title: Benzodiazepine treatment induces subtype-specific changes in GABA(A) receptor trafficking and decreases synaptic inhibition.
abstract: Benzodiazepines potentiate γ-aminobutyric acid type A receptor (GABA(A)R) activity and are widely prescribed to treat anxiety, insomnia, and seizure disorders. Unfortunately, clinical use of benzodiazepines (BZs) is severely limited by tolerance. The mechanisms leading to BZ tolerance are unknown. BZs bind at the interface between an α and γ subunit of GABA(A)Rs, preferentially enhancing synaptic receptors largely composed of α(1-3, 5), β3, and γ2 subunits. Using confocal imaging and patch-clamp approaches, we show that treatment with the BZ flurazepam decreases GABA(A)R surface levels and the efficacy of neuronal inhibition in hippocampal neurons. A dramatic decrease in surface and total levels of α2 subunit-containing GABA(A)Rs occurred within 24 h of flurazepam treatment, whereas GABA(A)Rs incorporating α1 subunits showed little alteration. The GABA(A)R surface depletion could be reversed by treatment with the BZ antagonist Ro 15-1788. Coincident with decreased GABA(A)R surface levels, flurazepam treatment reduced miniature inhibitory postsynaptic current amplitude, which returned to control levels with acute Ro 15-1788 treatment. GABA(A)R endocytosis and insertion rates were unchanged by flurazepam treatment. Treatment with leupeptin restored flurazepam lowered receptor surface levels, strongly suggesting that flurazepam increases lysosomal degradation of GABA(A)Rs. Together, these data suggest that flurazepam exposure enhances degradation of α2 subunit-containing GABA(A)Rs after their removal from the plasma membrane, leading to a reduction in inhibitory synapse size and number along with a decrease in the efficacy of synaptic inhibition. These reported subtype-specific changes in GABA(A)R trafficking provide significant mechanistic insight into the initial neuroadaptive responses occurring with BZ treatment.
authors.affiliation: Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Tija C
authors.initials: TC
authors.lastname: Jacob
authors.forename: Guido
authors.initials: G
authors.lastname: Michels
authors.forename: Liliya
authors.initials: L
authors.lastname: Silayeva
authors.forename: Julia
authors.initials: J
authors.lastname: Haydon
authors.forename: Francesca
authors.initials: F
authors.lastname: Succol
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Moss
mesh_terms: D000818:Animals; D001569:Benzodiazepines / Q000494:pharmacology*; D001665:Binding Sites; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects; D053444:Inhibitory Postsynaptic Potentials / Q000187:drug effects; D007976:Leupeptins / Q000494:pharmacology; D009433:Neural Inhibition / Q000187:drug effects*; D009474:Neurons / Q000187:drug effects / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000378:metabolism*; D013569:Synapses / Q000187:drug effects / Q000502:physiology*
chemical_list: D007976:Leupeptins; D021122:Protein Subunits; D011963:Receptors, GABA-A; D001569:Benzodiazepines; C032854:leupeptin