scholarly journals Reconstitution and functional characterization of ion channels from nanodiscs in lipid bilayers

2018 ◽  
Vol 150 (4) ◽  
pp. 637-646 ◽  
Author(s):  
Laura-Marie Winterstein ◽  
Kerri Kukovetz ◽  
Oliver Rauh ◽  
Daniel L. Turman ◽  
Christian Braun ◽  
...  
Keyword(s):  
Ion Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Functional Characterization ◽  
In Vitro Translation ◽  
Target Membrane ◽  
Electrophysiological Characterization ◽  
Channel Properties

Recent studies have shown that membrane proteins can be efficiently synthesized in vitro before spontaneously inserting into soluble nanoscale lipid bilayers called nanodiscs (NDs). In this paper, we present experimental details that allow a combination of in vitro translation of ion channels into commercially available NDs followed by their direct reconstitution from these nanobilayers into standard bilayer setups for electrophysiological characterization. We present data showing that two model K+ channels, Kcv and KcsA, as well as a recently discovered dual-topology F− channel, Fluc, can be reliably reconstituted from different types of NDs into bilayers without contamination from the in vitro translation cocktail. The functional properties of Kcv and KcsA were characterized electrophysiologically and exhibited sensitivity to the lipid composition of the target DPhPC bilayer, suggesting that the channel proteins were fully exposed to the target membrane and were no longer surrounded by the lipid/protein scaffold. The single-channel properties of the three tested channels are compatible with studies from recordings of the same proteins in other expression systems. Altogether, the data show that synthesis of ion channels into NDs and their subsequent reconstitution into conventional bilayers provide a fast and reliable method for functional analysis of ion channels.

Reconstitution and Electrophysiological Characterization of Ion Channels in Lipid Bilayers

2018 ◽  
Vol 81 (1) ◽  
pp. e37 ◽  
Author(s):  
Dan A. Klaerke ◽  
Maria de los Angeles Tejada ◽  
Vibeke Grøsfjeld Christensen ◽  
Mette Lassen ◽  
Per Amstrup Pedersen ◽  
...  
Keyword(s):  
Ion Channels ◽  
Lipid Bilayers ◽  
Electrophysiological Characterization

Molecular and functional characterization of VDAC2 purified from mammal spermatozoa

2009 ◽  
Vol 29 (6) ◽  
pp. 351-362 ◽  
Author(s):  
Viviana A. Menzel ◽  
M. Carolina Cassará ◽  
Roland Benz ◽  
Vito De Pinto ◽  
Angela Messina ◽  
...  
Keyword(s):  
Molecular Mass ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Functional Characterization ◽  
Membrane Conductance ◽  
Peptide Sequencing ◽  
Blue Staining ◽  
Voltage Dependent Anion Channel ◽  
Bovine Spermatozoa

VDAC (voltage-dependent anion channel) is the pore-forming protein located in the outer mitochondrial membrane. In higher eukaryotes, three genes encode VDAC. Nevertheless, the knowledge of VDAC isoforms is mainly restricted to VDAC1, the only isoform that has been characterized from living tissues to date. We have highly enriched the isoform VDAC2 using as starting material bovine spermatozoa. VDAC2 was obtained in the hydroxyapatite/celite pass-through of sperm proteins solubilized with Triton X-100. This fraction showed in SDS/PAGE two major bands and one faint band in the molecular mass range of 30–35 kDa. Two-dimensional electrophoresis resolved these bands in ten spots with various Coomassie Blue staining intensities. Western-blot analysis with antibodies monospecific for each isoform and MS peptide sequencing showed that the main protein resolved in electrophoresis was VDAC2 with minor contaminations of the other isoforms. Proteomic analysis of the higher molecular mass VDAC2 protein allowed the coverage of the whole protein with the exception of the tripeptide A24AR26. In the same material, the presence of two possible amino acid substitutions (T88 to L88 and A97 to Q97) was revealed. Reconstitution of VDAC2 pores in planar lipid bilayers showed typical features of mitochondrial porins. Stepwise increases in membrane conductance were observed with a predominant conductance of approx. 3.5 nS (nanoSiemens) in 1 M KCl. Very often, small short-lived fluctuations were observed with single-channel conductance of approx. 1.5 nS. Bovine spermatozoa VDAC2 was anion selective and showed voltage dependence. The present study is the first work to report the purification and characterization of VDAC2 from a mammalian tissue.

scholarly journals Molecular and Functional Characterization of Neurogenin-2 Induced Human Sensory Neurons

2020 ◽  
Vol 14 ◽  
Author(s):  
Amy J. Hulme ◽  
Jeffrey R. McArthur ◽  
Simon Maksour ◽  
Sara Miellet ◽  
Lezanne Ooi ◽  
...  
Keyword(s):  
Ion Channels ◽  
Sensory Neurons ◽  
Molecular Basis ◽  
Functional Characterization ◽  
Transcript Level ◽  
Peripheral Neuropathies ◽  
Sensory Physiology ◽  
Depth Analysis

Sensory perception is fundamental to everyday life, yet understanding of human sensory physiology at the molecular level is hindered due to constraints on tissue availability. Emerging strategies to study and characterize peripheral neuropathies in vitro involve the use of human pluripotent stem cells (hPSCs) differentiated into dorsal root ganglion (DRG) sensory neurons. However, neuronal functionality and maturity are limited and underexplored. A recent and promising approach for directing hPSC differentiation towards functionally mature neurons involves the exogenous expression of Neurogenin-2 (NGN2). The optimized protocol described here generates sensory neurons from hPSC-derived neural crest (NC) progenitors through virally induced NGN2 expression. NC cells were derived from hPSCs via a small molecule inhibitor approach and enriched for migrating NC cells (66% SOX10+ cells). At the protein and transcript level, the resulting NGN2 induced sensory neurons (NGN2iSNs) express sensory neuron markers such as BRN3A (82% BRN3A+ cells), ISLET1 (91% ISLET1+ cells), TRKA, TRKB, and TRKC. Importantly, NGN2iSNs repetitively fire action potentials (APs) supported by voltage-gated sodium, potassium, and calcium conductances. In-depth analysis of the molecular basis of NGN2iSN excitability revealed functional expression of ion channels associated with the excitability of primary afferent neurons, such as Nav1.7, Nav1.8, Kv1.2, Kv2.1, BK, Cav2.1, Cav2.2, Cav3.2, ASICs and HCN among other ion channels, for which we provide functional and transcriptional evidence. Our characterization of stem cell-derived sensory neurons sheds light on the molecular basis of human sensory physiology and highlights the suitability of using hPSC-derived sensory neurons for modeling human DRG development and their potential in the study of human peripheral neuropathies and drug therapies.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

2021 ◽  
Vol 9 (5) ◽  
pp. 1107
Author(s):  
Wonho Choi ◽  
Yoshihiro Yamaguchi ◽  
Ji-Young Park ◽  
Sang-Hyun Park ◽  
Hyeok-Won Lee ◽  
...  
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Physiological Function ◽  
Functional Characterization ◽  
Site Directed Mutagenesis ◽  
In Vitro Assays ◽  
Cell Growth Inhibition ◽  
The Right

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Electrophysiological characterization and computational models of HVC neurons in the zebra finch

2013 ◽  
Vol 110 (5) ◽  
pp. 1227-1245 ◽  
Author(s):  
Arij Daou ◽  
Matthew T. Ross ◽  
Frank Johnson ◽  
Richard L. Hyson ◽  
Richard Bertram
Keyword(s):  
Computational Models ◽  
Premotor Cortex ◽  
Brain Slices ◽  
Ionic Currents ◽  
Proper Name ◽  
Using Data ◽  
Electrophysiological Characterization

The nucleus HVC (proper name) within the avian analog of mammal premotor cortex produces stereotyped instructions through the motor pathway leading to precise, learned vocalization by songbirds. Electrophysiological characterization of component HVC neurons is an important requirement in building a model to understand HVC function. The HVC contains three neural populations: neurons that project to the RA (robust nucleus of arcopallium), neurons that project to Area X (of the avian basal ganglia), and interneurons. These three populations are interconnected with specific patterns of excitatory and inhibitory connectivity, and they fire with characteristic patterns both in vivo and in vitro. We performed whole cell current-clamp recordings on HVC neurons within brain slices to examine their intrinsic firing properties and determine which ionic currents are responsible for their characteristic firing patterns. We also developed conductance-based models for the different neurons and calibrated the models using data from our brain slice work. These models were then used to generate predictions about the makeup of the ionic currents that are responsible for the different responses to stimuli. These predictions were then tested and verified in the slice using pharmacological manipulations. The model and the slice work highlight roles of a hyperpolarization-activated inward current ( Ih), a low-threshold T-type Ca2+ current ( ICa-T), an A-type K+ current ( IA), a Ca2+-activated K+ current ( ISK), and a Na+-dependent K+ current ( IKNa) in driving the characteristic neural patterns observed in the three HVC neuronal populations. The result is an improved characterization of the HVC neurons responsible for song production in the songbird.

scholarly journals NFB-01. FUNCTIONAL CHARACTERIZATION OF ATRX LOSS IN NF1-ASSOCIATED GLIOMA AND MPNST

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii417-iii418
Author(s):  
Ming Yuan ◽  
Karlyne Reilly ◽  
Christine Pratilas ◽  
Christopher Heaphy ◽  
Fausto Rodriguez
Keyword(s):  
Cell Lines ◽  
Functional Characterization ◽  
Tert Promoter ◽  
Aggressive Tumors ◽  
Sirna Knockdown ◽  
Nih 3T3 ◽  
Vitro Effect ◽  
Murine Glioma

Abstract To identify the biologic relevance of ATRX loss in NF1-associated gliomagenesis, we studied the effects of Atrx loss using four previously characterized Nf1+/-Trp53+/- murine glioma lines. Lines 130G#3 and 158D#8 (corresponding to grade IV and III gliomas, respectively) displayed preserved ATRX protein expression compared to NIH-3T3 cells. We studied the effects of Atrx knockdown in these two lines in the presence and absence of the TERT inhibitor, BIRBR1532. Using a telomere-specific FISH assay, we identified increased signal intensity after Atrx knockdown, only in the presence of the TERT inhibitor. These features are reminiscent of ALT, although there were no significant alterations in cell growth. Next, we studied the effect of ATRX loss in MPNST lines ST88-14, NF90-8, STS-26T. These cell lines all expressed ATRX and DAXX. However, STS-26T contained a TERT promoter mutation and ST88-14 had a known SNP in the TERT promoter, while NF90-8 had no alterations. ATRX siRNA knockdown showed no significant effects in cell proliferation or apoptosis. However, ATRX knockdown resulted in rare ultra-bright foci, indicative of ALT. Next, we studied the in vitro effect of the ATR inhibitor VE-821 in MPNST cell lines. Only NF90-8 (lacking TERT alterations) demonstrated a decrease in growth after ATRX knockdown and VE-821 treatment. However, ATRX knockdown alone did not affect sensitivity to carboplatin. Our findings further support a role for ATRX loss with subsequent ALT activation in a biologic subset of NF1-associated malignancies, thereby opening an opportunity for therapeutic targeting of these aggressive tumors using specific classes of drugs.

Functional Characterization of GABAA Receptor Ligands In Vitro

2004 ◽  
pp. 85-94
Author(s):  
Bjarke Ebert ◽  
Sally Anne Thompson ◽  
Signe Í. Stórustovu ◽  
Keith A. Wafford
Keyword(s):  
Gabaa Receptor ◽  
Functional Characterization ◽  
Receptor Ligands

scholarly journals Biochemical and Functional Characterization of Anthocyanidin Reductase (ANR) from Mangifera indica L.

Molecules ◽  
2018 ◽  
Vol 23 (11) ◽  
pp. 2876 ◽  
Author(s):  
Lin Tan ◽  
Mei Wang ◽  
Youfa Kang ◽  
Farrukh Azeem ◽  
Zhaoxi Zhou ◽  
...  
Keyword(s):  
Enzyme Assay ◽  
Molecular Mechanisms ◽  
Mangifera Indica ◽  
Functional Characterization ◽  
Citrate Buffer ◽  
Anthocyanidin Reductase ◽  
High Amino Acid ◽  
Optimum Ph

Mango (Mangifera indica L.) is abundant in proanthocyanidins (PAs) that are important for human health and plant response to abiotic stresses. However, the molecular mechanisms involved in PA biosynthesis still need to be elucidated. Anthocyanidin reductase (ANR) catalyzes a key step in PA biosynthesis. In this study, three ANR cDNAs (MiANR1-1,1-2,1-3) were isolated from mango, and expressed in Escherichia coli. In vitro enzyme assay showed MiANR proteins convert cyanidin to their corresponding flavan-3-ols, such as (−)-catechin and (−)-epicatechin. Despite high amino acid similarity, the recombinant ANR proteins exhibited differences in enzyme kinetics and cosubstrate preference. MiANR1-2 and MiANR1-3 have the same optimum pH of 4.0 in citrate buffer, while the optimum pH for MiANR1-1 is pH 3.0 in phosphate buffer. MiANR1-1 does not use either NADPH or NADH as co-substrate while MiANR1-2/1-3 use only NADPH as co-substrate. MiANR1-2 has the highest Km and Vmax for cyanidin, followed by MiANR1-3 and MiANR1-1. The overexpression of MiANRs in ban mutant reconstructed the biosynthetic pathway of PAs in the seed coat. These data demonstrate MiANRs can form the ANR pathway, leading to the formation of two types of isomeric flavan-3-ols and PAs in mango.

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