Receptor Pharmacology & Drug Delivery
Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21746
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Item Characterizing the amiloride potentiation site in the GABAA ρ1 receptor(2015-03) Snell, Heather D.; Gonzales, Eric B.γ- amino butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate brain, and targets the ionotropic GABAA receptors. GABAC, or GABAA rho, is a subclass of GABAA receptors composed entirely of rho (ρ) subunits and are located on the axonal terminal of retinal bipolar cells, where it not only exhibits a tonic inhibitory current, but also regulates the GABAA and other GABAA rho synaptic currents (Jones et al 2011). GABAA-rho exhibits unique properties, such as insensitivity to select antagonists of the heteromeric GABAA receptors (Korpi et al., 2002). A group of ligands, which possess a guanidine group, have been shown to influence GABAA receptors. This includes the acid sensing ion channel (ASIC) ligand, amiloride. Our previous work elucidated the intrinsic activity of the guanidine compound amiloride as having an allosteric modulatory effect on the human GABAA rho1 receptor, but the exact mechanism, or site of interaction, remains unknown. Homology modeling of amiloride interacting with ethanol sensitive GLIC, a bacterial ligand gated ion channel of known structure, has yielded possible residues that might form the amiloride site in the GABAA rho1 subunits of the receptor. We hypothesize mutating these residues in the GABAA rho1 receptor will eliminate the allosteric modulatory effect of amiloride, and thus reveal the site of interaction with the receptor. Point mutations will be introduced through polymerase chain reaction (PCR), and whole cell electrophysiology will be utilized to assess the intrinsic activity of amilorde following introduction of the mutated residue. Our findings suggest that there are functional, as well as therapeutic, implications for the use of guanidino compounds in targeting the GABA-A rho1 receptor mediated activity. This site of action could be a unique allosteric binding site in the GABAA rho1 receptor, and thus could be utilized as a target for therapeutics not only for the GABAA rho receptor family, but also other subunits in the GABAA receptor family.Item Acid-sensing ion channel proton sensitivity is modulated by a guanidine containing dietary supplement(2015-03) Agharkar, Amruta S.; Smith, Rachel N.; Gonzales, Eric B.Acid-sensing ion channels (ASICs) belong to the epithelial/degenerin family. ASICs are sodium selective and are sensitive to extracellular protons specifically those following ischemia and injury. The ASIC1a subtype has been implicated centrally in the neurodegeneration following ischemic stroke while ASIC3 is involved in pain sensation and is expressed peripherally. Protons and inflammatory mediators can activate or modulate ASIC1a and ASIC3, suggesting that ASICs can be a pharmacological target for ischemic stroke and pain. The large extracellular domain of ASICs offers multiple sites for interacting with protons and guanidinium group containing compounds. Guanidinium compounds such as 2-guanidine-4-methylquinazoline (GMQ), amiloride, and agmatine are known to modulate the electrophysiological properties of ASICs. Here we identified a dietary supplement, GL-001 that shares molecular similarity to these ASIC ligands and modulate ASICs. We utilize whole-cell patch-clamp electrophysiology to determine the interaction of GL-001 with endogenous human ASIC1a (hASIC1a) and transiently expressed rat ASIC3 (rASIC3). Our data suggests that GL-001 reduces the hASIC1a pH sensitivity at physiologically relevant supplement concentrations consistent with suggested dietary supplementation. The rASIC3 peak current amplitude and steady-state current is reduced in the presence of GL-001. In the absence of extracellular calcium, GL-001 reduces the rASIC3 proton sensitivity by shifting pH-activation profile to lower pH. This suggests that the effect of GL-001 on rASIC3 is calcium dependent. Future studies will focus on determining the effect of GL-001 on the rASIC3 window current and other ASIC3 properties to resolve the mechanism of action of the GL-001 influence on channel activity.