Methods for negating the impact of zinc contamination to allow characterization of positive allosteric modulators of glycine receptors

Casey I Gallagher; David P Bishop; Thomas E Lockwood; Tristan Rawling; Robert J Vandenberg

doi:10.3389/fnmol.2024.1392715

Methods for negating the impact of zinc contamination to allow characterization of positive allosteric modulators of glycine receptors

Front Mol Neurosci. 2024 Jun 24:17:1392715. doi: 10.3389/fnmol.2024.1392715. eCollection 2024.

Authors

Casey I Gallagher¹, David P Bishop², Thomas E Lockwood², Tristan Rawling³, Robert J Vandenberg¹

Affiliations

¹ Discipline of Pharmacology, School of Medical Sciences, University of Sydney, Sydney, NSW, Australia.
² Hyphenated Mass Spectrometry Laboratory, Faculty of Science, University of Technology Sydney, Broadway, NSW, Australia.
³ School of Mathematical and Physical Sciences, Faculty of Science, The University of Technology Sydney, Sydney, NSW, Australia.

Abstract

Zinc is a ubiquitous contaminant in many buffers, purified products and common labware that has previously been suggested to impact on the results of functional GlyR studies and may inadvertently cause the effectiveness of some GlyR modulators to be over-estimated. This could greatly impact the assessment of potential drug-candidates and contribute to the reduced effectiveness of compounds that reach clinical stages. This is especially true for GlyR modulators being developed for pain therapeutics due to the changes in spinal zinc concentrations that have been observed during chronic pain conditions. In this study we use two-electrode voltage clamp electrophysiology to evaluate the metal chelators tricine and Ca-EDTA, and show that tricine produces inhibitory effects at GlyRα₁ that are not mediated by zinc. We also utilized the zinc insensitive W170S mutation as a tool to validate metal chelators and confirm that zinc contamination has not impacted the examination of lipid modulators previously developed by our lab. This study helps to further develop methods to negate the impact of contaminating zinc in functional studies of GlyRs which should be incorporated into future studies that seek to characterize the activity of novel modulators at GlyRs.

Keywords: bioactive lipid; chelator; drug development; glycine receptor; positive allosteric modulator; tricine; zinc.

Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. The study was supported by the NHMRC APP144429.