Abstract

The GABA type A receptor (GABA_AR) is expressed ubiquitously throughout the brain and is a target for many therapeutic agents, including general anesthetics and benzodiazepines, which enhance receptor function by increasing the open probability (P_o) of the ion channel. It is commonplace for in vitro studies of receptor pharmacological characteristics to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification, which results in chloride flow in the opposite direction, compared with in vivo conditions. This critical difference is usually overlooked because the GABA_AR has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is nonlinear with respect to P_o. Specifically, we found that currents were outwardly rectifying at low P_o and linear at high P_o. We confirmed the correlation between P_o and rectification with a partial agonist, piperidine-4-sulfonic acid, and a gating-impaired mutation, α1(L277A); both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted under altered chloride gradient conditions, which suggests that rectification is linked to the direction of chloride flux. Finally, our results showed that the degree of potentiation by general anesthetics (etomidate, propofol, and isoflurane) was greater at negative membrane potentials. Traditional in vitro experiments thus overestimate the action of positive allosteric modulators of the GABA_AR. Our results show that the direction of the driving force on the permeant ion, as well as P_o, must be considered together for a complete understanding of drug actions on ligand-gated ion channels.