Redox modulation of GABA A receptors obscured by Zn 2+ complexation

Abstract

Redox reagents are thought to modulate γ-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 α1β2 H267A and α1β2γ2 receptors, which are both less sensitive to Zn 2+ and H + modulation, ablated the potentiating effect of the reducing agent, dithiothreitol (DTT) seen for α1β2 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 α1β2 but not α1β2 H267A or α1β2γ2 receptors. A Zn 2+ chelating agent, tricine, potentiated GABA currents for the αβ 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 α1β2 H267A or α1β2γ2 receptors but did prevent the potentiation by 2 mM DTT and reduced the potentiation caused by 10 mM DTT on α1β2 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 αβ and αβγ subunit receptors.