Abstract

The amino-terminal extracellular domain of the subunits comprising the gamma-aminobutyric acid (GABA) receptor contains two cysteine residues (designated at relative positions 1 and 15) separated by 13 amino acids. These two cysteines (presumably disulfide bonded) are located approximately 150 amino acids from the amino terminus. There is significant homology in the amino acid sequence of this cysteine loop both between the different subunits of the GABA receptor and with subunits of other members of this ligand-gated ion channel superfamily (nicotinic acetylcholine- and glycine-activated ion channels). A number of highly conserved amino acids within the cysteine loop have been postulated to play a role in agonist binding. Here, using site-directed mutagenesis and oocyte expression, we have examined the effects of mutating amino acids comprising the cysteine loop on the activation of recombinant GABA channels composed of rat alpha 1, beta 2 and gamma 2 subunits. Preventing the formation of the putative cysteine-cysteine disulfide bond in any of the subunits, by mutating the cysteine at position 15 to serine, prevented the functional expression of that subunit. For example, coexpression of gamma C15S with wild-type alpha and beta subunits resulted in GABA-activated currents with properties identical to those of GABA-activated currents from coexpression of alpha and beta subunits alone. These properties included sensitivity to activation by GABA (similar EC50 values), blockade by Zn2+, and lack of modulation by the benzodiazepine diazepam. We also mutated conserved amino acids in the beta subunit that had been specifically proposed to form the GABA binding site (beta R6, beta Y8, and beta D11). These mutations (as well as several others within or adjacent to the cysteine loop) produced either a very moderate effect or no effect on GABA sensitivity, suggesting that these particular amino acids do not play a key role in activation of the GABA channel. The data presented in this study support a role for the cysteine loop in subunit assembly, rather than channel activation.