Abstract

The properties of the human α₁β₁γ-aminobutyric acid (GABA)_A receptors were investigated after mutation of a highly conserved leucine residue at the 9′ position in the second membrane-spanning region (TM2). The role of this residue in α₁ and β₁ subunits was examined by mutating the 9′ leucine to phenylalanine, tyrosine, or alanine. The mutations were in either the α₁ subunit (α∗β), the β₁ subunit (αβ∗), or in both subunits (α∗β∗), and the receptors were expressed in Sf9 cells. Our results show that the rate of desensitization is increased as the size and hydrophobicity of the 9′ residue in the α₁ subunit is increased: Y, F > L > A, T. Mutation of L9′ in only the β₁ subunit (αβ∗) to either phenylalanine or tyrosine increased the EC₅₀ value for GABA at least 100 times, but the EC₅₀ was unchanged in αβ∗ alanine mutants. In the 9′ α₁ mutants (α∗β, α∗β∗) the GABA EC₅₀ was minimally affected. In α∗β and α∗β∗, but not αβ∗, the peak currents evoked by millimolar concentrations of GABA were greatly reduced. The reduction in currents could only be partially accounted for by decreased expression of the receptors These findings suggest different roles for the two types of subunits in GABA activation and later desensitization of α₁β₁ receptors. In addition, an increase in the resting membrane conductance was recorded in alanine but not in phenylalanine and tyrosine mutants, indicating that the side chain size at the 9′ position is a major determinant of current flow in the closed conformation.