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Please use this identifier to cite or link to this item: http://hdl.handle.net/10277/133

Authors: Cherubino, Francesca
Internal Tutor: BOSSI, ELENA
Title: Electrophysiological characterization and drug interaction in glycine and GABA transporters.
Abstract: Neurotransmitter transporters are involved in various neurological disorders and are believed to be involved in the etiology of some mood disorders. Furthermore many of these molecules represent the target of therapeutical agents used in neurological pathologies or of drugs of abuse. In contrast to their medical and social relevance, the study of neurotransmitter transporters is not as advanced as the study of channels and other molecules involved in the synaptic transmission. Due to the coupling of the uptake of the substrate with transmembrane movement of the driver ion(s), whose electrochemical gradient represents the energy source of the process, most cotransporters generate electrical currents during their activity. For this reason it is possible to express the transporter RNA in heterologous systems and investigate their electric properties with electrophysiological techniques. The electrophysiological and biophysical properties of glycine transporters GlyT1b and GlyT2a expressed in Xenopus oocytes were investigated with two electrode voltage clamp. Integration of the transients isolated by subtraction of the traces in presence from those in absence of saturating glycine confirmed the intramembrane nature of the charge movement underlying the process. The sigmoidal Q/V relationships is much steeper for GlyT2a compared to GlyT1b, in agreement with different Na: Cl: glycine stoichiometry of the two transporters. The transient currents are best fitted by the sum of two exponentials, with the slow time constant (τslow) being larger for GlyT2a and in the order of tens of milliseconds. The apparent affinity for glycine is in the micromolar range and voltage-dependent, decreasing at positive potentials for both transporters. A simple, three-state model, is sufficient to explain the main features of GlyT1b operation, including the voltage-dependent affinity, while for GlyT2a a more complex scheme is required. The effects of the tricyclic antidepressant imipramine on the activity of the GABA cotransporter GAT-1 have been studied with electrophysiological and confocal microscopy experiments on Xenopus oocytes transfected with GAT-1 cRNA. Imipramine at concentrations 10 – 1000 μM progressively inhibits the transient (presteady-state) currents in absence of GABA, as well as the currents associated to the neurotransmitter translocation. In contrast, the GAT-1-related uncoupled current, best seen in presence of Li+, appears to be unaffected by the drug. Redistribution studies using GAT1-GFP constructs on transfected HEK293 cells indicate no evident internalization of the transporter upon imipramine exposure. The drug also appears to inhibit a K+ M-like current endogenously present in the oocytes. The effect of TCA on GAT-1 mutant K448E were investigated also. Lysine 448 of GAT-1 corresponds to aspartate 401 of LeuT, a residue found to be involved in the TCA binding. Desipramine is the antidepressant that shows the most powerful inhibition of transport and presteady state currents on the mutant compared to the wild type transporter. The result confirms the ability of TCAs to act on multiple targets, and the involvement of specific aminoacidic residues in substrate and inhibitor binding.
Issue Date: 2009
Language: en
Doctoral course: Neurobiologia
Academic cycle: 21
Publisher: Università degli Studi dell'Insubria
Citation: Cherubino, F.Electrophysiological characterization and drug interaction in glycine and GABA transporters. (Doctoral Thesis, Università degli Studi dell'Insubria, 2009).

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