Zinc interactions with allosteric modulators at the glycine receptor

Cornelison, Garrett Lee
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The glycine receptor (GlyR) is a ligand-gated ion channel member of the Cys-loop receptor superfamily, responsible for inhibitory neurotransmission in the brain and spinal cord. Zinc is a potent allosteric modulator of GlyR function, enhancing GlyR activity at low nM to 10[mu]M concentrations while inhibiting GlyR activity at higher concentrations. We investigated sources of contaminating zinc, identifying low nM levels of zinc in ultrapure H₂O, powdered reagents used in the preparation of common electrophysiological buffers, and in polystyrene pipets. These low levels of zinc were capable of enhancing GlyR function. These findings suggest that without checking for this effect using a zinc-chelator such as tricine, one cannot assume that responses elicited by glycine applied alone are not necessarily also partially due to some level of allosteric modulation by zinc. Taurine-activated GlyR may have a role in the rewarding effects of drugs of abuse. Zinc is found at GlyR-potentiating concentrations throughout the nervous system, so we examined the combinatorial effects of zinc with drugs of abuse on taurine-activated GlyR to mimic in vivo conditions. Whole cell recordings revealed that zinc potentiation of saturating taurine-generated currents decreased further potentiation by drugs of abuse, indicating no synergistic effects on efficacy when receptors are saturated with taurine as may be seen during synaptic events in vivo. Finally, we utilized phage display to identify novel peptide modulators of the GlyR. We tested 26 peptides against [alpha1beta] GlyRs, identifying peptides with various levels of activity on GlyR function. We demonstrated that these modulators were zinc-dependent, as their effects on GlyR activity were abolished in the presence of the zinc-chelating agent tricine. Together, these data indicate the importance of accounting for the effects of zinc when studying the function of the GlyR, as even low levels of zinc that can be found as contaminants in labware and buffers can affect GlyR function and responses to various allosteric modulators, including drugs of abuse.