scholarly journals Whole-GUV patch-clamping

2016 ◽  
Vol 114 (2) ◽  
pp. 328-333 ◽  
Author(s):  
Matthias Garten ◽  
Lars D. Mosgaard ◽  
Thomas Bornschlögl ◽  
Stéphane Dieudonné ◽  
Patricia Bassereau ◽  
...  
Keyword(s):  
Ion Transport ◽  
Ion Channel ◽  
High Resistance ◽  
Membrane Properties ◽  
Membrane Tension ◽  
Patch Clamping ◽  
Membrane Composition ◽  
Residual Solvent ◽  
Voltage Dependent

Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the “whole-GUV” patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

Patch-clamping of the inner mitochondrial membrane reveals a voltage-dependent ion channel

Nature ◽  
1987 ◽  
Vol 330 (6147) ◽  
pp. 498-500 ◽  
Author(s):  
M. Catia Sorgato ◽  
Bernhard U. Keller ◽  
Walter Stühmer
Keyword(s):  
Ion Channel ◽  
Mitochondrial Membrane ◽  
Patch Clamping ◽  
Inner Mitochondrial Membrane ◽  
Voltage Dependent

scholarly journals Modeling membrane nanotube morphology: the role of heterogeneity in composition and material properties

2018 ◽  
Author(s):  
Haleh Alimohamadi ◽  
Ben Ovryn ◽  
Padmini Rangamani
Keyword(s):  
Membrane Properties ◽  
Membrane Tension ◽  
Membrane Mechanics ◽  
Membrane Composition ◽  
Tube Radius ◽  
Cylindrical Tubes ◽  
Dynamic Structures ◽  
Protein Density ◽  
Membrane Protein Interaction

AbstractMembrane nanotubes have been identified as dynamic structures for cells to connect over long distances. Nanotubes typically appear as thin and cylindrical tubes, but they may also have a beaded architecture along the tube. In this paper, we study the role of membrane mechanics in governing the architecture of these tubes and show that the formation of beadlike structures along the nanotubes can result from local heterogeneities in the membrane either due to protein aggregation or due to membrane composition. We present numerical results that predict how membrane properties, protein density, and local tension compete to create a phase space that governs the morphology of a nanotube. We also find that there is an energy barrier that prevents two beads from fusing. These results suggest that the membrane-protein interaction, membrane composition, and membrane tension closely govern the tube radius, number of beads, and the bead morphology.

scholarly journals Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording

2015 ◽  
Vol 113 (4) ◽  
pp. 1275-1282 ◽  
Author(s):  
Reid R. Harrison ◽  
Ilya Kolb ◽  
Suhasa B. Kodandaramaiah ◽  
Alexander A. Chubykin ◽  
Aimei Yang ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Ion Channel ◽  
Voltage Clamp ◽  
Single Cells ◽  
Patch Clamping ◽  
Patch Clamp Recording ◽  
Current Clamp ◽  
Low Levels

Patch clamping is a gold-standard electrophysiology technique that has the temporal resolution and signal-to-noise ratio capable of reporting single ion channel currents, as well as electrical activity of excitable single cells. Despite its usefulness and decades of development, the amplifiers required for patch clamping are expensive and bulky. This has limited the scalability and throughput of patch clamping for single-ion channel and single-cell analyses. In this work, we have developed a custom patch-clamp amplifier microchip that can be fabricated using standard commercial silicon processes capable of performing both voltage- and current-clamp measurements. A key innovation is the use of nonlinear feedback elements in the voltage-clamp amplifier circuit to convert measured currents into logarithmically encoded voltages, thereby eliminating the need for large high-valued resistors, a factor that has limited previous attempts at integration. Benchtop characterization of the chip shows low levels of current noise [1.1 pA root mean square (rms) over 5 kHz] during voltage-clamp measurements and low levels of voltage noise (8.2 μV rms over 10 kHz) during current-clamp measurements. We demonstrate the ability of the chip to perform both current- and voltage-clamp measurement in vitro in HEK293FT cells and cultured neurons. We also demonstrate its ability to perform in vivo recordings as part of a robotic patch-clamping system. The performance of the patch-clamp amplifier microchip compares favorably with much larger commercial instrumentation, enabling benchtop commoditization, miniaturization, and scalable patch-clamp instrumentation.

scholarly journals Electrospun Bioresorbable Membrane Eluting Chlorhexidine for Dental Implants

Polymers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 66 ◽  
Author(s):  
Pierre Pouponneau ◽  
Ophélie Perrey ◽  
Céline Brunon ◽  
Carol Grossiord ◽  
Nicolas Courtois ◽  
...  
Keyword(s):  
Drug Release ◽  
Release Kinetics ◽  
Gingival Tissue ◽  
Membrane Composition ◽  
Cell Penetration ◽  
Electrospun Membrane ◽  
Γ Irradiation ◽  
Breaking Strain ◽  
Residual Solvent

To prevent the uncontrolled development of a pathogenic biofilm around a dental implant, an antimicrobial drug-release electrospun membrane, set up between the implant and the gingival tissue, was developed by taking several technical, industrial and regulatory specifications into account. The membrane formulation is made of a blend of poly(l-lactic–co–gycolic acid) (PLGA, 85:15) and poly(l-lactic acide–co–ɛ-caprolactone) (PLC, 70:30) copolymers with chlorhexidine diacetate (CHX) complexed with β-cyclodextrin (CD). The amount of residual solvent, the mechanical properties and the drug release kinetics were tuned by the copolymers’ ratio, between 30% and 100% of PLC, and a CHX loading up to 20% w/w. The membranes were sterilized by γ-irradiation without significant property changes. The fiber′s diameter was between 600 nm and 3 µm, depending on the membrane composition and the electrospinning parameters. CHX was released in vitro over 10 days and the bacterial inhibitory concentration, 80 µg·mL−1, was reached within eight days. The optimal membrane, PGLA/PLC/CHX-CD (60%/40%/4%), exhibited a breaking strain of 50%, allowing its safe handling. This membrane and a membrane without CHX-CD were implanted subcutaneous in a rat model. The cell penetration remained low. The next step will be to increase the porosity of the membrane to improve the dynamic cell penetration and tissue remodeling.

Modulation of regenerative membrane properties by stimulation of metabotropic glutamate receptors in rat deep dorsal horn neurons

1996 ◽  
Vol 76 (4) ◽  
pp. 2794-2798 ◽  
Author(s):  
V. Morisset ◽  
F. Nagy
Keyword(s):  
Glutamate Receptors ◽  
Dorsal Horn ◽  
Metabotropic Glutamate Receptors ◽  
Firing Frequency ◽  
Membrane Properties ◽  
Cervical Region ◽  
Metabotropic Glutamate ◽  
Dorsal Horn Neurons ◽  
Voltage Dependent

1. Intracellular recordings were obtained from 111 dorsal horn neurons in lamina V, in an in vitro transverse spinal cord slice preparation of the cervical region from young rats. 2. Of these neurons, 28% showed voltage-dependent plateau potentials, mainly underlain by a tetrodotoxin-resistant dihydropyridine-sensitive Ca2+ current. When depolarized, neurons with plateau properties produced accelerating firing frequency, afterdischarge, and bistability. They also exhibited windup of action potentials when stimulated by repetitive intracellular injections of current. 3. Glutamate being the main excitatory transmitter released by primary afferents, we also considered the effects of specific agonists of metabotropic glutamate receptors and showed that they modulate positively (induce or enhance) plateau properties in the deep dorsal horn neurons.

The actions of hydrogen sulfide on dorsal raphe serotonergic neurons in vitro

1993 ◽  
Vol 70 (1) ◽  
pp. 81-96 ◽  
Author(s):  
S. B. Kombian ◽  
R. J. Reiffenstein ◽  
W. F. Colmers
Keyword(s):  
Hydrogen Sulfide ◽  
Outward Current ◽  
Rapid Onset ◽  
Dorsal Raphe ◽  
Membrane Properties ◽  
Delayed Rectifier ◽  
Inward Current ◽  
Voltage Dependent ◽  
Dorsal Raphé

1. The actions of hydrogen sulfide (HS-) on membrane and synaptic properties of dorsal raphe (DR) serotonergic cells were studied in the in vitro brain stem slice preparation, using intracellular sharp microelectrode and whole-cell recording techniques. 2. Sulfide produced two reversible, concentration-dependent effects on resting membrane properties of DR cells: (1) 14% responded to HS- with a slow onset hyperpolarization or an outward current accompanied by an conductance increase in voltage clamp (holding potential = -60 mV; monophasic outward cell) or (2) 39% responded with a rapid-onset depolarization corresponding to a weakly voltage-dependent inward current showing little or no change in conductance between -115 and -40 mV (monophasic inward cell). In addition, 29.5% showed both the above effects, responding first with a rapid-onset depolarization and then a sustained hyperpolarization. Such cells had membrane currents very similar to those seen in the monophasic inward and outward cells (biphasic cells). Finally, 17.5% of DR cells had no measurable postsynaptic membrane response to HS-. 3. The outward current induced in the presence of HS- had a reversal potential of about -90 mV when recorded either with 2 M KCl or 145 mM potassium gluconate in the pipette and was accompanied by an increase in conductance, suggesting that it is caused by an elevated conductance to K+. 4. This current was sensitive to the removal of external Ca2+ and blockade by Cd2+, suggesting that it is activated by an elevation in internal [Ca2+]. It was also blocked by apamin or Ba2+ and Cs+, both of which revealed an underlying inward current. The outward current was insensitive to the application of a large variety of antagonists to other known voltage- and calcium-dependent K+ channels. Elevation of intracellular ATP using a patch pipette did not prevent the activation of the outward current. 5. HS- reversibly suppressed a voltage-dependent outward current activated in the voltage range of -50 to -40 mV. This current was also blocked by 10 mM tetraethylammonium, suggesting that HS- suppresses the delayed rectifier in DR cells. 6. The inward current could be observed in the presence of HS- not only in monophasic inward cells but also in monophasic outward or biphasic cells whose outward current was selectively blocked. This inward current was sensitive to the removal of extracellular Ca2+, or the the application of relatively low concentrations of Cd2+, suggesting that it is carried by Ca2+. Both these manipulations also blocked the outward current in monophasic outward or biphasic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Deep Mutational Scan of a cardiac sodium channel voltage sensor

2019 ◽  
Author(s):  
Andrew M. Glazer ◽  
Brett M. Kroncke ◽  
Kenneth A. Matreyek ◽  
Tao Yang ◽  
Yuko Wada ◽  
...  
Keyword(s):  
Ion Channel ◽  
Sodium Channel ◽  
High Throughput ◽  
High Throughput Sequencing ◽  
Voltage Sensor ◽  
Patch Clamping ◽  
Loss Of Function ◽  
Pathogenic Variants ◽  
Cardiac Sodium Channel

AbstractVariants in ion channel genes have classically been studied in low-throughput by patch clamping. Deep Mutational Scanning (DMS) is a complementary approach that can simultaneously assess function of thousands of variants. We have developed and validated a method to perform a DMS of variants in SCN5A, which encodes the major voltage-gated sodium channel in the heart. We created a library of nearly all possible variants in a 36 base region of SCN5A in the S4 voltage sensor of domain IV and stably integrated the library into HEK293T cells. In preliminary experiments, challenge with three drugs (veratridine, brevetoxin, and ouabain) could discriminate wildtype channels from gain and loss of function pathogenic variants. High-throughput sequencing of the pre- and post-drug challenge pools was used to count the prevalence of each variant and identify variants with abnormal function. The DMS scores identified 40 putative gain of function and 33 putative loss of function variants. For 8/9 variants, patch clamping data was consistent with the scores. These experiments demonstrate the accuracy of a high-throughput in vitro scan of SCN5A variant function, which can be used to identify deleterious variants in SCN5A and other ion channel genes.

Inhibition of dihydropyridine-sensitive calcium entry in hypoxic relaxation of airway smooth muscle

1995 ◽  
Vol 268 (2) ◽  
pp. L201-L206 ◽  
Author(s):  
C. Vannier ◽  
T. L. Croxton ◽  
L. S. Farley ◽  
C. A. Hirshman
Keyword(s):  
Smooth Muscle ◽  
Airway Smooth Muscle ◽  
Muscle Tone ◽  
Bay K 8644 ◽  
O2 Concentration ◽  
Voltage Dependent ◽  
Progressive Hypoxia ◽  
Carbamyl Choline

Hypoxia dilates airways in vivo and reduces active tension of airway smooth muscle in vitro. To determine whether hypoxia impairs Ca2+ entry through voltage-dependent channels (VDC), we tested the ability of dihydropyridines to modulate hypoxia-induced relaxation of KCl- and carbamyl choline (carbachol)-contracted porcine bronchi. Carbachol- or KCl-contracted bronchial rings were exposed to progressive hypoxia in the presence or absence of 1 microM BAY K 8644 (an L-type-channel agonist). In separate experiments, rings were contracted with carbachol or KCl, treated with nifedipine (a VDC antagonist), and finally exposed to hypoxia. BAY K 8644 prevented hypoxia-induced relaxation in KCl-contracted bronchi. Nifedipine (10(-5) M) totally relaxed KCl- contracted bronchi. Carbachol-contracted bronchi were only partially relaxed by nifedipine but were completely relaxed when the O2 concentration of the gas was reduced from 95 to 0%. These data indicate that hypoxia can reduce airway smooth muscle tone by limiting entry of Ca2+ through a dihydropyridine-sensitive pathway, but that other mechanisms also contribute to hypoxia-induced relaxation of carbachol-contracted bronchi.

scholarly journals Rare Gain-of-Function KCND3 Variant Associated with Cerebellar Ataxia, Parkinsonism, Cognitive Dysfunction, and Brain Iron Accumulation

2021 ◽  
Vol 22 (15) ◽  
pp. 8247
Author(s):  
Cheng-Tsung Hsiao ◽  
Thomas F. Tropea ◽  
Ssu-Ju Fu ◽  
Tanya M. Bardakjian ◽  
Pedro Gonzalez-Alegre ◽  
...  
Keyword(s):  
Cerebellar Ataxia ◽  
Iron Accumulation ◽  
Molecular Spectra ◽  
Loss Of Function ◽  
Brain Iron ◽  
Gain Of Function ◽  
Progressive Cerebellar Ataxia ◽  
Voltage Dependent ◽  
Window Current

Loss-of-function mutations in the KV4.3 channel-encoding KCND3 gene are linked to neurodegenerative cerebellar ataxia. Patients suffering from neurodegeneration associated with iron deposition may also present with cerebellar ataxia. The mechanism underlying brain iron accumulation remains unclear. Here, we aim to ascertain the potential pathogenic role of KCND3 variant in iron accumulation-related cerebellar ataxia. We presented a patient with slowly progressive cerebellar ataxia, parkinsonism, cognitive impairment, and iron accumulation in the basal ganglia and the cerebellum. Whole exome sequencing analyses identified in the patient a heterozygous KCND3 c.1256G>A (p.R419H) variant predicted to be disease-causing by multiple bioinformatic analyses. In vitro biochemical and immunofluorescence examinations revealed that, compared to the human KV4.3 wild-type channel, the p.R419H variant exhibited normal protein abundance and subcellular localization pattern. Electrophysiological investigation, however, demonstrated that the KV4.3 p.R419H variant was associated with a dominant increase in potassium current amplitudes, as well as notable changes in voltage-dependent gating properties leading to enhanced potassium window current. These observations indicate that, in direct contrast with the loss-of-function KCND3 mutations previously reported in cerebellar ataxia patients, we identified a rare gain-of-function KCND3 variant that may expand the clinical and molecular spectra of neurodegenerative cerebellar disorders associated with brain iron accumulation.

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