Pulsed electron spin resonance resolves the coordination site of Cu²(+) ions in α1-glycine receptor.
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.
Department of Chemistry, University of Pittsburgh, Pennsylvania, USA.
Sharon
S
Ruthstein
Katherine M
KM
Stone
Timothy F
TF
Cunningham
Ming
M
Ji
Michael
M
Cascio
Sunil
S
Saxena
D000818:Animals; D001665:Binding Sites; D003300:Copper; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D018009:Receptors, Glycine / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D018009:Receptors, Glycine; D003300:Copper