Location of a High Affinity Zn2+ Binding Site in the Channel of α1β1 γ-Aminobutyric AcidA Receptors
- 1Center for Molecular Recognition (J.H., M.H.A.) and the Departments of 2Physiology and Cellular Biophysics (J.H., M.H.A.) and 3Medicine (M.H.A.), Columbia University, New York, New York 10032
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.
Footnotes
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Send reprint requests to: Dr. Myles Akabas, Center for Molecular Recognition, Columbia University, 630 West 168th Street, Box 7, New York, NY 10032. E-mail: ma14{at}columbia.edu
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This work was supported in part by National Institutes of Health Grants NS30808 and DK51794. M.H.A. is the recipient of an Established Scientist Award from the American Heart Association, New York City Affiliate.
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A preliminary report of this work has appeared in abstract form [Horenstein J and Akabas MH (1997) Soc Neurosci Abstr23:112]. While preparing the manuscript, we were informed of a preliminary report describing similar results of mutation of the aligned residue in the β3 subunit, β3His292A [Wooltorton JRA, McDonald BJ, Moss SJ, and Smart TG (1997) Br J Pharmacol122(suppl):38P]. A full version of this work was subsequently published in J Physiol (Lond)505:633–640 (1997).
- Abbreviations:
- GABAA
- γ-aminobutyric acid type A receptor
- ACh
- acetylcholine
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- CFFR
- Ca2+-free frog Ringer’s solution
- GABA
- γ-aminobutyric acid
- MTS
- methanethiosulfonate
- MTSES−
- methanethiosulfonate ethylsulfonate
- MTSET+
- methanethiosulfonate ethyltrimethylammonium
- pCMBS
- p-chloromercuribenzenesulfonate
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- Received November 10, 1997.
- Accepted January 16, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
