Current Protocols in Pharmacology最新文献_第2页

Studying Nicotinic Acetylcholine Receptors Using the IonFlux™ Microfluidic-Based Automated Patch-Clamp System with Continuous Perfusion and Fast Solution Exchange. 使用基于IonFlux™微流体的自动膜片钳系统研究烟碱乙酰胆碱受体，具有连续灌注和快速溶液交换。

Current Protocols in Pharmacology Pub Date : 2020-03-01 DOI: 10.1002/cpph.73

Ali Yehia, Haiyang Wei

{"title":"Studying Nicotinic Acetylcholine Receptors Using the IonFlux™ Microfluidic-Based Automated Patch-Clamp System with Continuous Perfusion and Fast Solution Exchange.","authors":"Ali Yehia, Haiyang Wei","doi":"10.1002/cpph.73","DOIUrl":"https://doi.org/10.1002/cpph.73","url":null,"abstract":"Automated patch‐clamp (APC) systems have become indispensable tools of drug‐discovery programs by allowing high‐throughput electrophysiology‐based screening of ion channel compounds. The recent development and introduction of microfluidics‐based APC systems have made it possible to study the interactions of ligand‐gated ion channels with pharmacological reagents, such as agonists, antagonists, or positive allosteric modulators (PAMs), with reliable pharmacological results comparable to those of the gold‐standard manual patch‐clamp technique while maintaining high‐throughput capacity. Many ligand‐gated ion channels exhibit rapid desensitization upon repetitive introduction of ligands; this loss of channel activity in the absence of pharmacological interaction poses a challenge for developing accurate, precise, and robust assays with high success rate, low run‐down, and reliable pharmacological results. Here we present procedures to study nicotinic acetylcholine receptors (nAChRs) with the IonFlux™, an automated patch‐clamp system with continuous flow and precise fluidic exchange; these procedures can also be generalized to the study of other ligand‐gated ion channels. We present protocols to study agonist, antagonist, and PAM activities on nAChRs, particularly the rapidly desensitizing nAChR α7 receptors. The data demonstrate that the IonFlux™ system is a fast, robust, and reliable platform for the study of nAChRs and other ligand‐gated ion channels, generating data that closely mimic those from manual patch‐clamp conditions. © 2020 by John Wiley & Sons, Inc.","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"88 1","pages":"e73"},"PeriodicalIF":0.0,"publicationDate":"2020-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.73","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37652793","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 5

Issue Information TOC 发布信息TOC

Current Protocols in Pharmacology Pub Date : 2019-12-22 DOI: 10.1002/cpph.53

{"title":"Issue Information TOC","authors":"","doi":"10.1002/cpph.53","DOIUrl":"10.1002/cpph.53","url":null,"abstract":"Cover: In Liu et al. (https://doi.org/10.1002/cpph.69), the image shows a comparison between conventional manual patch clamp and automated electrophysiology. (A) Schematic of manual patch clamp recording (left panel) under whole-cell configuration. A single cell is recorded using a glass recording pipette. Compounds can be applied to the extracellular solution (blue), whereas the artificial intracellular solution (green) is contained in the pipette. Right panel: voltage pulse protocol and representative transient inward Nav1.7 channel current. (B) Schematic of planar array–based automated electrophysiology experiment. A single cell is recorded through a micropore in a single well (upper left) from a chip containing 384 wells (upper right). Both extracellular (blue) and intracellular (green) solutions can be changed by perfusion. Lower panel: representative result from one Syncropatch run. Wells were color coded based on seal resistance. Resistance, cells: green, >0.5 GΩ, 350/384; blue, 100 to 500 MΩ, 30/384; white, <100 MΩ, 4/384. Current traces within each well were autoscaled for maximum display.\u0000\u0000 <figure>\u0000 <div><picture>\u0000 <source></source></picture>\u0000 </div>\u0000 </figure>","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-12-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.53","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46009676","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Role of High‐Throughput Electrophysiology in Drug Discovery 高通量电生理学在药物发现中的作用

Current Protocols in Pharmacology Pub Date : 2019-12-01 DOI: 10.1002/cpph.69

Chang Liu, Tianbo Li, Jun Chen

引用次数: 16

Assays for Modulators of Ryanodine Receptor (RyR)/Ca²⁺ Release Channel Activity for Drug Discovery for Skeletal Muscle and Heart Diseases. Ryanodine受体(RyR)/Ca2+释放通道活性调节剂用于骨骼肌和心脏病药物发现的测定。

Current Protocols in Pharmacology Pub Date : 2019-12-01 DOI: 10.1002/cpph.71

Takashi Murayama, Nagomi Kurebayashi

{"title":"Assays for Modulators of Ryanodine Receptor (RyR)/Ca2+ Release Channel Activity for Drug Discovery for Skeletal Muscle and Heart Diseases.","authors":"Takashi Murayama, Nagomi Kurebayashi","doi":"10.1002/cpph.71","DOIUrl":"https://doi.org/10.1002/cpph.71","url":null,"abstract":"The ryanodine receptor (RyR) is a Ca2+ release channel that is present in the sarcoplasmic reticulum and endoplasmic reticulum (ER) and that plays a central role in excitation-contraction coupling in skeletal and cardiac muscle. Hyperactivation of RyR by genetic mutations or posttranslational modification can cause various skeletal muscle and arrhythmogenic heart diseases. Inhibitors of RyR are therefore expected to be potential drugs for treatment of such diseases. This article describes assays to evaluate RyR channel activity, including an ER Ca2+ measurement assay that is compatible with high-throughput screening and a [3 H]-ryanodine binding assay that provides a quantitative measure of RyR channel activity as a second screen for compound hits. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: [Ca2+ ]ER assay for ryanodine receptor (RyR) channel activity Support Protocol 1: Determination of dose dependence and isoform selectivity of RyR inhibitors using [Ca2+ ]ER assay Basic Protocol 2: [3 H]-Ryanodine binding assay for RyR channel activity Support Protocol 2: Isolation of microsomes from RyR-expressing HEK293 cells.","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"87 1","pages":"e71"},"PeriodicalIF":0.0,"publicationDate":"2019-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.71","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"37454872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 15

A Simple Assay to Evaluate the Function of Human Connexin Hemichannels Expressed in Escherichia coli that Can Be Used for Drug Discovery and Mutant Analysis 一种评价大肠杆菌中表达的人类连接蛋白半通道功能的简单方法，可用于药物发现和突变分析

Current Protocols in Pharmacology Pub Date : 2019-10-04 DOI: 10.1002/cpph.68

Mariana C. Fiori, Luis G. Cuello, Guillermo A. Altenberg

{"title":"A Simple Assay to Evaluate the Function of Human Connexin Hemichannels Expressed in Escherichia coli that Can Be Used for Drug Discovery and Mutant Analysis","authors":"Mariana C. Fiori, Luis G. Cuello, Guillermo A. Altenberg","doi":"10.1002/cpph.68","DOIUrl":"10.1002/cpph.68","url":null,"abstract":"Abnormally increased activity of connexin hemichannels contributes to cell damage in many disorders, including deafness, stroke, and cardiac infarct, and therefore hemichannels constitute a potentially important therapeutic target. Unfortunately, the available hemichannel inhibitors are not specific and most are toxic. The absence of a simple and cost-effective screening assay has made the discovery of hemichannel inhibitors difficult. Here, we present an optimized assay where human connexins are expressed in genetically modified Escherichia coli cells deficient in potassium uptake (LB2003 cells). These cells cannot grow in low-potassium medium, and hemichannel function is assayed by the reversion of the no-growth phenotype. Since functional hemichannels are permeable to potassium, they allow for its uptake and cell growth. The simple reading of bacterial growth in low-potassium medium distinguishes functional hemichannels (growth) from those inhibited (no growth). This assay is simple, robust, inexpensive, and reliable, and is easily scaled to high-throughput multiwell platforms. © 2019 by John Wiley & Sons, Inc.Basic Protocol 1: Preparation of competent LB2003 cells resistant to kanamycinBasic Protocol 2: Growth complementation assaySupport Protocol: Evaluation of cytotoxic effects of potential connexin hemichannel inhibitors","PeriodicalId":10871,"journal":{"name":"Current Protocols in Pharmacology","volume":"87 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2019-10-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1002/cpph.68","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"42647058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 2

Issue Information TOC 发布信息TOC

Current Protocols in Pharmacology Pub Date : 2019-09-20 DOI: 10.1002/cpph.52

引用次数: 0

Natural Products as a Foundation for Drug Discovery 天然产物是药物发现的基础

Current Protocols in Pharmacology Pub Date : 2019-08-26 DOI: 10.1002/cpph.67

John A. Beutler

引用次数: 22

Transgenic Tumor Models for Evaluating CAR T-Cell Immunotherapies 评价CAR - t细胞免疫疗法的转基因肿瘤模型

Current Protocols in Pharmacology Pub Date : 2019-08-16 DOI: 10.1002/cpph.66

Fernando Aranda, Miguel Barajas, Eduardo Huarte

引用次数: 0