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Potassium Channels

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Cover of 'Potassium Channels'

Table of Contents

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    Book Overview
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    Chapter 1 Manipulating Potassium Channel Expression and Function in Hippocampal Neurons by In Utero Electroporation
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    Chapter 2 Studying KCNQ1 Mutation and Drug Response in Type 1 Long QT Syndrome Using Patient-Specific Induced Pluripotent Stem Cell-Derived Cardiomyocytes
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    Chapter 3 Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation
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    Chapter 4 Nonsense-Mediated mRNA Decay of hERG Mutations in Long QT Syndrome
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    Chapter 5 Probing Subunits Interactions in KATP Channels Using Photo-Crosslinking via Genetically Encoded p-Azido-l-phenylalanine
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    Chapter 6 Hyper-SUMOylation of K+ Channels in Sudden Unexplained Death in Epilepsy: Isolation and Primary Culture of Dissociated Hippocampal Neurons from Newborn Mice for Subcellular Localization
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    Chapter 7 Simultaneous Real-Time Measurement of the β-Cell Membrane Potential and Ca2+ Influx to Assess the Role of Potassium Channels on β-Cell Function
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    Chapter 8 Methods for Characterizing Disease-Associated ATP-Sensitive Potassium Channel Mutations
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    Chapter 9 Thallium Flux Assay for Measuring the Activity of Monovalent Cation Channels and Transporters
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    Chapter 10 Nuclear Magnetic Resonance Approaches for Characterizing Protein-Protein Interactions
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    Chapter 11 Studying Mechanosensitivity of Two-Pore Domain K+ Channels in Cellular and Reconstituted Proteoliposome Membranes
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    Chapter 12 Migration of PIP2 on KCNQ2 Surface Revealed by Molecular Dynamics Simulations
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    Chapter 13 Studying Structural Dynamics of Potassium Channels by Single-Molecule FRET
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    Chapter 14 Patch-Clamp Recordings of the KcsA K+ Channel in Unilamellar Blisters
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    Chapter 15 Combinatorial Assembly of Lumitoxins
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    Chapter 16 Characterization of MC4R Regulation of the Kir7.1 Channel Using the Tl+ Flux Assay
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    Chapter 17 Stopped-Flow Fluorometric Ion Flux Assay for Ligand-Gated Ion Channel Studies
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    Chapter 18 In Vivo Analysis of Potassium Channelopathies: Loose Patch Recording of Purkinje Cell Firing in Living, Awake Zebrafish
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    Chapter 19 Site-Directed Unnatural Amino Acid Mutagenesis to Investigate Potassium Channel Pharmacology in Xenopus laevis Oocytes
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    Chapter 20 Random Spherically Constrained Single-Particle (RSC) Method to Study Voltage-Gated Ion Channels
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    Chapter 21 CW-EPR Spectroscopy and Site-Directed Spin Labeling to Study the Structural Dynamics of Ion Channels
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    Chapter 22 Ion Binding to Transport Proteins using Isothermal Titration Calorimetry
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    Chapter 23 Building Atomic Models of the Ion Channels Based on Low Resolution Electron Microscopy Maps and Homology Modeling
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    Chapter 24 Studying Kv Channels Function using Computational Methods
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    Chapter 25 Erratum to: Ion Binding to Transport Proteins using Isothermal Titration Calorimetry
Attention for Chapter 3: Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation
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Chapter title
Monitoring Changes in the Abundance of Endogenously Expressed ATP-Sensitive Potassium (KATP) Channels in the Plasma Membrane Using Surface Biotinylation
Chapter number 3
Book title
Potassium Channels
Published in
Methods in molecular biology, January 2018
DOI 10.1007/978-1-4939-7362-0_3
Pubmed ID
Book ISBNs
978-1-4939-7361-3, 978-1-4939-7362-0
Authors

Jing-Syuna Ruan, Pei-Chun Chen

Abstract

The conductance of KATP channel activity can be regulated by gating and/or surface expression. Gating analysis is usually performed by electrophysiological recording. Analysis of surface KATP channel expression levels requires cell fractionation, protein separation, and quantification. Cell fractionation involves time-consuming high-speed centrifugation steps and skilled techniques for taking out specific layers. Moreover, contamination of intracellular membranes can confound results. Although commercial kits have been developed to make it easier for scientists, qualities of these kits vary which can give rise to variable results. Detection of membrane proteins using surface biotinylation technique consists of labeling cell surface proteins with a biotin reagent before lysing the cells, and isolating these tagged proteins by NeutrAvidin pulldown. Then, the samples are subjected to SDS-PAGE separation, transferred to PVDF membranes, and probed with specific antibodies. Quantification of cell surface expression is accomplished by densitometric measurement of the bands corresponding to the protein of interest and subsequent normalization by a membrane protein (as control). This alternative method for detecting expression of surface protein is relatively easy in steps and more economical in comparison to other methods such as cell fractionation.

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