Effects of Mutating Leucine to Threonine in the M2 Segment of α 1 and β 1 Subunits of GABA A α 1 β 1 Receptors

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Abstract
The conserved leucine residues at the 9′ positions in the M2 segments of α1 (L264) and β1 (L259) subunits of the human GABAA receptor were replaced with threonine. Normal or mutant α1 subunits were co-expressed with normal or mutant β1 subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting'' chloride conductance than cells expressing wild-type α1β1 receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABAA channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β1 with mutated α1 subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α1β1 receptors. In cells expressing mutated β1 subunits with wild-type or mutated α1 subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABAA receptors in the plasmalemma because the cells bound [3H]-muscimol. It was concluded that in GABAA channels containing the L9′T mutation in the β1 subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABAA receptor in the absence of ligand.

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