Redox modulation of GABAA receptors obscured by Zn2+ complexation.
Redox reagents are thought to modulate gamma-Aminobutyric acid type A (GABA(A)) receptors by regulating the redox state of the N-terminal disulphide bridge. Examining the redox sensitivity of recombinant GABA(A) receptors in human embryonic kidney cells, using whole-cell patch clamp techniques, revealed that alpha1beta2(H267A) and alpha1beta2gamma2 receptors, which are both less sensitive to Zn(2+) and H(+) modulation, ablated the potentiating effect of the reducing agent, dithiothreitol (DTT) seen for alpha1beta2 receptors. This effect could result from disruption to the redox signal transduction pathway or be due to DTT chelating Zn(2+) from its H267 inhibitory binding site, consequently potentiating GABA-activated currents in alpha1beta2 but not alpha1beta2(H267A) or alpha1beta2gamma2 receptors. A Zn(2+) chelating agent, tricine, potentiated GABA currents for the alphabeta constructs and vertically displaced GABA dose-response curves, suggesting that these receptors are subject to some inhibition by basal Zn(2+). Tricine, did not affect the GABA currents of either alpha1beta2(H267A) or alpha1beta2gamma2 receptors but did prevent the potentiation by 2 mM DTT and reduced the potentiation caused by 10 mM DTT on alpha1beta2 receptors. Thus, at low concentrations of DTT, a substantial component of the potentiation probably occurs via Zn(2+) chelation from H267 in the ion channel. In contrast, at higher DTT concentrations, it is more likely to be acting as a redox agent, which modulates both alphabeta and alphabetagamma subunit receptors.
Wilkins
M E
ME
Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.
Smart
T G
TG
D000494:Allosteric Regulation / Q000187:drug effects / Q000235:genetics / Q000502:physiology; D000818:Animals; D018929:Cell Culture Techniques; D002614:Chelating Agents / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction / Q000187:drug effects; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000145:classification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D015032:Zinc / Q000494:pharmacology*
D002614:Chelating Agents; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; D015032:Zinc; D004229:Dithiothreitol; D005998:Glycine; C100184:tricine