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Single channel patch clamp

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https://www.readbyqxmd.com/read/28511915/altered-ionic-currents-and-amelioration-by-igf-1-and-pacap-in-motoneuron-derived-cells-modelling-sbma
#1
Aura M Jiménez Garduño, Leon J Juárez-Hernández, María J Polanco, Laura Tosatto, Daniela Michelatti, Daniele Arosio, Manuela Basso, Maria Pennuto, Carlo Musio
Spinal and bulbar muscular atrophy (SBMA), also known as Kennedy's disease, is a motor neuron disease caused by the expansion of a polymorphic CAG tandem repeat encoding a polyglutamine (polyQ) tract in the androgen receptor (AR) gene. SBMA is triggered by the binding of mutant AR to its natural ligands, testosterone and dihydrotestosterone (DHT). To investigate the neuronal alterations of motor neuron cell models of SBMA, we applied patch-clamp methods to verify how polyQ expansions in the AR alter cell ionic currents...
May 10, 2017: Biophysical Chemistry
https://www.readbyqxmd.com/read/28495885/proteolytic-cleavage-of-the-hydrophobic-domain-in-the-cav%C3%AE-2%C3%AE-1-subunit-improves-assembly-and-activity-of-cardiac-cav1-2-channels
#2
Emilie Segura, Benoîte Bourdin, Marie-Philippe Tétreault, Julie Briot, Bruce G Allen, Gaétan Mayer, Lucie Parent
Voltage-gated L-type CaV1.2 channels in cardiomyocytes exist as heteromeric complexes with the pore-forming CaVα1, CaVβ, and CaVα2δ1 subunits. The full complement of subunits is required to reconstitute the native-like properties of L-type Ca(2+) currents but the molecular determinants responsible for the formation of the heteromeric complex are still being studied. Enzymatic treatment with PI-PLC, a phospholipase C specific for the cleavage of glycosyl-phosphatidylinositol (GPI)-anchored proteins, disrupted plasma membrane localization of the cardiac CaVα2δ1 prompting us to investigate deletions of its hydrophobic transmembrane domain...
May 11, 2017: Journal of Biological Chemistry
https://www.readbyqxmd.com/read/28483800/xe991-and-linopirdine-are-state-dependent-inhibitors-for-kv7-kcnq-channels-that-favor-activated-single-subunits
#3
Derek L Greene, Seungwoo Kang, Naoto Hoshi
M-channel inhibitors, especially XE991, are increasingly being used in animal experiments. However, insufficient characterization of XE991 at times confounds the interpretation of results when using this compound. Here, we demonstrate that XE991 and linopirdine are state-dependent inhibitors that favor the activated-subunit of neuronal Kv7/KCNQ channels. We performed patch clamp experiments on homomeric Kv7.2 or heteromeric Kv7.2/3 channels expressed in Chinese hamster ovary cells in order to characterize XE991 and linopirdine...
May 8, 2017: Journal of Pharmacology and Experimental Therapeutics
https://www.readbyqxmd.com/read/28483598/the-mechano-sensitivity-of-cardiac-atp-sensitive-potassium-channels-is-mediated-by-intrinsic-mgatpase-activity
#4
Mohammad Fatehi, Christian C Carter, Nermeen Youssef, Peter E Light
Cardiac ATP-sensitive K(+) (KATP) channel activity plays an important cardio-protective role in regulating excitability in response to metabolic stress. Evidence suggests that these channels are also mechano-sensitive and therefore may couple KATP channel activity to increased cardiac workloads. However, the molecular mechanism that couples membrane stretch to channel activity is not currently known. We hypothesized that membrane stretch may alter the intrinsic MgATPase activity of the cardiac KATP channel resulting in increased channel activation...
May 5, 2017: Journal of Molecular and Cellular Cardiology
https://www.readbyqxmd.com/read/28472069/detection-of-trpv4-channel-current-like-activity-in-fawn-hooded-hypertensive-fhh-rat-cerebral-arterial-muscle-cells
#5
Debebe Gebremedhin, David X Zhang, Dorothee Weihrauch, Nnamdi N Uche, David R Harder
The transient receptor potential vallinoid type 4 (TRPV4) is a calcium entry channel known to modulate vascular function by mediating endothelium-dependent vasodilation. The present study investigated if isolated cerebral arterial myocytes of the Fawn Hooded hypertensive (FHH) rat, known to display exaggerated KCa channel current activity and impaired myogenic tone, express TRPV4 channels at the transcript and protein level and exhibit TRPV4-like single-channel cationic current activity. Reverse transcription polymerase chain reaction (RT-PCR), Western blot, and immunostaining analysis detected the expression of mRNA transcript and translated protein of TRPV4 channel in FHH rat cerebral arterial myocytes...
2017: PloS One
https://www.readbyqxmd.com/read/28468944/enac-activity-is-regulated-by-calpain-2-proteolysis-of-marcks-proteins
#6
Darrice S Montgomery, Ling Yu, Zinah M Ghazi, Tiffany L Thai, Otor Al-Khalili, He-Ping Ma, Douglas C Eaton, Abdel A Alli
We previously demonstrated a role for the myristoylated alanine-rich C kinase substrate (MARCKS) to serve as an adaptor protein in the anionic phospholipid phosphate-dependent regulation of the epithelial sodium channel (ENaC). Both MARCKS and ENaC are regulated by proteolysis. Calpains are a family of ubiquitously expressed intracellular Ca(2+)-dependent cysteine proteases involved in signal transduction. Here we examine the role of calpain-2 in regulating MARCKS and ENaC in cultured renal epithelial cells and in the mouse kidney...
May 3, 2017: American Journal of Physiology. Cell Physiology
https://www.readbyqxmd.com/read/28436159/the-mechanism-research-of-hypoxia-induced-increased-late-sodium-current-resulting-in-intracellular-ca-2-overload-in-ventricular-myocytes
#7
Chen Fu, Jie Hao, Mengliu Zeng, Yejia Song, Wanzhen Jiang, Peihua Zhang, Antao Luo, Zhenzhen Cao, Luiz Belardinelli, Jihua Ma
Hypoxia leads to augmentation of late sodium current (INa.L ) and cellular Na(+) loading, increased reverse Na(+) -Ca(2+) exchange current (reverse INCX ) and intracellular Ca(2+) loading in rabbit ventricular myocytes. The purpose of this study was to determine the intracellular signal transduction pathways involved in the modulation of INa.L during hypoxia in ventricular myocytes. Whole-cell and cell-attached patch clamp techniques were used to record INa.L , and the whole-cell mode was also used to record reverse INCX and to study intercellular signal transduction mechanisms that mediate the increased INa...
April 24, 2017: Experimental Physiology
https://www.readbyqxmd.com/read/28417339/expression-and-function-of-the-small-conductance-ca-2-activated-k-channel-is-decreased-in-urinary-bladder-smooth-muscle-cells-from-female-guinea-pig-with-partial-bladder-outlet-obstruction
#8
Ning Li, Honglin Ding, Xiaoning He, Zizheng Li, Yili Liu
PURPOSE: Overactive bladder (OAB), usually accompanied by partial bladder outlet obstruction (PBOO), is associated with detrusor overactivity (DO) which is related to the increased urinary bladder smooth muscle (UBSM) cells excitability. Small-conductance Ca(2+)-activated K(+) (SK) channels play a constitutive regulatory role of UBSM excitability and contractility. PBOO is associated with the decreased SK channels mRNA expression and the attenuated regulative effect of SK channels on UBSM contractility...
April 17, 2017: International Urology and Nephrology
https://www.readbyqxmd.com/read/28414903/nanopore-logic-operation-with-dna-to-rna-transcription-in-a-droplet-system
#9
Masayuki Ohara, Masahiro Takanoue, Ryuji Kawano
This paper describes an AND logic operation with amplification and transcription from DNA to RNA using T7 RNA polymerase. All four operations, (0 0) to (1 1), with an enzyme reaction can be performed simultaneously using four-droplet devices that are directly connected to a patch-clamp amplifier. The output RNA molecule is detected using a biological nanopore at the single-molecule level. Channel current recordings can be obtained using the enzyme solution. The integration of DNA logic gates into electrochemical devices is necessary to obtain output information in a human-recognizable form...
April 17, 2017: ACS Synthetic Biology
https://www.readbyqxmd.com/read/28388364/piezo2-expression-in-mechanosensitive-dental-primary-afferent-neurons
#10
J Won, H Vang, P R Lee, Y H Kim, H W Kim, Y Kang, S B Oh
Mechanosensitive ion channels have been suggested to be expressed in dental primary afferent (DPA) neurons to transduce the movement of dentinal fluid since the proposal of hydrodynamic theory. Piezo2, a mechanosensitive, rapidly inactivating (RI) ion channel, has been recently identified in dorsal root ganglion (DRG) neurons to mediate tactile transduction. Here, we examined the expression of Piezo2 in DPA neurons by in situ hybridization, single-cell reverse transcriptase polymerase chain reaction, and whole-cell patch-clamp recordings...
April 1, 2017: Journal of Dental Research
https://www.readbyqxmd.com/read/28379974/non-invasive-phenotyping-and-drug-testing-in-single-cardiomyocytes-or-beta-cells-by-calcium-imaging-and-optogenetics
#11
Yu-Fen Chang, Connor N Broyles, Frances A Brook, Mark J Davies, Cameron W Turtle, Takeharu Nagai, Matthew J Daniels
Identification of drug induced electrical instability of the heart curtails development, and introduction, of potentially proarrhythmic drugs. This problem usually requires complimentary contact based approaches such as patch-clamp electrophysiology combined with field stimulation electrodes to observe and control the cell. This produces data with high signal to noise but requires direct physical contact generally preventing high-throughput, or prolonged, phenotyping of single cells or tissues. Combining genetically encoded optogenetic control and spectrally compatible calcium indicator tools into a single adenoviral vector allows the analogous capability for cell control with simultaneous cellular phenotyping without the need for contact...
2017: PloS One
https://www.readbyqxmd.com/read/28334404/calcium-homeostasis-modulator-1-like-currents-in-rat-fungiform-taste-cells-expressing-amiloride-sensitive-sodium-currents
#12
Albertino Bigiani
Salt reception by taste cells is still the less understood transduction process occurring in taste buds, the peripheral sensory organs for the detection of food chemicals. Although there is evidence suggesting that the epithelial sodium channel (ENaC) works as sodium receptor, yet it is not clear how salt-detecting cells signal the relevant information to nerve endings. Taste cells responding to sweet, bitter, and umami substances release ATP as neurotransmitter through a nonvesicular mechanism. Three different channel proteins have been proposed as conduit for ATP secretion: pannexin channels, connexin hemichannels, and calcium homeostasis modulator 1 (CALHM1) channels...
May 1, 2017: Chemical Senses
https://www.readbyqxmd.com/read/28333338/drug-discovery-for-male-subfertility-using-high-throughput-screening-a-new-approach-to-an-unsolved-problem
#13
Sarah J Martins da Silva, Sean G Brown, Keith Sutton, Louise V King, Halil Ruso, David W Gray, Paul G Wyatt, Mark C Kelly, Christopher L R Barratt, Anthony G Hope
STUDY QUESTION: Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility? SUMMARY ANSWER: High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility. WHAT IS KNOWN ALREADY: There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform...
May 1, 2017: Human Reproduction
https://www.readbyqxmd.com/read/28327344/direct-activation-of-ca-2-and-voltage-gated-potassium-channels-of-large-conductance-by-anandamide-in-endothelial-cells-does-not-support-the-presence-of-endothelial-atypical-cannabinoid-receptor
#14
Alexander I Bondarenko, Olga Panasiuk, Iryna Okhai, Fabrizio Montecucco, Karim J Brandt, Francois Mach
Endocannabinoid anandamide induces endothelium-dependent relaxation commonly attributed to stimulation of the G-protein coupled endothelial anandamide receptor. The study addressed the receptor-independent effect of anandamide on large conductance Ca(2+)-dependent K(+) channels expressed in endothelial cell line EA.hy926. Under resting conditions, 10µM anandamide did not significantly influence the resting membrane potential. In a Ca(2+)-free solution the cells were depolarized by ~10mV. Further administration of 10µM anandamide hyperpolarized the cells by ~8mV...
March 19, 2017: European Journal of Pharmacology
https://www.readbyqxmd.com/read/28299825/fingolimod-gilenya-%C3%A2-in-multiple-sclerosis-bradycardia-atrioventricular-blocks-and-mild-effect-on-the-qtc-interval-something-to-do-with-the-l-type-calcium-channel
#15
Sylvie Pilote, Chantale Simard, Benoit Drolet
Cardiac arrhythmias and ECG abnormalities including bradycardia, prolongation of the QT interval, and atrioventricular (AV) conduction blocks have been extensively observed with fingolimod, the first marketed oral drug for treating the relapsing-remitting form of multiple sclerosis. This study was aiming to further elucidate the effects of fingolimod on cardiac electrophysiology at three different levels: (i) in vitro, (ii) ex vivo, and (iii) in vivo. (i) Patch-clamp experiments in whole cell configuration were performed on Cav 1...
March 16, 2017: Fundamental & Clinical Pharmacology
https://www.readbyqxmd.com/read/28293175/two-photon-na-imaging-reports-somatically-evoked-action-potentials-in-rat-olfactory-bulb-mitral-and-granule-cell-neurites
#16
Tiffany Ona-Jodar, Niklas J Gerkau, S Sara Aghvami, Christine R Rose, Veronica Egger
Dendrodendritic synaptic interactions are a hallmark of neuronal processing in the vertebrate olfactory bulb. Many classes of olfactory bulb neurons including the principal mitral cells (MCs) and the axonless granule cells (GCs) dispose of highly efficient propagation of action potentials (AP) within their dendrites, from where they can release transmitter onto each other. So far, backpropagation in GC dendrites has been investigated indirectly via Ca(2+) imaging. Here, we used two-photon Na(+) imaging to directly report opening of voltage-gated sodium channels due to AP propagation in both cell types...
2017: Frontiers in Cellular Neuroscience
https://www.readbyqxmd.com/read/28261794/nicotinic-acetylcholine-receptors-at-the-single-channel-level
#17
REVIEW
Cecilia Bouzat, Steven M Sine
Over the past four decades, the patch clamp technique and nicotinic acetylcholine (nACh) receptors have established an enduring partnership. Like all good partnerships, each partner has proven significant in its own right, while their union has spurred innumerable advances in life science research. A member and prototype of the superfamily of pentameric ligand-gated ion channels, the nACh receptor is a chemo-electric transducer, binding nerve-released ACh and rapidly opening its channel to cation flow to elicit cellular excitation...
March 5, 2017: British Journal of Pharmacology
https://www.readbyqxmd.com/read/28202726/two-tonoplast-mate-proteins-function-as-turgor-regulating-chloride-channels-in-arabidopsis
#18
Haiwen Zhang, Fu-Geng Zhao, Ren-Jie Tang, Yuexuan Yu, Jiali Song, Yuan Wang, Legong Li, Sheng Luan
The central vacuole in a plant cell occupies the majority of the cellular volume and plays a key role in turgor regulation. The vacuolar membrane (tonoplast) contains a large number of transporters that mediate fluxes of solutes and water, thereby adjusting cell turgor in response to developmental and environmental signals. We report that two tonoplast Detoxification efflux carrier (DTX)/Multidrug and Toxic Compound Extrusion (MATE) transporters, DTX33 and DTX35, function as chloride channels essential for turgor regulation in Arabidopsis Ectopic expression of each transporter in Nicotiana benthamiana mesophyll cells elicited a large voltage-dependent inward chloride current across the tonoplast, showing that DTX33 and DTX35 each constitute a functional channel...
March 7, 2017: Proceedings of the National Academy of Sciences of the United States of America
https://www.readbyqxmd.com/read/28197715/vacuolar-ion-channels-in-the-liverwort-marchantia-polymorpha-influence-of-ion-channel-inhibitors
#19
Mateusz Koselski, Kazimierz Trebacz, Halina Dziubinska
Potassium-permeable slow activating vacuolar channels (SV) and chloride-permeable channels in the vacuole of the liverwort Marchantia polymorpha were characterized in respect to calcium dependence, selectivity, and pharmacology. The patch-clamp method was used in the study of ion channel activity in the vacuoles from the liverwort Marchantia polymorpha. The whole-vacuole recordings allowed simultaneous observation of two types of currents-predominant slow activated currents recorded at positive voltages and fast activated currents recorded at negative voltages...
May 2017: Planta
https://www.readbyqxmd.com/read/28197552/altered-channel-conductance-states-and-gating-of-gabaa-receptors-by-a-pore-mutation-linked-to-dravet-syndrome
#20
Ciria C Hernandez, Weijing Kong, Ningning Hu, Yujia Zhang, Wangzhen Shen, Laurel Jackson, Xiaoyan Liu, Yuwu Jiang, Robert L Macdonald
We identified a de novo missense mutation, P302L, in the γ-aminobutyric acid type A (GABAA) receptor γ2 subunit gene GABRG2 in a patient with Dravet syndrome using targeted next-generation sequencing. The mutation was in the cytoplasmic portion of the transmembrane segment M2 of the γ2 subunit that faces the pore lumen. GABAA receptor α1 and β3 subunits were coexpressed with wild-type (wt) γ2L or mutant γ2L(P302L) subunits in HEK 293T cells and cultured mouse cortical neurons. We measured currents using whole-cell and single-channel patch clamp techniques, surface and total expression levels using surface biotinylation and Western blotting, and potential structural perturbations in mutant GABAA receptors using structural modeling...
January 2017: ENeuro
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