Electronic Thesis/Dissertation
Using TBPS To Identify Functionally Distinct GABAA Receptors In The Rodent CNS Open Access
The GABAA receptor is a pentameric anion permeable Cys-loop receptor that can be formed from several combinations of α(1-6), β(1-3), γ(1-3), ρ(1-3), δ, ε or π subunits and is the site of action for treatment of epilepsy, anxiety, insomnia and anesthesia. Mutations in the GABAA receptor result in deficits of channel function and loss of inhibitory neurotransmission, resulting in seizures. GABAA receptor-mediated inhibition is either phasic, mediated by synaptic αβγ receptors or tonic, mediated by GABA-activation of high affinity extrasynaptic αβδ receptors or spontaneous agonist-independent gating of different subtypes. The ultimate goal of this study was to develop the GABAA receptor-specific radioligand [35S]t-butylbicyclophosphorothionate ([35S]TBPS) as a probe to identify functionally distinct GABAA receptors. TBPS and picrotoxin (PIC) are non-competitive GABAA receptor antagonists that bind within the second transmembrane domain (TM2). Accessibility to their binding sites is dependent on whether the receptor is resting, activated or desensitized. Putative activation and desensitization "gates" within the TM2 control the flow of ions through the GABAA receptor's ion channel. GABA modulation of [35S]TBPS binding differs between receptor subtypes, indicating that their distinctive channel properties may influence accessibility to the TM2 binding site. [35S]TBPS is primarily used to localize GABAA receptors in the brain and I hypothesized that the differential binding of [35S]TBPS to various brain regions is dependent on the functional channel properties dictated by the expression of specific subunits in each brain region and that [35S]TBPS could be used as a tool to detect functional deficits associated with neurological disorders such as epilepsy.The in vivo mutant γ2(K289M) subunit, associated with generalized epilepsy with febrile seizures and corresponding synthetic α1(K278M) mutant cause deficits in GABA efficacy and diminish spontaneous gating. Using patch-clamp electrophysiology, I examined the ability of TBPS and PIC to block wild-type and mutant receptors containing the epilepsy subunits and established that the binding site for TBPS lies above the GABAA receptor activation gate and below the desensitization gate, indicating that biphasic modulation of [35S]TBPS binding by GABA represents channel activation (enhancement of binding) and desensitization (inhibition of binding). Together, these findings demonstrated that the modulation of [35S]TBPS binding to different receptor populations by GABA represented the functional properties attributed by distinct subunits.I have also demonstrated that [35S]TBPS can be used to detect receptors containing different wild-type α, β and auxiliary γ, δ or ε subunits on the basis of their sensitivities to selective ligands that preferentially modulate specific GABAA receptor subtypes. Experiments with the mutant γ2(K289M) and &alpha1;(K278M) subunits demonstrated that [35S]TBPS binding enables the detection of functional deficits in GABAA receptors affected by mutations associated with epilepsy. Taken together, my findings provide a greater understanding of the contribution of GABAA function in the differential binding of [35S]TBPS. These findings pave the way for the use of [35S]TBPS binding to detect functionally distinct GABAA receptors in different brain regions and in the brains of individuals suffering from epilepsy.
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Othman_gwu_0075A_10930.pdf | 2018-01-16 | Open Access |
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