Disrupted Cl − homeostasis contributes to reductions in the inhibitory efficacy of diazepam during hyperexcited states

The K + ‐ Cl − cotransporter type 2 is the major Cl − extrusion mechanism in most adult neurons. This process in turn leads to Cl − influx upon activation of γ‐aminobutyric acid type A ( GABA A ) receptors and the canonical hyperpolarising inhibitory postsynaptic potential. Several neurological disorders are treated with drugs that target and enhance GABA A receptor signaling, including the commonly used benzodiazepine diazepam and the anesthetic propofol. Some of these disorders are also associated with deficits in GABA A signaling and become less sensitive to therapeutic drugs that target GABA A receptors. To date, it is unknown if alterations in the neuronal Cl − gradient affect the efficacies of diazepam and propofol. We therefore used the in vitro model of glutamate‐induced hyperexcitability to test if alterations in the Cl − gradient affect the efficacy of GABA A modulators. We exclusively utilised the gramicidin perforated‐patch‐clamp configuration to preserve the endogenous Cl − gradient in rat neurons. Brief exposure to glutamate reduced the inhibitory efficacy of diazepam within 5 min, which was caused by the collapse of the Cl − gradient, and not due to reductions in GABA A receptor number. Unlike diazepam, propofol retained its efficacy by shunting the membrane conductance despite the glutamate‐induced appearance of depolarising GABA A ‐mediated currents. Similarly, pharmacological inhibition of K + ‐ Cl − cotransporter type 2 by furosemide disrupted Cl − homeostasis and reduced the efficacy of diazepam but not propofol. Collectively our results suggest that pathological hyperexcitable conditions could cause the rapid accumulation of intracellular Cl − and the appearance of depolarising GABA A ‐mediated currents that would decrease the efficacy of diazepam.