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

IF 3.5 3区医学 Q2 NEUROSCIENCES

Frontiers in Molecular Neuroscience Pub Date : 2024-06-24 DOI:10.3389/fnmol.2024.1392715

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

{"title":"Methods for negating the impact of zinc contamination to allow characterization of positive allosteric modulators of glycine receptors","authors":"Casey I. Gallagher, David P. Bishop, Thomas E. Lockwood, Tristan Rawling, Robert J. Vandenberg","doi":"10.3389/fnmol.2024.1392715","DOIUrl":null,"url":null,"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.","PeriodicalId":12630,"journal":{"name":"Frontiers in Molecular Neuroscience","volume":"23 1","pages":""},"PeriodicalIF":3.5000,"publicationDate":"2024-06-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Frontiers in Molecular Neuroscience","FirstCategoryId":"3","ListUrlMain":"https://doi.org/10.3389/fnmol.2024.1392715","RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q2","JCRName":"NEUROSCIENCES","Score":null,"Total":0}

引用次数: 0

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.

查看原文本刊更多论文

消除锌污染影响以确定甘氨酸受体正异位调节剂特征的方法

锌是许多缓冲液、纯化产品和普通实验室器皿中无处不在的污染物，以前曾被认为会影响功能性 GlyR 研究的结果，并可能无意中导致某些 GlyR 调节剂的有效性被高估。这可能会极大地影响对潜在候选药物的评估，并导致进入临床阶段的化合物疗效降低。由于在慢性疼痛病症中观察到脊髓锌浓度的变化，这对于开发用于疼痛治疗的 GlyR 调节剂来说尤其如此。在这项研究中，我们使用双电极电压钳电生理学来评估金属螯合剂三尖杉碱和 Ca-EDTA，结果表明三尖杉碱对 GlyRα1 产生的抑制作用不是由锌介导的。我们还利用对锌不敏感的 W170S 突变作为验证金属螯合剂的工具，并证实锌污染没有影响我们实验室之前开发的脂质调节剂的检验。这项研究有助于进一步开发在 GlyRs 功能研究中消除锌污染影响的方法，这些方法应被纳入今后寻求表征新型调制剂在 GlyRs 上活性的研究中。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Frontiers in Molecular Neuroscience NEUROSCIENCES-

CiteScore

5.70

自引率

2.10%

发文量

669

审稿时长

14 weeks

期刊介绍： Frontiers in Molecular Neuroscience is a first-tier electronic journal devoted to identifying key molecules, as well as their functions and interactions, that underlie the structure, design and function of the brain across all levels. The scope of our journal encompasses synaptic and cellular proteins, coding and non-coding RNA, and molecular mechanisms regulating cellular and dendritic RNA translation. In recent years, a plethora of new cellular and synaptic players have been identified from reduced systems, such as neuronal cultures, but the relevance of these molecules in terms of cellular and synaptic function and plasticity in the living brain and its circuits has not been validated. The effects of spine growth and density observed using gene products identified from in vitro work are frequently not reproduced in vivo. Our journal is particularly interested in studies on genetically engineered model organisms (C. elegans, Drosophila, mouse), in which alterations in key molecules underlying cellular and synaptic function and plasticity produce defined anatomical, physiological and behavioral changes. In the mouse, genetic alterations limited to particular neural circuits (olfactory bulb, motor cortex, cortical layers, hippocampal subfields, cerebellum), preferably regulated in time and on demand, are of special interest, as they sidestep potential compensatory developmental effects.