Thymol is a monoterpene that specifically interacts with synaptic neural functions in neuronal GABA-operated Cl(-) channels. Here we explore the effects of thymol, and propofol as positive control, on t-[(35)S]butylbicyclophosphorothionate ([(35)S]TBPS) binding in primary cultures of cortical neurons. The study includes a meaningful analysis of the effect of various exposure buffers, and their correlation with GABA released from cells, chloride influx through the GABA(A) receptor and GABA transporter activity. Cell viability was also determined. Thymol and propofol inhibited the binding of [(35)S]TBPS to cells exposed to Tris-citrate-NaCl buffer whereas a biphasic effect was observed in HEPES solution. The different effects of the two buffers analysed are due to the higher capacity of Tris-citrate-NaCl buffer to induce the release of endogenous GABA facilitating the binding of [(35)S]TBPS to its recognition site at the GABA(A) receptor. Released GABA in the presence of this buffer was inhibited by the neuronal GABA transporter inhibitor SKF 100330-A. Tris-citrate-NaCl buffer also induced a chloride influx, which was reverted by picrotoxinin. TBPS binding in living cells is facilitated by GABA released from the cells, which in turn activates basal GABA(A) receptor activity. The results deepen on the allosteric mechanism of thymol as positive modulator of the GABA(A) receptor. Furthermore, we corroborate [(35)S]TBPS binding as an important test to verify the capacity of drugs to act on and recognize a site at the GABA(A) receptor.

2005-12-01·Molecular Pharmacology3区 · 医学

Identification and Selective Inhibition of the Channel Mode of the Neuronal GABA Transporter 1

3区 · 医学

Article

作者: Stephan Michael Krause ; Wolfgang Schwarz

The function of GAT1, the transporter for the inhibitory neurotransmitter GABA, is characterized by expression in Xenopus laevis oocytes and measurements of GABA-induced uptake of [3H]GABA, 22Na+, and 36Cl-, and GABA-evoked currents under voltage-clamp conditions. N-[4,4-Diphenyl-3-butenyl]-nipecotic acid (SKF-89976-A), a specific inhibitor of GAT1, is used in our system as a pharmacological tool. The GABA-evoked current can be decomposed into a transport current, which is coupled to the GABA uptake, and a transmitter-gated current, which is uncoupled from the GABA uptake. The transport current results from a fixed stoichiometry of 1 GABA/2 Na+/1 Cl- transported during each cycle, as determined by radioactive tracer flux measurements. The transmitter-gated current is mediated by an Na+-conductance pathway. As a competitive inhibitor for GABA uptake, SKF-89976-A can separate the two current components. The GABA uptake is blocked with a K(I) value of approximately 7 microM, whereas the uncoupled transmitter-gated current is inhibited with a K(I) value of approximately 0.03 microM. Thus, the results of this study not only identify the transport mode and the channel mode of GAT1 but also raise the possibility of separating these components in a physiological environment.

2004-04-01·Journal of Biological Chemistry2区 · 生物学

The Aqueous Accessibility in the External Half of Transmembrane Domain I of the GABA Transporter GAT-1 Is Modulated by Its Ligands

2区 · 生物学

Article

作者: Baruch I. Kanner ; Estelle R. Bennett ; Yonggang Zhou

The sodium- and chloride-dependent gamma-aminobutyric acid (GABA) transporter GAT-1 is the first identified member of a family of transporters, which maintain low synaptic neurotransmitter levels and thereby enable efficient synaptic transmission. To obtain evidence for the idea that the highly conserved transmembrane domain I (TMD I) participates in the permeation pathway, we have determined the impact of impermeant methanethiosulfonate (MTS) reagents on cysteine residues engineered into this domain. As a background the essentially insensitive but fully active C74A mutant has been used. Transport activity of mutants with a cysteine introduced cytoplasmic to glycine 63 is largely unaffected and is resistant to the impermeant MTS reagents. Conversely, transport activity in mutants extracellular to glycine 63 is strongly impacted. Nevertheless, transport activity could be measured in all but three mutants: G65C, N66C, and R69C. In each of the six active cysteine mutants the activity is highly sensitive to the impermeant MTS reagents. This sensitivity is potentiated by sodium in L64C, F70C, and Y72C, but is protected in V67C and P71C. GABA protects in L64C, W68C, F70C, and P71C. The non-transportable GABA analogue SKF100330A also protects in L64C, W68C, and P71C as well as V67C, but strikingly potentiates inhibition in F70C. Although cysteine substitution in this region may have perturbed the native structure of GAT-1, our observations, taken together with the recently published accessibility study on the related serotonin transporter (Henry, L. K., Adkins, E. M., Han, Q., and Blakely, R. D. (2003) J. Biol. Chem. 278, 37052-37063), suggest that the extracellular part of TMD I is conformationally sensitive, lines the permeation pathway, and forms a more extended structure than expected from a membrane-embedded alpha-helix.

100 项与 SKF-100330-A 相关的药物交易

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