scholarly journals Mechanism of modulation of AMPA receptors by TARP-γ8

2019 ◽  
Vol 152 (1) ◽  
Author(s):  
Elisa Carrillo ◽  
Sana A. Shaikh ◽  
Vladimir Berka ◽  
Ryan J. Durham ◽  
Douglas B. Litwin ◽  
...  
Keyword(s):  
Single Molecule ◽  
Ampa Receptors ◽  
Single Channel ◽  
Regulatory Proteins ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
High Conductance State ◽  
Tightly Coupled ◽  
And Function ◽  
High Conductance

Fast excitatory synaptic transmission in the mammalian central nervous system is mediated by glutamate-activated α-amino-5-methyl-3-hydroxy-4-isoxazole propionate (AMPA) receptors. In neurons, AMPA receptors coassemble with transmembrane AMPA receptor regulatory proteins (TARPs). Assembly with TARP γ8 alters the biophysical properties of the receptor, producing resensitization currents in the continued presence of glutamate. Using single-channel recordings, we show that under resensitizing conditions, GluA2 AMPA receptors primarily transition to higher conductance levels, similar to activation of the receptors in the presence of cyclothiazide, which stabilizes the open state. To study the conformation associated with these states, we have used single-molecule FRET and show that this high-conductance state exhibits tighter coupling between subunits in the extracellular parts of the receptor. Furthermore, the dwell times for the transition from the tightly coupled state to the decoupled states correlate to longer open durations of the channels, thus correlating conformation and function at the single-molecule level.

scholarly journals Gating Transitions in Bacterial Ion Channels Measured at 3 μs Resolution

2004 ◽  
Vol 124 (2) ◽  
pp. 151-161 ◽  
Author(s):  
George Shapovalov ◽  
Henry A. Lester
Keyword(s):  
Ion Channels ◽  
Single Molecule ◽  
Time Course ◽  
Single Channel ◽  
Low Noise ◽  
State Transitions ◽  
Channel Measurements ◽  
Clear Trend ◽  
Single Molecule Level ◽  
High Conductance

Ion channels of high conductance (>200 pS) are widespread among prokaryotes and eukaryotes. Two examples, the Escherichia coli mechanosensitive ion channels Ec-MscS and Ec-MscL, pass currents of 125–300 pA. To resolve temporal details of conductance transitions, a patch-clamp setup was optimized for low-noise recordings at a time resolution of 3 μs (10–20 times faster than usual). Analyses of the high-resolution recordings confirm that Ec-MscL visits many subconductance states and show that most of the intersubstate transitions occur more slowly than the effective resolution of 3 μs. There is a clear trend toward longer transition times for the larger transitions. In Ec-MscS recordings, the majority of the observed full conductance transitions are also composite. We detected a short-lived (∼20 μs) Ec-MscS substate at 2/3 of full conductance; transitions between 2/3 and full conductance did not show fine structure and had a time course limited by the achieved resolution. Opening and closing transitions in MscS are symmetrical and are not preceded or followed by smaller, rapid currents (“anticipations” or “regrets”). Compared with other, lower-conductance channels, these measurements may detect unusually early states in the transitions from fully closed to fully open. Increased temporal resolution at the single-molecule level reveals that some elementary steps of structural transitions are composite and follow several alternative pathways, while others still escape resolution. High-bandwidth, low-noise single-channel measurements may provide details about state transitions in other high-conductance channels; and similar procedures may also be applied to channel- and nanopore-based single-molecule DNA measurements.

Quantitative single molecule FRET efficiencies using TIRF microscopy

2015 ◽  
Vol 184 ◽  
pp. 131-142 ◽  
Author(s):  
Lasse L. Hildebrandt ◽  
Søren Preus ◽  
Victoria Birkedal
Keyword(s):  
Single Molecule ◽  
Structural Information ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Molecular Complexes ◽  
Distance Information ◽  
Single Molecule Level ◽  
Single Molecule Fret ◽  
Wide Range ◽  
Intermolecular Distances

Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as other correction factors can compensate for inaccurate values of these parameters. We give here guidelines, that can be used through tools within the iSMS software (http://www.isms.au.dk), for determining quantitative FRET and assess uncertainties linked with the procedure. Our results provide insights into the experimental parameters governing quantitative FRET determination, which is essential for obtaining accurate structural information from a wide range of biomolecules.

scholarly journals Synaptic plasticity through activation of GluA3-containing AMPA-receptors

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Maria C Renner ◽  
Eva HH Albers ◽  
Nicolas Gutierrez-Castellanos ◽  
Niels R Reinders ◽  
Aile N van Huijstee ◽  
...  
Keyword(s):  
Ampa Receptors ◽  
Adrenergic Receptors ◽  
Pyramidal Neurons ◽  
Synaptic Potentiation ◽  
High Conductance State ◽  
Hippocampal Synapses ◽  
Β Adrenergic Receptors ◽  
Camp Levels ◽  
Conductance State ◽  
High Conductance

Excitatory synaptic transmission is mediated by AMPA-type glutamate receptors (AMPARs). In CA1 pyramidal neurons of the hippocampus two types of AMPARs predominate: those that contain subunits GluA1 and GluA2 (GluA1/2), and those that contain GluA2 and GluA3 (GluA2/3). Whereas subunits GluA1 and GluA2 have been extensively studied, the contribution of GluA3 to synapse physiology has remained unclear. Here we show in mice that GluA2/3s are in a low-conductance state under basal conditions, and although present at synapses they contribute little to synaptic currents. When intracellular cyclic AMP (cAMP) levels rise, GluA2/3 channels shift to a high-conductance state, leading to synaptic potentiation. This cAMP-driven synaptic potentiation requires the activation of both protein kinase A (PKA) and the GTPase Ras, and is induced upon the activation of β-adrenergic receptors. Together, these experiments reveal a novel type of plasticity at CA1 hippocampal synapses that is expressed by the activation of GluA3-containing AMPARs.

scholarly journals How Microbes Use Force To Control Adhesion

2020 ◽  
Vol 202 (12) ◽  
Author(s):  
Albertus Viljoen ◽  
Johann Mignolet ◽  
Felipe Viela ◽  
Marion Mathelié-Guinlet ◽  
Yves F. Dufrêne
Keyword(s):  
Single Molecule ◽  
Molecular Mechanisms ◽  
Flow Chamber ◽  
Microbial Adhesion ◽  
Single Molecule Level ◽  
Force Microscopy ◽  
Atomic Force ◽  
Adhesive Interactions ◽  
And Function ◽  
Mechanisms Of Adhesion

ABSTRACT Microbial adhesion and biofilm formation are usually studied using molecular and cellular biology assays, optical and electron microscopy, or laminar flow chamber experiments. Today, atomic force microscopy (AFM) represents a valuable addition to these approaches, enabling the measurement of forces involved in microbial adhesion at the single-molecule level. In this minireview, we discuss recent discoveries made applying state-of-the-art AFM techniques to microbial specimens in order to understand the strength and dynamics of adhesive interactions. These studies shed new light on the molecular mechanisms of adhesion and demonstrate an intimate relationship between force and function in microbial adhesins.

scholarly journals Broken force dispersal network in tip-links by the mutations at the Ca2+-binding residues induces hearing-loss

2019 ◽  
Vol 476 (16) ◽  
pp. 2411-2425 ◽  
Author(s):  
Jagadish P. Hazra ◽  
Amin Sagar ◽  
Nisha Arora ◽  
Debadutta Deb ◽  
Simerpreet Kaur ◽  
...  
Keyword(s):  
Hearing Loss ◽  
Inner Ear ◽  
Single Molecule ◽  
Force Sensor ◽  
Force Sensors ◽  
Single Molecule Level ◽  
Conformational Variability ◽  
Atomic Force ◽  
Wide Range ◽  
And Function

Abstract Tip-link as force-sensor in hearing conveys the mechanical force originating from sound to ion-channels while maintaining the integrity of the entire sensory assembly in the inner ear. This delicate balance between structure and function of tip-links is regulated by Ca2+-ions present in endolymph. Mutations at the Ca2+-binding sites of tip-links often lead to congenital deafness, sometimes syndromic defects impairing vision along with hearing. Although such mutations are already identified, it is still not clear how the mutants alter the structure-function properties of the force-sensors associated with diseases. With an aim to decipher the differences in force-conveying properties of the force-sensors in molecular details, we identified the conformational variability of mutant and wild-type tip-links at the single-molecule level using FRET at the endolymphatic Ca2+ concentrations and subsequently measured the force-responsive behavior using single-molecule force spectroscopy with an Atomic Force Microscope (AFM). AFM allowed us to mimic the high and wide range of force ramps (103–106 pN s−1) as experienced in the inner ear. We performed in silico network analysis to learn that alterations in the conformations of the mutants interrupt the natural force-propagation paths through the sensors and make the mutant tip-links vulnerable to input forces from sound stimuli. We also demonstrated that a Ca2+ rich environment can restore the force-response of the mutant tip-links which may eventually facilitate the designing of better therapeutic strategies to the hearing loss.

scholarly journals Single Molecule FRET Studies of Protein Conformational Landscapes: 3 Prototypic Examples for the Relation Between Conformational Dynamics and Function

2011 ◽  
Vol 100 (3) ◽  
pp. 474a-475a
Author(s):  
Markus Richert ◽  
Dymitro Rodnin ◽  
Carola S. Hengstenberg ◽  
Thomas Peulen ◽  
Alessandro Valeri ◽  
...  
Keyword(s):  
Single Molecule ◽  
Conformational Dynamics ◽  
Single Molecule Fret ◽  
Dynamics And Function ◽  
And Function

scholarly journals Pausing controls branching between productive and non-productive pathways during initial transcription

2017 ◽  
Author(s):  
David Dulin ◽  
David L. V. Bauer ◽  
Anssi M. Malinen ◽  
Jacob J. W. Bakermans ◽  
Martin Kaller ◽  
...  
Keyword(s):  
Single Molecule ◽  
Transcription Initiation ◽  
Rna Synthesis ◽  
Molecular Mechanisms ◽  
Dependent Manner ◽  
Single Molecule Fret ◽  
Bacterial Rna ◽  
Tightly Coupled ◽  
Relationship Of ◽  
The Relationship

AbstractTranscription in bacteria is controlled by multiple molecular mechanisms that precisely regulate gene expression. Recently, initial RNA synthesis by the bacterial RNA polymerase (RNAP) has been shown to be interrupted by pauses; however, the pausing determinants and the relationship of pausing with productive and abortive RNA synthesis remain poorly understood. Here, we employed single-molecule FRET and biochemical analysis to disentangle the pausing-related pathways of bacterial initial transcription. We present further evidence that region σ3.2 constitutes a barrier after the initial transcribing complex synthesizes a 6-nt RNA (ITC6), halting transcription. We also show that the paused ITC6 state acts as a checkpoint that directs RNAP, in an NTP-dependent manner, to one of three competing pathways: productive transcription, abortive RNA release, or a new unscrunching/scrunching pathway that blocks transcription initiation. Our results show that abortive RNA release and DNA unscrunching are not as tightly coupled as previously thought.

scholarly journals Prokaryotic Argonaute from Archaeoglobus fulgidus interacts with DNA as a homodimer

2020 ◽  
Author(s):  
Edvardas Golovinas ◽  
Danielis Rutkauskas ◽  
Elena Manakova ◽  
Marija Jankunec ◽  
Arunas Silanskas ◽  
...  
Keyword(s):  
Nucleic Acid ◽  
Single Molecule ◽  
Full Length ◽  
Archaeoglobus Fulgidus ◽  
Piwi Domain ◽  
Single Molecule Fret ◽  
Dna Loop ◽  
Domains Of Life ◽  
Monomeric Proteins ◽  
And Function

ABSTRACTBackgroundArgonaute (Ago) proteins are found in all three domains of life. The best characterized group is eukaryotic Argonautes (eAgos), which are the core of RNA interference. The best understood prokaryotic Ago (pAgo) proteins are full-length pAgos. They are monomeric proteins, all composed of four major structural/functional domains (N, PAZ, MID and PIWI) and thereby closely resemble eAgos. It is believed that full-length pAgos function as prokaryotic antiviral systems, with the PIWI domain performing cleavage of invading nucleic acids. However, the majority of identified pAgos are shorter and catalytically inactive (encode just MID and inactive PIWI domains), thus their action mechanism and function remain unknown.ResultsIn this work we focus on AfAgo, a short pAgo protein encoded by an archaeon Archaeoglobus fulgidus. We find that in all previously solved AfAgo structures, its two monomers form substantial dimerization interfaces involving the C-terminal β-sheets. Led by this finding, we have employed various biochemical and biophysical assays, including single-molecule FRET, SAXS and AFM, to test the possible dimerization of AfAgo. SAXS results confirm that WT AfAgo, but not the dimerization surface mutant AfAgoΔ, forms a homodimer both in the apo-form and when bound to a nucleic acid. Single molecule FRET and AFM studies demonstrate that the dimeric WT AfAgo binds two ends of a linear DNA fragment, forming a relatively stable DNA loop.ConclusionOur results show that contrary to other characterized Ago proteins, AfAgo is a stable homodimer in solution, which is capable of simultaneous interaction with two DNA molecules. This finding broadens the range of currently known Argonaute-nucleic acid interaction mechanisms.

scholarly journals Biased cytochrome P450-mediated metabolism via small-molecule ligands binding P450 oxidoreductase

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Simon Bo Jensen ◽  
Sara Thodberg ◽  
Shaheena Parween ◽  
Matias E. Moses ◽  
Cecilie C. Hansen ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Single Molecule ◽  
Cytochromes P450 ◽  
Conformational Sampling ◽  
P450 Oxidoreductase ◽  
Single Molecule Fret ◽  
Biased Signaling ◽  
Small Molecule Ligands ◽  
Cell Assays ◽  
And Function

AbstractMetabolic control is mediated by the dynamic assemblies and function of multiple redox enzymes. A key element in these assemblies, the P450 oxidoreductase (POR), donates electrons and selectively activates numerous (>50 in humans and >300 in plants) cytochromes P450 (CYPs) controlling metabolism of drugs, steroids and xenobiotics in humans and natural product biosynthesis in plants. The mechanisms underlying POR-mediated CYP metabolism remain poorly understood and to date no ligand binding has been described to regulate the specificity of POR. Here, using a combination of computational modeling and functional assays, we identify ligands that dock on POR and bias its specificity towards CYP redox partners, across mammal and plant kingdom. Single molecule FRET studies reveal ligand binding to alter POR conformational sampling, which results in biased activation of metabolic cascades in whole cell assays. We propose the model of biased metabolism, a mechanism akin to biased signaling of GPCRs, where ligand binding on POR stabilizes different conformational states that are linked to distinct metabolic outcomes. Biased metabolism may allow designing pathway-specific therapeutics or personalized food suppressing undesired, disease-related, metabolic pathways.

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