TY - JOUR T1 - Location of a High Affinity Zn<sup>2+</sup> Binding Site in the Channel of α1β1 γ-Aminobutyric Acid<sub>A</sub> Receptors JF - Molecular Pharmacology JO - Mol Pharmacol SP - 870 LP - 877 VL - 53 IS - 5 AU - Jeffrey Horenstein AU - Myles H. Akabas Y1 - 1998/05/01 UR - http://molpharm.aspetjournals.org/content/53/5/870.abstract N2 - Zn2+ inhibits currents through γ-aminobutyric acid (GABA)A receptors. Its affinity depends on the subunit composition; α1β1 receptors are inhibited with high affinity (IC50 = 0.54 μm). We sought to identify the residues that form this high affinity Zn2+ binding site. β1His267 aligns with α1Ser272, 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 α1β1 H267S was reduced by 300-fold (IC50 = 161 μm). Addition of a histidine at the aligned position in α1 creates a receptor, α1S272Hβ1, that should have five channel-lining histidines; the Zn2+ affinity was increased 20-fold (IC50 = 0.025 μm). Shifting the position of the histidine from the β1 subunit to the aligned position in α1 with the two mutants α1S272Hβ1H267S reduced the affinity (IC50 = 26 μm) compared with wild-type. We infer that the high affinity Zn2+ binding site involves β1His267 from at least two subunits. For two histidines to interact with a Zn2+ ion, the α carbons must be separated by &lt;13 Å. 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 β1His267 implies that the charge-selectivity filter, the structure that discriminates between anions and cations, is located at a more cytoplasmic position than β1His267; this is consistent with our previous work that showed that positively charged sulfhydryl-specific reagents reacted with an engineered cysteine residue as cytoplasmic as α1T261C. The American Society for Pharmacology and Experimental Therapeutics ER -