title: Spontaneous and gamma-aminobutyric acid (GABA)-activated GABA(A) receptor channels formed by epsilon subunit-containing isoforms.
abstract: A new gamma-aminobutyric acid (GABA)A receptor (GABAR) subunit class, epsilon, has recently been cloned and shown to form functional channels when coexpressed with both alpha and beta subunits. We report that the combination of alpha1beta3epsilon subunit subtypes expressed in L929 cells produced functional chloride ion channels that were both spontaneously active and gated by the application of extracellular GABA. When cells were voltage-clamped at -75 mV in the whole-cell configuration, holding currents of 50 to 300 pA associated with increased noise were consistently recorded. The application of pentobarbital and loreclezole, which increase GABAR currents, increased the holding current, whereas the application of zinc and picrotoxin, which reduce GABAR currents, reduced the holding current in a concentration-dependent manner. Coexpression of alpha1beta3gamma2L, alpha1beta3delta, alpha1epsilon, beta3epsilon, alpha1beta3, or epsilon subtypes did not produce holding currents that were sensitive to picrotoxin (30 microM). Cells expressing alpha1 beta3epsilon subtypes had concentration-dependent GABAR currents that were potentiated by pentobarbital, loreclezole, and lanthanum and inhibited by zinc and furosemide. Spontaneous and GABAR single-channel currents from alpha1beta3epsilon receptors had single-channel conductances of approximately 24 pS. The biophysical properties and the effects of allosteric modulators were similar for spontaneous and evoked GABAR currents, suggesting that a single GABAR isoform was responsible for both currents. These data extend the pharmacological characterization of epsilon-containing GABARs and demonstrate that incorporation of the epsilon subunit permits spontaneous channel gating while preserving the structural information necessary for GABA sensitivity.
authors.affiliation: Neuroscience Program, University of Michigan, Ann Arbor, Michigan, USA.
authors.forename: T R
authors.initials: TR
authors.lastname: Neelands
authors.forename: J L
authors.initials: JL
authors.lastname: Fisher
authors.forename: M
authors.initials: M
authors.lastname: Bianchi
authors.forename: R L
authors.initials: RL
authors.lastname: Macdonald
mesh_terms: D000818:Animals; D002460:Cell Line; D004305:Dose-Response Relationship, Drug; D005665:Furosemide / Q000494:pharmacology; D007811:Lanthanum / Q000494:pharmacology; D051379:Mice; D010424:Pentobarbital / Q000494:pharmacology; D020033:Protein Isoforms / Q000187:drug effects; D051381:Rats; D011963:Receptors, GABA-A / Q000187:drug effects* / Q000502:physiology; D014230:Triazoles / Q000494:pharmacology; D015032:Zinc / Q000494:pharmacology; D005680:gamma-Aminobutyric Acid / Q000494:pharmacology*
chemical_list: D020033:Protein Isoforms; D011963:Receptors, GABA-A; D014230:Triazoles; D005680:gamma-Aminobutyric Acid; C066440:loreclezole; D007811:Lanthanum; D005665:Furosemide; D010424:Pentobarbital; D015032:Zinc