LOCATION OF A HIGH-AFFINITY ZN2-SITE IN THE CHANNEL OF ALPHA-1-BETA-1GAMMA-AMINOBUTYRIC ACID(A) RECEPTORS( BINDING)

Zn2+ inhibits currents through gamma-aminobutyric acid (GABA)(A) recep tors. Its affinity depends on the subunit composition; alpha 1 beta 1 receptors are inhibited with high affinity (IC50 = 0.54 mu M) We sough t to identify the residues that form this high affinity Zn2+ binding s ite. beta His267 aligns with alpha 1Ser272, a residue near the extrace llular end of the M2 membrane-spanning segment that we previously demo nstrated to be exposed in the channel. The Zn2+ affinity of alpha 1 be ta 1 H267S was reduced by 300-fold (IC50 = 161 mu M). Addition of a hi stidine at the aligned position in alpha 1 creates a receptor, alpha 1 S272H beta 1, that should have five channel-lining histidines; the Zn2 + affinity was increased 20-fold (IC50 = 0.025 mu M). Shifting the pos ition of the histidine from the beta 1 subunit to the aligned position in alpha 1 with the two mutants alpha 1S272H beta 1H267S reduced the affinity (IC50 = 26 mu M) compared with wild-type We infer that the hi gh affinity Zn2+ binding site involves beta 1His267 from at least two subunits, For two histidines to interact with a Zn2+ ion, the a carbon s must be separated by <13 Angstrom. This limits the separation of the subunits and provides a constraint on the possible quaternary structu res of the channel. The ability of a divalent cation to penetrate from the extracellular end of the channel to beta 1His267 implies that the charge-selectivity filter, the structure that discriminates between a nions and cations, is located at a more cytoplasmic position than beta 1His267; this is consistent with our previous work that showed that p ositively charged sulfhydryl-specific reagents reacted with an enginee red cysteine residue as cytoplasmic as alpha 1T261C.