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Optical Control of Ligand-Gated Ion Channels

In the vibrant field of optogenetics, optics and genetic targeting are combined to commandeer cellular functions, such as the neuronal action potential, by optically stimulating light-sensitive ion channels expressed in the cell membrane. One broadly applicable manifestation ...

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Production and Validation of Recombinant Adeno-Associated Virus for Channelrhodopsin Expression in Neurons

Recent discovery of the light-activated ion channel, channelrhodopsin (ChR), has provided researchers a powerful and convenient tool to manipulate the membrane potential of specific cells with light. With genetic targeting of these channels and illumination of light to a specific l ...

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Patch-Clamping Drosophila Sensory Neurons

Electrophysiological studies provide essential clues about the regulation and physiological function of ion channel proteins. Probing ion channel activity in vivo, though, often is challenging. This can limit the usefulness of such model organisms as Drosophila for electrophy ...

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Ca2+ Imaging as a Tool to Assess TRP Channel Function in Murine Distal Nephrons

Transient receptor potential (TRP) channels are expressed in almost every segment of renal nephron from the glomerulus to the inner medullary collecting duct. Serving as a route for Ca2+ entry from the intratubular space into cells in response to external cues, TRP channels modulate water– ...

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Single-Channel Analysis of TRPC Channels in the Podocytes of Freshly Isolated Glomeruli

One of the most important functions of the kidney is the filtration of the blood that takes place in the glomeruli. Glomerular epithelial cells (podocytes) have several functions, including regulation of the filtration process and glomerular basement membrane turnover. Dysfunction ...

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Recording Ion Channels in Isolated, Split-Opened Tubules

Ion channels play key roles in physiology. They function as protein transducers able to transform stimuli and chemical gradients into electrical signals. They also are critical for cell signaling and play a particularly important role in epithelial transport acting as gateways for the m ...

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Studying Ion Channels in Human Erythrocytes by Direct and Indirect Means

Mature human or mouse erythrocytes functionally express an unexpected diversity of ion channels that endows these small enucleated cells with a toolkit for electrosignaling. Being largely dormant under resting conditions, these ion channels enable erythrocytes to quickly res ...

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M-Current Recording from Acute DRG Slices

Electrophysiological recordings from an acutely sliced preparation provide information on ionic currents and excitability of native neurons under near physiological conditions. Although this technique is commonly used on central nervous system structures such as spinal ...

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Recording Dendritic Ion Channel Properties and Function from Cortical Neurons

Dendrites emerging from the cell bodies of neurons receive the majority of synaptic inputs. They possess a plethora of ion channels that are essential for the processing of these synaptic signals. To fully understand how dendritic ion channels influence neuronal information processi ...

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Isotope Labeling Strategies for Analysis of an Ion Channel Cytoplasmic Domain by NMR Spectroscopy

As large, multimeric, integral membrane proteins, ion channels pose technical challenges to analysis by NMR spectroscopy. Here we present a strategy to overcome some of these technical hurdles, using a representative ion channel modulatory domain, the regulator of K+ conductance (RCK) ...

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Analysis of Ca2+-Binding Sites in the MthK RCK Domain by X-Ray Crystallography

Regulator of K+ conductance (RCK) domains form a conserved class of ligand-binding domains that control the activity of a variety of prokaryotic and eukaryotic K+ channels. Structural analysis of these domains by X-ray crystallography has provided insight toward mechanisms underly ...

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Cysteine-Based Cross-Linking Approach to Study Inter-domain Interactions in Ion Channels

Cysteine contains a highly reactive thiol group and therefore under oxidizing conditions a disulfide bond can form between a pair of cysteines that are juxtaposed in the close vicinity, which can be only reversed by reducing agents. These attributes have been elegantly exploited to study the ...

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Site-Directed Mutagenesis to Study the StructureFunction Relationships of Ion Channels

Ion channels mediate a wide variety of physiological processes by forming small pores across the membranes that allow regulated flow of ions into or out of the cell. The primary linear sequences of ion channel proteins, like any proteins, are composed by 20 different amino acids, each of which is dete ...

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Generation of Antibodies That Are Externally Acting Isoform-Specific Inhibitors of Ion Channels

There is demand for isoform-specific ion channel inhibitors as tools to investigate the biology of �endogenous ion channels and validate them as targets in drug discovery programs. There is also hope that such inhibitors may be new therapeutic agents or provide the foundation for such agent ...

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Imaging and Quantification of Recycled KATP Channels

This chapter describes immunochemistry-based methods to investigate recycling of membrane proteins at the cell surface. Two methods are described, one qualitative and the other quantitative. Both methods consist of two rounds of extracellular antibody capture. Firstly, a prima ...

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The Use of Dansyl-Calmodulin to Study Interactions with Channels and Other Proteins

Steady-state fluorescence spectroscopy is a biophysical technique widely employed to characterize �interactions between proteins in vitro. Only a few proteins naturally fluoresce in cells, but by covalently attaching fluorophores virtually all proteins can be monitored. O ...

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Frster Resonance Energy Transfer-Based Imaging at the Cell Surface of Live Cells

Understanding the molecular mechanisms of protein–protein interactions at the cell surface of living cells is fundamental to identifying the nature of cellular processes. Here, we discuss how fluorescence-based approaches have been successfully developed to visualize prot ...

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Using Total Internal Reflection Fluorescence Microscopy to Observe Ion Channel Trafficking and Assembly

Ion channels are integral membrane proteins that allow the flow of ions across membranes down their electrochemical gradients and are a major determinant of cellular excitability. They play an important role in a variety of biological processes as diverse as insulin release from beta cel ...

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Recording Macroscopic Currents in Large Patches from Xenopus Oocytes

The excised inside-out patch clamp technique gives rapid access to the intracellular surface of the plasma membrane while measuring channel activity. This way the effects of intracellular regulators of ion channels or transporters can be studied in isolation, in the absence of most of the c ...

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Recording of Ion Channel Activity in Planar Lipid Bilayer Experiments

Planar lipid bilayer is an electrophysiological technique that enables study of functional activities of ion channels, porins, and other pore-forming molecular complexes. The main purpose of this method is to monitor ion channels’ behavior at the single molecule level in the artifici ...

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