Spontaneous and γ-Aminobutyric Acid (GABA)-Activated GABAAReceptor Channels Formed by ε Subunit-Containing Isoforms

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Abstract
A new γ-aminobutyric acid (GABA)A receptor (GABAR) subunit class, ε, has recently been cloned and shown to form functional channels when coexpressed with both α and β subunits. We report that the combination of α1β3ε 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 α1β3γ2L, α1β3δ, α1ε, β3ε, α1β3, or ε subtypes did not produce holding currents that were sensitive to picrotoxin (30 μM). Cells expressing α1 β3ε 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 α1β3ε receptors had single-channel conductances of ∼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 ε-containing GABARs and demonstrate that incorporation of the ε subunit permits spontaneous channel gating while preserving the structural information necessary for GABA sensitivity.