Location of a high affinity Zn2+ binding site in the channel of alpha1beta1 gamma-aminobutyric acidA receptors.

Zn2+ inhibits currents through gamma-aminobutyric acid (GABA)A receptors. Its affinity depends on the subunit composition; alpha1beta1 receptors are inhibited with high affinity (IC50 = 0.54 micro M). We sought to identify the residues that form this high affinity Zn2+ binding site. beta1His267 aligns with alpha1Ser272, a residue near the extracellular end of the M2 membrane-spanning segment that we previously demonstrated to be exposed in the channel. The Zn2+ affinity of alpha1beta1 H267S was reduced by 300-fold (IC50 = 161 micro M). Addition of a histidine at the aligned position in alpha1 creates a receptor, alpha1S272Hbeta1, that should have five channel-lining histidines; the Zn2+ affinity was increased 20-fold (IC50 = 0.025 micro M). Shifting the position of the histidine from the beta1 subunit to the aligned position in alpha1 with the two mutants alpha1S272Hbeta1H267S reduced the affinity (IC50 = 26 micro M) compared with wild-type. We infer that the high affinity Zn2+ binding site involves beta1His267 from at least two subunits. For two histidines to interact with a Zn2+ ion, the alpha carbons must be separated by <13 A. This limits the separation of the subunits and provides a constraint on the possible quaternary structures of the channel. The ability of a divalent cation to penetrate from the extracellular end of the channel to beta1His267 implies that the charge-selectivity filter, the structure that discriminates between anions and cations, is located at a more cytoplasmic position than beta1His267; this is consistent with our previous work that showed that positively charged sulfhydryl-specific reagents reacted with an engineered cysteine residue as cytoplasmic as alpha1T261C.