title: Disrupted Cl(-) homeostasis contributes to reductions in the inhibitory efficacy of diazepam during hyperexcited states.
abstract: 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 (GABAA ) receptors and the canonical hyperpolarising inhibitory postsynaptic potential. Several neurological disorders are treated with drugs that target and enhance GABAA receptor signaling, including the commonly used benzodiazepine diazepam and the anesthetic propofol. Some of these disorders are also associated with deficits in GABAA signaling and become less sensitive to therapeutic drugs that target GABAA 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 GABAA 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 GABAA receptor number. Unlike diazepam, propofol retained its efficacy by shunting the membrane conductance despite the glutamate-induced appearance of depolarising GABAA -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 GABAA -mediated currents that would decrease the efficacy of diazepam.
authors.affiliation: Department of Neuroscience, Tufts University, Boston, MA, USA.
authors.forename: Tarek Z
authors.initials: TZ
authors.lastname: Deeb
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Nakamura
authors.forename: Greg D
authors.initials: GD
authors.lastname: Frost
authors.forename: Paul A
authors.initials: PA
authors.lastname: Davies
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Moss
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D003975:Diazepam / Q000494:pharmacology*; D005665:Furosemide / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology*; D018698:Glutamic Acid / Q000494:pharmacology; D006706:Homeostasis; D009118:Muscimol / Q000494:pharmacology; D009433:Neural Inhibition / Q000187:drug effects*; D015742:Propofol / Q000494:pharmacology; D017966:Pyramidal Cells / Q000187:drug effects* / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D049994:Sodium Potassium Chloride Symporter Inhibitors / Q000494:pharmacology; D027981:Symporters / Q000187:drug effects / Q000378:metabolism*; D000096922:K Cl- Cotransporters
chemical_list: D002712:Chlorides; D003975:Diazepam; D005665:Furosemide; D018757:GABA Modulators; D018698:Glutamic Acid; D009118:Muscimol; D015742:Propofol; D049994:Sodium Potassium Chloride Symporter Inhibitors; D027981:Symporters; D000096922:K Cl- Cotransporters
keywords: K+-Cl− cotransporter type 2; glutamate; neurons; propofol; rat