Mutating the highly conserved second membrane-spanning region 9 ' leucine residue in the alpha(1) or beta(1) subunit produces subunit-specific changes in the function of human alpha(1)beta(1) gamma-aminobutyric acid(A) receptors

The properties of the human alpha(1)beta(1) gamma-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). T he role of this residue in alpha(1) and beta(1) subunits was examined by mu tating the 9' leucine to phenylalanine, tyrosine, or alanine. The mutations were in either the alpha(1) subunit (alpha*beta), the beta(1) subunit (alp ha beta*), or in both subunits (alpha*beta*), and the receptors were expres sed in Sf9 cells. Our results show that the rate of desensitization is incr eased as the size and hydrophobicity of the 9' residue in the alpha(1) subu nit is increased: Y, F > L > A, T. Mutation of L9' in only the beta(1) subu nit (alpha beta*) to either phenylalanine or tyrosine increased the EC50 va lue for GABA at least 100 times, but the EC50 was unchanged in alpha beta* alanine mutants. In the 9' alpha(1) mutants (alpha*beta, alpha*beta*) the G ABA EC50 was minimally affected. In alpha*beta and alpha*beta*, but not alp ha beta*, the peak currents evoked by millimolar concentrations of GABA wer e greatly reduced. The reduction in currents could only be partially accoun ted for by decreased expression of the receptors These findings suggest dif ferent roles for the two types of subunits in GABA activation and later des ensitization of alpha(1)beta(1) receptors. In addition, an increase in the resting membrane conductance was recorded in alanine but not in phenylalani ne and tyrosine mutants, indicating that the side chain size at the 9' posi tion is a major determinant of current flow in the closed conformation.