Browsing by Author "Cornelison, Garrett Lee"
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Item Characterization of the interactions of zinc with known and novel allosteric modulators of glycine receptor function(2016-12) Cornelison, Garrett Lee; Mihic, S. John; Aldrich, Richard W; Harris, R. Adron; Stavchansky, Salomon AThe glycine receptor is a member of the Cys-loop receptor superfamily of ligand-gated ion channels and is implicated as a possible therapeutic target for the treatment of diseases such as alcoholism and inflammatory pain. In humans, four glycine receptor subtypes (α1, α2, α3, and β) co-assemble to form pentameric channel proteins as either α homomers or αβ heteromers. To date, few agents have been identified that can selectively modulate the glycine receptor, especially those possessing subtype specificity. We used a cell-based method of phage display panning, coupled with two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes, to identify novel heptapeptide modulators of the α1β glycine receptor. Peptides were identified that act with selectivity on α1β and α3β compared to α2β glycine receptors. In addition, peptide activity at the glycine receptor decreased when zinc was chelated by tricine, similar to previous observations of a decrease in ethanol’s enhancing actions at the receptor in the absence of zinc. Zinc is an allosteric modulator of glycine receptor function, enhancing the effects of glycine at nanomolar to low micromolar concentrations, and inhibiting its effects at higher concentrations. As zinc is present physiologically at various concentrations within this range, it is capable of influencing glycine receptor function, including modulation by other pharmacological agents; however, the magnitude of this effect and its possible relevance are not known. I therefore investigated the utility of previously-described “zinc-enhancement insensitive” α1 glycine receptor mutants D80A, D80G, and W170S to probe for interactions between zinc and other allosteric modulators at the glycine receptor. Interestingly, I found that only the W170S mutation conferred complete abolishment of zinc enhancement across a variety of agonist and zinc concentrations. Using α1 W170S receptors, I established that in addition to ethanol, zinc also interacts with inhaled drugs of abuse, but not volatile anesthetics, to synergistically enhance channel function. Additionally, I determined that this interaction is abolished at higher zinc concentrations, when receptor-enhancing bindings sites are saturated, suggesting a mechanism by which modulators such as ethanol and inhalants are capable of increasing receptor affinity for zinc in addition to enhancing channel function on their own.Item Zinc interactions with allosteric modulators at the glycine receptor(2014-08) Cornelison, Garrett Lee; Mihic, S. JohnThe 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.