Effects of mutating leucine to threonine in the M2 segment of alpha(1) and beta(1) subunits of GABA(A) alpha(1)beta(1) receptors

The conserved leucine residues at the 9' positions in the M2 segments of alpha(1) (L264) and beta(1) (L259) subunits of the human GABA(A) receptor were replaced with threonine. Normal or mutant alpha(1) subunits were coexpressed with normal or mutant beta(1) 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 alpha(1) beta(1) receptors. This chloride conductance was blocked by 10 mM penicillin, a recognized blocker of GABA(A) channels, but not by bicuculline (100 mu M) or picrotoxin (100 mu M) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 mu M) In cells expressing wild-type beta(1) with mutated alpha(1) subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type alpha(1) beta(1) receptors. In cells expressing mutated beta(1) subunits with wild-type or mutated alpha(1) subunits (alpha beta(L9'T) and alpha(L9'T)beta(L9'T)), no response to GABA could be elicited: this was not due to an absence of GABA(A) receptors in the plasmalemma because the cells bound [H-3]-muscimol. It was concluded that in GABA(A) channels containing the L9'T mutation in the beta(1) 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 GABA(A) receptor in the absence of ligand.