Pulsed electron spin resonance resolves the coordination site of Cu 2+ ions in α1-glycine receptor

Sharon Ruthstein, University of Pittsburgh
Katherine M. Stone, University of Pittsburgh
Timothy F. Cunningham, University of Pittsburgh
Ming Ji, University of Pittsburgh
Michael Cascio, Duquesne University
Sunil Saxena, Duquesne University


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 Zn 2+ site. The opposing modulatory actions of these cations at a shared binding site highlight the sensitive allosteric nature of GlyR. © 2010 by the Biophysical Society.