Browsing by Subject "GABA"
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Item Carisoprodol's Pharmacological Properties at GABAA Receptors, In-vitro and In-silico Studies(2021-05) Claudio, Maria del Carmen; Sumien, Nathalie; Huang, Ren-Qi; Gonzales, Eric B.; Dillon, Glenn H.; Mathew, Porunelloor A.Carisoprodol (CSP) is a centrally-acting prescription muscle relaxant that can directly activate, allosterically modulate and inhibit GABAA receptors. The GABAA receptor is a pentameric chloride ion channel in the cys-loop receptor family. The mechanism of GABAA's inhibitory role in the central nervous system lies in the resulting hyperpolarized state of the cell following chloride ion influx upon ligand binding. GABAA receptors are the target of many different clinically prescribed compounds because of the role they play in regulating the central nervous system. We used pharmacological and computational approaches to investigate the underlying mechanism mediating carisoprodols effects at GABAA receptors, with the ultimate goal of generating a new subunit selective compound related to the structure of carisoprodol and gaining a more thorough understanding of the molecular interaction governing carisoprodol's pharmacoglogical effects. Our evaluation of novel compounds related to the structure of carisoprodol did not yield promising leads, though the potential still remains for development of a novel carisoprodol-related compound with a unique selectivity profile. Probe for a binding site mediating carisoprodols postive modulatory effects and evaluation of additional novel compounds was eventually hindered by time. Our investigaiton into carisoprodol's direct gating effects involved a previously reported single amino acid residue, L415, located at the top of the fourth transmembrane domain (TM4) in the ɑ1 subunit of the GABAA receptor that is critical to carisoprodol's direct gating. Whether the residue is involved in a carisoprodol binding site remained unsolved. In studies probing for a binding site for carisprodol's direct activation of GABAA receptors, promising computaitonal docking data was not able to be validated with elecrophysiology and site-directed mutatgenesis studies, indicating that the residues revealed in docking studies do not form a binding pocket for carisprodol's direct activation effect. Our site directed mutagenesis, electrophysiology and molecular dynamic simulation studies to investigate carisoprodol's inhibitory effects at GABAA receptors revealed a binding site at the Cl- channel pore, in a mechanism similar to picrotoxin, providing a mechanism of action for carisoprodol's inhibitory effects at GABAA receptors.Item Characterization of the interactions of guanidine compounds with the human GABA-A ρ1 receptor(2015-12-01) Snell, Heather D.; Gonzales, Eric B.; Dillon, Glenn H.; Singh, MeharvanThis dissertation investigates the activity of guanidine compounds GMQ, and amiloride and its derivatives on the human GABA-A ρ1 receptor, compounds classified as antagonists for the heteromeric GABA-A αβγ receptor. The GABA-A ρ receptor possesses many differences in kinetics, expression, and pharmacology from the heteromeric GABA-A αβγ receptors. Many GABA-A αβγ receptors ligands interact differently, or fail to interact with, the GABA-A ρ receptor. Thus the activity of these guanidine compounds on the GABA-A ρ1 receptor remains unknown. Based on the differential pharmacology displayed by the GABA-A ρ receptors, we propose that GMQ and amiloride would interact with the GABA-A ρ1 receptor as agonists, different from their activity on the heteromeric GABA-A αβγ receptors. Importantly, our data demonstrates GMQ and amiloride interacts with the GABA-A ρ receptors as negative and positive allosteric modulators, respectively. The 15’ residue of the second transmembrane domain of the GABA-A ρ1 receptor is important in the positive allosteric modulatory mechanism, and the accessibility of the guanidine group on the guanidine compound is integral in the positive allosteric modulation mechanisms of amiloride and its derivative 5- (N,N-Hexamethylene) amiloride (HMA). The investigation of novel compounds that interact with the GABA-A ρ receptor differently from GABA-A αβγ receptor would contribute to a better understanding of the GABA-A ρ receptor structure and the production of novel therapeutics specific for the GABA-A ρ receptor. Particularly, the GABA-A ρ receptor is implicated in retinal hypoxic disorders such as diabetic retinopathy. These guanidine compounds could be utilized as a back-bone for the production of compounds that could alleviate the pathologies caused by advanced stages of diabetic retinopathy.Item EFFECT OF WATER DEPRIVATION ON KCC2 EXPRESSION IN HYPOTHALAMIC VASOPRESSIN NEURONS IN RAT(2013-04-12) Knapp, BlaynePurpose: Argininge Vasopressin (AVP) is a neurohypophyseal hormone that contributes to body fluid homeostasis by regulating plasma fluid and electrolyte composition. It is released from the posterior pituitary (PP) by magnocellular neurosecretory cells (MNCs) located within the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus. The regulation of AVP release is critical for maintaining blood volume and blood pressure, and although the molecular mechanisms of AVP regulation are not fully understood, we do know that it is partially due to the vital balance of synaptic excitatory and inhibitory inputs that determine the relationship between plasma osmolality and AVP release. Dysregulation of this system and the resulting disturbances of water and electrolyte balance can lead to increased morbidity associated with disease states such as heart and liver failure. This is why it is critical for us to elucidate the molecular mechanisms involved with its physiological control and its inappropriate release in disease states. Models of water deprivation represent a physiological challenge that requires sustained release of AVP from the PP into circulation as a means of maintaining body fluid homeostasis. We attempt to address the gaps that remain controversial or unexplained in our current understanding of AVP regulation by measuring changes in the expression of extruder KCC2 in water deprived and euhydrated adult male rats and measuring its effects on inhibitory GABAergic neurotransmission in AVP MNCs. Methods: Using laser capture microdissection (LCM), AVP MNCs were harvested from the SON and PVN of euhydrated and 48 hour water deprived adult male rats and reverse transcriptase polymerase chain reaction (RT-PCR) studies were used to test for changes in KCC2 message. Western blot protocols were used to measure changes in protein expression. Results: We observed a significant elevation in KCC2 mRNA expression in AVP cells of the SON (WD 2.5 ± 0.52; Control 1.0 ± 0.06, p<0.05) but not in the PVN (WD 3.5 ± 1.7; Control 1.0 ± 0.1). Immunofluorescence demonstrated the colocalization of KCC2 and AVP in the SON. Conclusions: Increased expression of KCC2 could be associated with decreased intracellular Cl- in AVP neurons in the SON, thereby serving to maintain or enhance the inhibitory tone of AVP neurons in the SON but not the PVN during WD.Item Effects of bile duct ligation on the inhibitory control of supraoptic vasopressin neurons(John Wiley & Sons, Inc., 2023-06-20) Aikins, Ato O.; Farmer, George E.; Little, Joel T.; Cunningham, J. ThomasDilutional hyponatremia due to increased plasma arginine vasopressin (AVP) is associated with liver cirrhosis. However, plasma AVP remains elevated despite progressive hypoosmolality. This study investigated changes to inhibitory control of supraoptic nucleus (SON) AVP neurons during liver cirrhosis. Experiments were conducted with adult male Sprague-Dawley rats. Bile duct ligation was used as a model of chronic liver cirrhosis. An adeno-associated virus containing a construct with an AVP promoter and either green fluorescent protein (GFP) or a ratiometric chloride indicator, ClopHensorN, was bilaterally injected into the SON of rats. After 2 weeks, rats received either BDL or sham surgery, and liver cirrhosis was allowed to develop for 4 weeks. In vitro, loose patch recordings of action potentials were obtained from GFP-labeled and unlabeled SON neurons in response to a brief focal application of the GABA(A) agonist muscimol (100 muM). Changes to intracellular chloride ([Cl]i) following muscimol application were determined by changes to the fluorescence ratio of ClopHensorN. The contribution of cation chloride cotransporters NKCC1 and KCC2 to changes in intracellular chloride was investigated using their respective antagonists, bumetanide (BU, 10 muM) and VU0240551 (10 muM). Plasma osmolality and hematocrit were measured as a marker of dilutional hyponatremia. The results showed reduced or absent GABA(A) -mediated inhibition in a greater proportion of AVP neurons from BDL rats as compared to sham rats (100% inhibition in sham vs. 47% in BDL, p = .001). Muscimol application was associated with increased [Cl]i in most cells from BDL as compared to cells from sham rats (chi(2) = 30.24, p < .001). NKCC1 contributed to the impaired inhibition observed in BDL rats (p < .001 BDL - BU vs. BDL + BU). The results show that impaired inhibition of SON AVP neurons and increased intracellular chloride contribute to the sustained dilutional hyponatremia in liver cirrhosis.Item PROGRESS TOWARDS CRYSTALLIZING A GABAA RECEPTOR(2013-04-12) Snell, HeatherPurpose: 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.