Springer protocols feed by Neuroscience 
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This is an RSS file. You can use it to subscribe to this data in your favourite RSS reader or to display this data on your own website or blog.Constructing a Rapid Solution Exchange System
The detailed study of ligand-gated channels often requires the reproducible, rapid, and temporally precise application of defined concentrations of ligands. There are numerous different systems for ligand application offering trade-offs between cost, ease of use, volume of compound needed, etc. When studying the extremely rapid kinetics of activation and desensitization in ionotropic ligand-gated receptors (iGluR), a prime consideration is how fast solutions can be exchanged. Of the available systems, rapid piezo-driven translation of parallel solutions streams over an outside-out patch offers the fastest and most versatil...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Assessing The Effects of Ligand-Binding Mutations to AMPA and Kainate Receptor Kinetics
When an agonist binds to the active site of a receptor channel, it interacts directly with a handful of key residues and indirectly with others to initiate a sequence of conformational steps that lead to channel opening. In the case of AMPA and KA receptors, they also lead to very rapid desensitization on a comparable time scale. Precisely which residues are most important to which processes, and how the receptor–ligand interactions impact various measures of channel function have yet to be fully elucidated. In this chapter I describe our approach to making single-point mutations in the ligand-binding domain and asse...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Analysis of Whole-Cell NMDA Receptor Currents
During receptor channel activation, the receptor goes through a ligand binding step followed by conformation steps leading to the open channel conformation. The probability of finding the receptor in a particular conformation is given by the apparent rate constants. The apparent rate constants describe the rate of transitions between conformational states and vary among different types of iGluR. To determine these parameters reflecting receptor channel function, several approaches have been developed. In this chapter we describe methods for the measurement of basic kinetic parameters of the NMDA receptor determined from no...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Calcium Imaging to Study NMDA Receptor-mediated Cellular Responses
We describe methods to accomplish particularly common [Ca2+]
i
imaging goals; however, these provide a versatile foundation that can be further developed into more complex approaches to acquire [Ca2+]
i
-dependent images with higher temporal or spatial resolution, and from more challenging preparations. (Source: Springer protocols feed by Neuroscience)
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Timing AMPA Receptor Activation with Laser-Pulse Photolysis
The use of laser light to liberate glutamate from “caged glutamate” for rapidly initiating AMPA receptor activation enables one to measure the rate of channel opening on the microsecond time scale. From a series of observed rate constants as a function of glutamate concentration, the channel-opening and channel-closing rate constants can be calculated. These kinetic constants define the basic gating property of AMPA receptors. They can also be used to characterize the mechanism of channel regulation by modulatory agents, and the structure-function relationship. Here, I describe the instrumentation of laser-puls...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Current Recording and Kinetic Analyses for Single AMPA Receptors
The kinetics of AMPA receptor activation, deactivation, and desensitization shape the synaptic current at a majority of synapses in the central nervous system; however, the sequence of molecular events that make up these receptors’ reaction mechanism is unknown. Information about reaction mechanism is best extracted from single-molecule observations, which for ion channels can be done in real time with the patch-clamp technique. AMPA receptors have complex single-channel behaviors, which have been difficult to organize into a comprehensive mechanism. Here we describe methodology to obtain lengthy recordings of curren...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Extracting Rate Constants for NMDA Receptor Gating from One-Channel Current Recordings
Like all neurotransmitter-gated channels, in response to agonist binding, ionotropic glutamate receptors produce electrical signals whose amplitudes and durations reflect intramolecular transitions between non-conducting (closed) and conducting (open) receptor conformations. Thus, delineating the reaction mechanism of synaptic channels represents an important step in understanding how information is transferred and processed in the nervous system. The recorded single-channel signal captures in real-time multiple series of discrete current amplitudes, whose complex duration distributions contain valuable information about t...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Expression, Purification, and Crystallization of Full Length Ionotropic Glutamate Receptors
Ionotropic glutamate receptors (iGluRs) are ligand-gated ion channels that mediate the majority of excitatory neurotransmission in brain and are implicated in nearly all aspects of central nervous system development and function. There is great clinical interest in iGluRs because their dysfunction is associated with chronic neurodegenerative conditions, psychiatric disorders, brain trauma and stroke. Despite physiological and pathophysiological importance, obtaining structural information about full-length iGluRs has proven to be a difficult task. To facilitate further structural/functional studies, we describe a detailed ...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Assaying the Energetics of NMDA Receptor Pore Opening
The NMDA receptor converts transient glutamate release into an electrical and biochemical signal that integrates neuronal activity and results in changes in synaptic connectivity and strength. A variety of approaches are used to address the linkage between neurotransmitter binding and ion channel opening, a key feature common to all ligand-gated ion channels. Φ-value or REFER analysis, an approach derived from chemical transition state theory, can characterize the energetics and dynamics of ion channel activation. Here, we describe an approach to apply Φ-value analysis to the unique features of NMDA receptor activi...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
LRET Methods for Investigating Conformational Changes in Functional Ionotropic Glutamate Receptors
Luminescence resonance energy transfer (LRET) is an invaluable tool to study the conformational changes of proteins induced by ligands or protein–protein interactions at an Ångstrom-level resolution. LRET experiments require a pair of fluorophores with overlapping spectra, where the emission of the “donor” overlaps with the excitation of the “acceptor” fluorophore. The efficiency of non-radiative energy transfer can be determined by measuring the sensitized emission of the acceptor fluorophore, upon exciting the donor fluorophore. The efficiency of transfer, in turn, can be correlated to...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Functional Detection of Novel Triheteromeric NMDA Receptors
Studies of higher order brain function, including learning and memory, require understanding of mechanisms that underlie changes in synaptic strength brought about by NMDA receptor activation. Studies with recombinant receptors have demonstrated that variation in subunit composition imparts functional diversity to NMDA receptors; however the exact makeup of native receptors is not known. Therefore, it is important to evaluate receptors functionally under minimal disruption, such as in acute brain slices, where pathways are relatively intact and potentially non-overlapping. NMDA receptors have traditionally been considered ...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Electron Microscopy Analysis of AMPA Receptors in Dendritic Spines
AMPA receptors are glutamate-gated ion channels that mediate most excitatory transmission in the brain and spinal cord. The type and number of AMPA receptors present at each synapse are regulated physiologically and determine the strength of the synaptic response. High-resolution imaging techniques such as electron microscopy (EM) can inform about the ultrastructural localization of AMPA receptors in native tissue; in addition, immunogold staining allows investigators to count the number of gold particles present in different subcellular compartments, and to evaluate quantitatively the localization of AMPA receptors within...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Electrophysiological Tagging of Ionotropic Glutamate Receptors
The vast majority of excitatory neurotransmission in the brain is mediated by glutamate and its ionotropic receptors, AMPA- and NMDA-type glutamate receptors. Study of the trafficking and synaptic incorporation of these receptors is critical to understanding the normal function and regulation of synapses as well as physiological processes like synaptic plasticity, and neuropathologies where glutamatergic synaptic transmission has been compromised. Here, we present a quantitative approach to estimate functional incorporation of these receptors into synapses or detect their presence in the surface of heterologous cells. The ...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
Counting NMDA Receptors at the Cell Surface
In the mammalian central nervous system, N-methyl-d-aspartate (NMDA) receptors mediate fast excitatory synaptic transmission. These receptors also play a key role in brain development, synaptic plasticity, and memory formation, and their dysregulation can cause neurological and/or psychiatric disorders. A variety of techniques have been applied to examine the molecular mechanisms that regulate the number of NMDA receptors at the cell surface. Here we provide a step-by-step guides for two commonly used techniques: a quantitative assay that measures both surface and total receptor pools, and a biotinylation assay that can ra...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
A Step-by-Step Guide to Single-Subunit Counting of Membrane-Bound Proteins in Mammalian Cells
Determining the composition and stoichiometry of membrane-bound proteins has been a perennial problem that has plagued biology for a long time. The most recurring issue is that composition and subunit stoichiometry is commonly inferred from bulk biochemical assays that can only shed light on the “averaged” makeup of the protein complex. However, recent studies have been able to circumvent this issue by studying the stoichiometry of individual protein complexes. The most common approach has been to express GFP-tagged subunits in Xenopus laevis oocytes and then manually count the number of photobleaching steps to...
Source: Springer protocols feed by Neuroscience - September 16, 2015 Category: Neuroscience Source Type: news
