector for insect cell expression. The construct obtained produced a histidine tagged protein with a molecular weight comparable to Hv1 protein. The generated Hv1 proton channel has been isolated and assessed for stability and monodispersity using size exclusion chromatography. Conclusions: Based on our preliminary data, the current Hv1 proton channel construct has potential as a protein crystallographic construct. Future experiments will focus on generating large quantities of purified and stable Hv1 proton channel and initiating protein crystallization trials.