Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor

Abstract

Herein, we identify the coordination environment of Cu²(+) in the human α1-glycine receptor (GlyR). GlyRs are members of the pentameric ligand-gated ion channel superfamily (pLGIC) that mediate fast signaling at synapses. Metal ions like Zn²(+) and Cu²(+) significantly modulate the activity of pLGICs, and metal ion coordination is essential for proper physiological postsynaptic inhibition by GlyR in vivo. Zn²(+) can either potentiate or inhibit GlyR activity depending on its concentration, while Cu²(+) is inhibitory. To better understand the molecular basis of the inhibitory effect we have used electron spin resonance to directly examine Cu²(+) coordination and stoichiometry. We show that Cu²(+) has one binding site per α1 subunit, and that five Cu²(+) can be coordinated per GlyR. Cu²(+) binds to E192 and H215 in each subunit of GlyR with a 40 μM apparent dissociation constant, consistent with earlier functional measurements. However, the coordination site does not include several residues of the agonist/antagonist binding site that were previously suggested to have roles in Cu²(+) coordination by functional measurements. Intriguingly, the E192/H215 site has been proposed as the potentiating Zn²(+) site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR.

Figure 1

Titration curve of [Cu²⁺] in WT-GlyR (at 20 K, ν = 9.21 GHz) and E192A-GlyR (at 20 K, ν = 9.76 GHz). The coordinated [Cu²⁺]/[GlyR] is plotted against number of equivalent [Cu²⁺] added. For WT-GlyR, the best fit (solid line) was obtained with a K_d = 40 μM, whereas for E192A-GlyR, the best fit corresponded to a K_d = 95 μM. The fits were determined using Eq. 1. The Cu²⁺-binding site in WT-GlyR can be fully saturated while the occupancy in E192A-GlyR is reduced to 2.5 Cu²⁺/receptor complex.

Figure 2

(A) FS-ESE spectra at 20 K for WT, H109N, T112A, H215A, and E192A-GlyR (at 20 K, ν = 9.69 GHz). (Inset) Suggested model for the Cu⁺² binding site in GlyR. (B) CW-ESR spectra for WT-GlyR, H215A-GlyR, and E192A-GlyR (at 5 K, ν = 9.406 GHz, modulation amplitude = 4.0 G, microwave power = 10 mW). (Dashed lines) Simulations obtained with the parameters described in the text. (Top inset) A_‖ splitting for each spectrum.

Figure 3

Time domain ESEEM signals for WT-GlyR and H215A-GlyR at τ = 140 ns. The ESEEM was acquired at 20 K, ν = 9.702 GHz, 3360 G, T increment, 16 ns, 1024 time points.

Figure 4

(A) Time domain ESEEM signals for WT-GlyR and H215A-GlyR at τ = 140 ns. Gray dotted lines are provided as guides. (B) Corresponding ESEEM spectra. In both panels the dashed lines represent the ESEEM simulations. The ESEEM data was acquired at 20 K, ν = 9.702 GHz, 3360 G, T increment, 16 ns, 1024 time points.

Figure 5

(A) ESEEM experimental spectra (solid line) of WT-GlyR and H215A-GlyR at τ = 150 ns. (B) ESEEM experimental spectra (solid line) of WT-GlyR and H215A-GlyR at τ = 200 ns. In both panels, the dashed lines represent the ESEEM simulations. The ESEEM data was acquired at 20 K, ν = 9.702 GHz, 3360 G, T increment, 12 ns, 512 time points.

Figure 6

Key sites hypothesized as metal ion binding are highlighted on the ECD model of GlyR (9). (Solid circle/blue circle online) Copper ion binding site (E192/H215). (Shaded circle/red circle online) Inhibitory Zn²⁺ site (H107/H109). (Dashed circle/green circle online) Strychnine antagonist binding site.