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The apparent voltage dependence of GABA A receptor activation and modulation is inversely related to channel open probability Abstract The GABA type A receptor (GABA A R) 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 pharmacology to use negative membrane holding potentials to mimic the resting potential of neurons and symmetrical chloride to eliminate Goldman rectification; resulting in chloride flow in the opposite direction of in vivo conditions. This critical difference is usually overlooked because the GABA A R has been reported to behave as an ohmic pore, but our results show that the current-voltage relationship is non-linear 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, both exhibited enhanced outward rectification. Furthermore, this correlation was independent of Goldman rectification and persisted in altered chloride gradient conditions, suggesting 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 will therefore over-estimate the action of positive allosteric modulators of the GABA A R. Our results show that the direction of driving force on the permeant ion, as well as, P O must be considered together in order to completely understand drug action on ligand-gated ion channels. In light of these differences we chose to study the current-voltage (IV) relationship of the GABA A R with full and partial agonists, in the presence of positive allosteric modulators and in receptors harboring mutations that hinder channel gating. We found that low P O conditions were associated with currents that exhibited outward rectification and likewise, increasing P O resulted in a linearization of the IV relationship. Our results also show that the degree of receptor enhancement by therapeutics was voltage dependent; as the membrane potential increased, and as it tended towards generating current flow in the physiological direction, so the amount of enhancement reduced. the magnitude of inward currents, in low P O conditions, resulting in outward rectification. At high P O , desensitization of outward currents was enhanced resulting in a linearization of the IV relationship. The different effects of gating and desensitization on the IV relationship suggest that these mechanisms are mediated by different channel structures. We have also shown that the degree of receptor enhancement by therapeutics was voltage dependent; in particular, potentiation was greater at negative membrane potentials. Finally, our results showed that the apparent voltage dependence of channel activation and modulation was actually mediated by the direction of chloride flux. Therefore, in order to properly understand receptor activation and modulation, we must take both the channel open probability and the direction of ion flux into account.