Abstract

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 <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.