Abstract
The divalent cation Zn(2+) is a potent potentiator at the strychnine-sensitive glycine receptor (GlyR). This occurs at nanomolar concentrations, which are the predicted endogenous levels of extracellular neuronal Zn(2+). Using structural modeling and functional mutagenesis, we have identified the molecular basis for the elusive Zn(2+) potentiation site on GlyRs and account for the differential sensitivity of GlyR alpha(1) and GlyR alpha(2) to Zn(2+) potentiation. In addition, juxtaposed to this Zn(2+) site, which is located externally on the N-terminal domain of the alpha subunit, another residue was identified in the nearby Cys loop, a region that is critical for receptor gating in all Cys loop ligand-gated ion channels. This residue acted as a key control element in the allosteric transduction pathway for Zn(2+) potentiation, enabling either potentiation or overt inhibition of receptor activation depending upon the moiety resident at this location. Overall, we propose that Zn(2+) binds to a site on the extracellular outer face of the GlyR alpha subunit and exerts its positive allosteric effect via an interaction with the Cys loop to increase the efficacy of glycine receptor gating.
Highlights
Zn2ϩ binding sites do provide realistic targets for identification, as these are traditionally compact, consisting of only 3– 4 residues, and their coordination chemistry is understood [9]
glycine receptor (GlyR) ␣1 and ␣2 Exhibit Distinct Sensitivities to Zn2ϩ Potentiation— The sensitivity of the GlyR ␣1 and ␣2 subtypes to Zn2ϩ potentiation was assessed from whole-cell recordings of half-maximal (EC50) responses to glycine obtained from transfected HEK cells maintained at Ϫ40 mV
GlyR ␣1 Thr-151 Is a Critical Control Element for Zn2ϩ Potentiation—Besides the classical Zn2ϩ binding moieties of the potentiating site, we identified a nearby polar residue at position 151 located in the Cys loop called L7 [27]
Summary
Zn2ϩ binding sites do provide realistic targets for identification, as these are traditionally compact, consisting of only 3– 4 residues, and their coordination chemistry is understood [9]. A previous study demonstrates that the mutation D80A in the extracellular domain of the GlyR ␣1 subunit ablated Zn2ϩ potentiation, and it is proposed that this residue partici-. An additional report indicates that the Zn2ϩ potentiation of responses to the partial agonist taurine, which binds to the same agonist site as glycine, is unaffected by mutating Asp-80. Zn2ϩ is released following neuronal stimulation [13, 14] and can modulate inhibitory neurotransmitter receptors at basal concentrations [15, 16]. We uncovered a prospective transduction residue for this site that is located in the Cys loop-gating domain, providing a plausible molecular pathway for Zn2ϩ potentiation of glycine receptor gating
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