Determinants of Zinc Potentiation on the α4 Subunit of Neuronal Nicotinic Receptors

Abstract

We have shown previously that the function of neuronal nicotinic acetylcholine receptors can be modulated by zinc. This modulation varies from potentiation to inhibition, depending on receptor subunit composition and zinc concentration, with the ␣4␤2 and ␣4␤4 receptors displaying the most dramatic potentiation. In this study, we used site-directed mutagenesis to identify glutamate 59 and histidine 162 on the rat ␣4 subunit as potential mediators of zinc potentiation. By modeling the extracellular domain of the receptor pentamer, we locate these residues to two subunit-subunit interfaces that alternate with the two acetylcholine-binding interfaces. Substitution of a cysteine at either position allows additional reduction of zinc potentiation upon treatment with the methanethiosulfonate reagents N-biotinoylaminoethyl methanethiosulfonate (MTSEAbiotin) and [2-(trimethylammonium)ethyl] methanethiosulfonate.Mutagenesis and methanethiosulfonate treatment are most effective at position 162, and the presence of zinc hinders the reaction of MTSEA-biotin with the substituted cysteine at this position, suggesting that ␣4His162 participates in forming a coordination site for zinc. Mutagenesis and methanethiosulfonate treatment are less effective at position 59, suggesting that whereas ␣4Glu59 may be near the zinc coordination site, it may not be participating in coordination of the zinc ion. It is noteworthy that the position of ␣4Glu59 within the neuronal nAChR is identical to that of a residue that lines the benzodiazepinebinding site on GABA A receptors. We suggest that the zinc potentiation sites on neuronal nAChRs are structurally and functionally similar to the benzodiazepine-binding sites on GABA A receptors.

The structural basis of function in Cys-loop receptors

“…This region coincides with residues that have been shown to bind Ca 2+ in AChBP (Brejc et al 2001). Zinc-binding sites have been located at subunit interfaces in nAChR and GlyR (Hsiao et al 2006 ;Nevin et al 2003), while in GABA A receptors, zinc binds to both the ECD and the pore (Dunne et al 2002 ;Fisher & Macdonald, 1998 ;Fisher, 2002 ;Horenstein & Akabas, 1998 ;Hosie et al 2003). Binding of these ions is likely to have important physiological consequences although these are not yet fully understood.…”

Non-Agonist-Binding Subunit Interfaces Confer Distinct Functional Signatures to the Alternate Stoichiometries of the 4 2 Nicotinic Receptor: An 4- 4 Interface Is Required for Zn2+ Potentiation

“…Although advances have been made in identifying Zn 2ϩ sites on GABA A (Hosie et al, 2003) and glycine (Miller et al, 2005a,b) receptors, corresponding sites on ␣4␤2 receptors have been relatively unexplored. A potentiating Zn 2ϩ -binding site has been proposed on ␣4␤4/␤2 nAChRs that involves Zn 2ϩ coordination by a histidine residue (␣4 H162 ) and a less direct, probably structural, contribution from ␣4 E59 (Hsiao et al, 2006). The site is likely to reside on the ␤2(ϩ)/ ␣4(Ϫ) subunit interfaces that alternate with the ACh-binding ␣4(ϩ)/␤2(Ϫ) subunit interfaces (Hsiao et al, 2006).…”

“…A potentiating Zn 2ϩ -binding site has been proposed on ␣4␤4/␤2 nAChRs that involves Zn 2ϩ coordination by a histidine residue (␣4 H162 ) and a less direct, probably structural, contribution from ␣4 E59 (Hsiao et al, 2006). The site is likely to reside on the ␤2(ϩ)/ ␣4(Ϫ) subunit interfaces that alternate with the ACh-binding ␣4(ϩ)/␤2(Ϫ) subunit interfaces (Hsiao et al, 2006). Given that ␤2(ϩ)/␣4(Ϫ) interfaces are present on both receptor forms, receptor stoichiometry should not influence Zn 2ϩ effects on ␣4␤2 receptors (Hsiao et al, 2006).…”

Potentiation and inhibition of glycine receptors by tutin

“…This is not unique to Cl À inhibitory channels because a 4 b 4 nACh receptors are also potentiated by interaction of Zn 2þ with residues located at alternating a 4 b 4 interfaces (Hsiao et al, 2006). Similarly, other studies have demonstrated the presence of residues important for the potentiation of GlyRs by extracellular acting ligands.…”