Journal articles on the topic 'Whole-cell patch clamping'
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Garten, Matthias, Lars D. Mosgaard, Thomas Bornschlögl, Stéphane Dieudonné, Patricia Bassereau, and Gilman E. S. Toombes. "Whole-GUV patch-clamping." Proceedings of the National Academy of Sciences 114, no. 2 (December 21, 2016): 328–33. http://dx.doi.org/10.1073/pnas.1609142114.
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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.
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Zhang, W., S. E. Nilson, and S. M. Assmann. "Isolation and Whole-Cell Patch Clamping of Arabidopsis Guard Cell Protoplasts." Cold Spring Harbor Protocols 2008, no. 6 (June 1, 2008): pdb.prot5014. http://dx.doi.org/10.1101/pdb.prot5014.
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Harrison, Reid R., Ilya Kolb, Suhasa B. Kodandaramaiah, Alexander A. Chubykin, Aimei Yang, Mark F. Bear, Edward S. Boyden, and Craig R. Forest. "Microchip amplifier for in vitro, in vivo, and automated whole cell patch-clamp recording." Journal of Neurophysiology 113, no. 4 (February 15, 2015): 1275–82. http://dx.doi.org/10.1152/jn.00629.2014.
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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.
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Yantorno, R. E., D. A. Carre, M. Coca-Prados, T. Krupin, and M. M. Civan. "Whole cell patch clamping of ciliary epithelial cells during anisosmotic swelling." American Journal of Physiology-Cell Physiology 262, no. 2 (February 1, 1992): C501—C509. http://dx.doi.org/10.1152/ajpcell.1992.262.2.c501.
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Anisosmotic cell swelling triggers a regulatory volume decrease (RVD) in cell lines derived from human nonpigmented ciliary epithelium. Measurements of cell volume have indicated that the RVD reflects activation of K+ and/or Cl- channels. We have begun to characterize the putative channels by whole cell patch clamping. The results obtained by altering the external K+ and Cl- concentrations and by adding 20-50 microM quinidine or 1 mM Ba2+ indicate that K+ conductances contribute substantially and Cl- conductances contribute very little to the total membrane conductance (GT) under baseline isotonic conditions. Reducing the external osmolality by 20-50% reversibly and reproducibly increased GT by an order of magnitude. Data obtained from ion substitutions and the channel blockers quinidine and 5-nitro-2-(3-phenylpropylamino)-benzoate indicate that most of the hypotonicity-induced conductance reflects stationary Cl(-)-channel activity. The contribution of new K(+)-channel activity was small at intracellular free Ca2+ concentrations of 10 or 200 nM. We conclude that the RVD triggered by bath hypotonicity primarily reflects increased Cl(-)-channel activity.
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Zhu, Michael X. "A well-known potassium channel plays a critical role in lysosomes." Journal of Cell Biology 216, no. 6 (May 16, 2017): 1513–15. http://dx.doi.org/10.1083/jcb.201704017.
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Whole-endolysosome patch clamping presents new opportunities to identify and characterize channels pivotal for these acidic organelles. In this issue (Wang et al., 2017. J. Cell Biol. https://doi.org/10.1083/jcb.201612123), the identification of a role for the large conductance calcium-activated potassium channel brings new thinking about regulation of lysosome membrane potential and function.
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Clark, B., and P. Mobbs. "Transmitter-operated channels in rabbit retinal astrocytes studied in situ by whole-cell patch clamping." Journal of Neuroscience 12, no. 2 (February 1, 1992): 664–73. http://dx.doi.org/10.1523/jneurosci.12-02-00664.1992.
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Holst, Gregory L., William Stoy, Bo Yang, Ilya Kolb, Suhasa B. Kodandaramaiah, Lu Li, Ulf Knoblich, et al. "Autonomous patch-clamp robot for functional characterization of neurons in vivo: development and application to mouse visual cortex." Journal of Neurophysiology 121, no. 6 (June 1, 2019): 2341–57. http://dx.doi.org/10.1152/jn.00738.2018.
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Patch clamping is the gold standard measurement technique for cell-type characterization in vivo, but it has low throughput, is difficult to scale, and requires highly skilled operation. We developed an autonomous robot that can acquire multiple consecutive patch-clamp recordings in vivo. In practice, 40 pipettes loaded into a carousel are sequentially filled and inserted into the brain, localized to a cell, used for patch clamping, and disposed. Automated visual stimulation and electrophysiology software enables functional cell-type classification of whole cell-patched cells, as we show for 37 cells in the anesthetized mouse in visual cortex (V1) layer 5. We achieved 9% yield, with 5.3 min per attempt over hundreds of trials. The highly variable and low-yield nature of in vivo patch-clamp recordings will benefit from such a standardized, automated, quantitative approach, allowing development of optimal algorithms and enabling scaling required for large-scale studies and integration with complementary techniques. NEW & NOTEWORTHY In vivo patch-clamp is the gold standard for intracellular recordings, but it is a very manual and highly skilled technique. The robot in this work demonstrates the most automated in vivo patch-clamp experiment to date, by enabling production of multiple, serial intracellular recordings without human intervention. The robot automates pipette filling, wire threading, pipette positioning, neuron hunting, break-in, delivering sensory stimulus, and recording quality control, enabling in vivo cell-type characterization.
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Zhang, Yanli, Thai Phung, James Dunlop, and Julie Dalziel. "hERG ion channel pharmacology: cell membrane liposomes in porous-supported lipid bilayers compared with whole-cell patch-clamping." European Biophysics Journal 41, no. 11 (August 31, 2012): 949–58. http://dx.doi.org/10.1007/s00249-012-0852-2.
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Kang, Jiesheng, Yongyi Luo, Michelle Searles, and David Rampe. "Observations on conducting whole-cell patch clamping of the hERG cardiac K+channel in pure human serum." Journal of Applied Toxicology 37, no. 4 (August 24, 2016): 445–53. http://dx.doi.org/10.1002/jat.3377.
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Mansell, S. A., C. L. R. Barratt, and S. M. Wilson. "Potassium channel in human sperm identified by whole cell patch clamping plays a role in normal sperm physiology." Fertility and Sterility 98, no. 3 (September 2012): S13. http://dx.doi.org/10.1016/j.fertnstert.2012.07.047.
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Li, Y., M. L. Day, and C. O'Neill. "235.PAF induced changes in intracellular Ca2+ and membrane potential in the 2-cell mouse embryo." Reproduction, Fertility and Development 16, no. 9 (2004): 235. http://dx.doi.org/10.1071/srb04abs235.
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Platelet-activating factor (PAF) is an autocrine survival factor for the preimplantation embryo. PAF induces a transient increase in intracellular Ca2+ ([Ca2+]i) in 2-cell embryos that is caused by the interdependent influx of external calcium and release of calcium from internal stores. A membrane current with L-type calcium channel properties is activated during PAF-induced calcium signalling. Since the L-type channel in many cell types is primarily voltage-gated we were interested to learn whether this was also the case in the 2-cell embryo. The present study investigated the relationship between the PAF-induced Ca2+ transient and changes in membrane potential (Em) in the 2-cell embryo. The perforated whole-cell patch-clamp technique was used to detect changes in Em and standard calcium imaging techniques were used to measure changes in [Ca2+]i in 2-cell embryos from QS mice. Embryos were first loaded with Fluo-3 and then pretreated with PAF:acetylhydrolase to degrade the embryo derived PAF before patch clamping. Whole-cell perforated patch-clamping was performed by inclusion of 240mg/ml Nystatin in the pipette solution. Changes in Em and [Ca2+]i were recorded simultaneously after treatment of the embryo with PAF. In 2-cell embryos PAF induced a change in Em, consisting of an initial small depolarisation of 2.4 � 0.2 mV (42 � 4 sec after addition of PAF) followed by one or more transient hyperpolarisations of -8 � 1 mV (100 � 9 sec after addition of PAF). Transient increases in [Ca2+]i paralleled the membrane hyperpolaristions and were initiated at 84 � 8 sec after addition of PAF. These responses to PAF were seen in 58% of 2-cell embryos (n = 52). It is not yet clear whether these changes in Em account for the activation of calcium influx through the L-type channel. The results show for the first time that the 2-cell embryo is an electrically active organism.
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Grinstein, S., R. Romanek, and O. D. Rotstein. "Method for manipulation of cytosolic pH in cells clamped in the whole cell or perforated-patch configurations." American Journal of Physiology-Cell Physiology 267, no. 4 (October 1, 1994): C1152—C1159. http://dx.doi.org/10.1152/ajpcell.1994.267.4.c1152.
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A number of methods have been developed to manipulate the intracellular pH (pHi) of intact cells. However, such methods are not applicable when cells are studied using the patch-clamp technique, due to the continuity of the cell interior with the recording pipette. The perfused-pipette method can be used to modify pHi in the whole cell configuration, but this approach is slow, technically demanding, and not useful in the case of the perforated-patch configuration. In this report, we introduce a simple procedure that enables the investigator to predictably and reversibly alter pHi in cells clamped in either the whole cell or perforated-patch modes. The method is based on the provision of a virtually unlimited reservoir of an intracellular H+ (equivalent) donor/acceptor system, by inclusion of large concentrations of permeable weak electrolytes in the pipette solution. This system not only provides a means for the imposition and maintenance of a chosen pHi but, by changing the external concentration of the weak electrolyte, enables the investigator to rapidly and reversibly change pHi or the transmembrane delta pH during the course of an experiment. The effectiveness of the procedure was validated in peritoneal macrophages by two methods: 1) direct measurement of pHi in single cells by fluorescence ratio determinations and 2) estimation of the reversal potential of H(+)-selective currents. The pHi clamping procedure is shown to be effective using either organic or inorganic weak bases in the whole cell configuration. In addition, because NH+4/NH3 can readily permeate the pores formed by nystatin or amphotericin, the method is also shown to apply to the perforated-patch configuration.
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Devor, D. C., and M. E. Duffey. "Carbachol induces K+, Cl-, and nonselective cation conductances in T84 cells: a perforated patch-clamp study." American Journal of Physiology-Cell Physiology 263, no. 4 (October 1, 1992): C780—C787. http://dx.doi.org/10.1152/ajpcell.1992.263.4.c780.
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We used the perforated patch-clamp technique to examine cell membrane ionic conductances in isolated cells of the human colonic secretory cell line, T84, during exposure to the muscarinic agonist carbachol. Carbachol (100 microM) induced both outward and inward currents when the patch pipette contained a normal intracellular-like solution, the bath contained a normal extracellular-like solution, and the cells were intermittently voltage clamped between K+ and Cl- equilibrium potentials. The outward current was identified as a K+ current that averaged 483 +/- 95 pA, while the inward current averaged 152 +/- 29 pA (n = 15). The outward and inward currents oscillated with a synchronous frequency of 0.036 +/- 0.006 Hz; however, the onset of the K+ current occurred an average of 457 +/- 72 ms before the onset of the inward current. When the pipette contained a high-NaCl solution, the bath contained a Na(+)-gluconate solution, and the cells were intermittently voltage clamped between Cl- and Na+ equilibrium potentials, carbachol induced both Cl- and nonselective cation currents. The Cl- current averaged 455 +/- 73 pA, while the nonselective cation current, averaged 336 +/- 54 pA (n = 14). No difference was observed in the onset of these two currents. These results indicate that carbachol induces three separate ionic conductances in T84 cells. We used the whole cell patch-clamp technique in a previous study of these cells [D. C. Devor, S. M. Simasko, and M. E. Duffey. Am. J. Physiol. 258 (Cell Physiol. 27): C318-C326, 1990] and found that carbachol induced only an oscillating membrane K+ conductance. Thus some unidentified component of the carbachol-sensitive signal transduction pathway is diffusible and may be lost during whole cell patch clamping.
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Tessier-Lavigne, M., D. Attwell, P. Mobbs, and M. Wilson. "Membrane currents in retinal bipolar cells of the axolotl." Journal of General Physiology 91, no. 1 (January 1, 1988): 49–72. http://dx.doi.org/10.1085/jgp.91.1.49.
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By whole-cell patch-clamping bipolar cells isolated from enzymatically dissociated retinae, we have studied the nonsynaptic ionic currents that may play a role in shaping the bipolar cell light response and in determining the level of voltage noise in these cells. Between -30 and -70 mV, the membrane current of isolated bipolar cells is time independent, and the input resistance is 1-2 G omega. Depolarization past -30 mV activates an outward current (in less than 100 ms), which then inactivates slowly (approximately 1 s). Inactivation of this current is removed by hyperpolarization over the range -20 to -80 mV. This current is carried largely by K ions. It is not activated by internal Ca2+. The membrane current of isolated bipolar cells is noisy, and the variance of this noise has a minimum between -40 and -60 mV. At its minimum, the standard deviation of the voltage noise produced by nonsynaptic membrane currents is at least 100 microV. The membrane currents of depolarizing bipolar cells in slices of retina were investigated by whole-cell patch-clamping. Their membrane properties were similar to those of isolated bipolar cells, but with a larger membrane capacitance and a smaller input resistance. Their membrane current noise also showed a minimum near -40 to -60 mV. The time-dependent potassium current in axolotl bipolar cells is not significantly activated in the physiological potential range and can therefore play little role in shaping the bipolar cells' voltage response to light. Differences in the waveform of the light response of bipolar cells and photoreceptors must be ascribed to shaping by the synapses between these cells. The noise minimum in the bipolar membrane current is near the dark potential of these cells, and this may be advantageous for the detection of weak signals by the bipolar cells.
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O'Toole, John F., William Schilling, Diana Kunze, Sethu M. Madhavan, Martha Konieczkowski, Yaping Gu, Liping Luo, Zhenzhen Wu, Leslie A. Bruggeman, and John R. Sedor. "ApoL1 Overexpression Drives Variant-Independent Cytotoxicity." Journal of the American Society of Nephrology 29, no. 3 (November 27, 2017): 869–79. http://dx.doi.org/10.1681/asn.2016121322.
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Coding variants in the APOL1 gene are associated with kidney diseases in African ancestral populations; yet, the underlying biologic mechanisms remain uncertain. Variant-dependent autophagic and cytotoxic cell death have been proposed as pathogenic pathways mediating kidney injury. To examine this possibility, we conditionally expressed APOL1-G0 (reference), -G1, and -G2 (variants) using a tetracycline-regulated system in HEK293 cells. Autophagy was monitored biochemically and cell death was measured using multiple assays. We measured intracellular Na+ and K+ content with atomic absorption spectroscopy and APOL1-dependent currents with whole-cell patch clamping. Neither reference nor variant APOL1s induced autophagy. At high expression levels, APOL1-G0, -G1, and -G2 inserted into the plasma membrane and formed pH-sensitive cation channels, causing collapse of cellular Na+ and K+ gradients, phosphorylation of p38 mitogen-activated protein kinase, and cell death, without variant-dependent differences. APOL1-G0 and -G2 exhibited similar channel properties in whole-cell patch clamp experiments. At low expression levels, neither reference nor variant APOL1s localized on the plasma membrane, Na+ and K+ gradients were maintained, and cells remained viable. Our results indicate that APOL1-mediated pore formation is critical for the trypanolytic activity of APOL1 and drives APOL1-mediated cytotoxicity in overexpression systems. The absence of cytotoxicity at physiologic expression levels suggests variant-dependent intracellular K+ loss and cytotoxicity does not drive kidney disease progression.
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Carré, David A., Claire H. Mitchell, Kim Peterson-Yantorno, Miguel Coca-Prados, and Mortimer M. Civan. "Similarity of A3-adenosine and swelling-activated Cl− channels in nonpigmented ciliary epithelial cells." American Journal of Physiology-Cell Physiology 279, no. 2 (August 1, 2000): C440—C451. http://dx.doi.org/10.1152/ajpcell.2000.279.2.c440.
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Chloride release from nonpigmented ciliary epithelial (NPE) cells is a final step in forming aqueous humor, and adenosine stimulates Cl− transport by these cells. Whole cell patch clamping of cultured human NPE cells indicated that the A3-selective agonist 1-deoxy-1-(6-[([3-iodophenyl]methyl)amino]-9H-purin-9-yl)- N-methyl-β-d-ribofuranuronamide (IB-MECA) stimulated currents ( I IB-MECA) by ∼90% at +80 mV. Partial replacement of external Cl−with aspartate reduced outward currents and shifted the reversal potential ( V rev) from −23 ± 2 mV to −0.0 ± 0.7 mV. Nitrate substitution had little effect. Perfusion with the Cl− channel blockers 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid inhibited the currents. Partial Cl− replacement with aspartate and NO3 −, and perfusion with NPPB, had similar effects on the swelling-activated whole cell currents ( I Swell). Partial cyclamate substitution for external Cl− inhibited inward and outward currents of both I IB-MECA and I Swell. Both sets of currents also showed outward rectification and inactivation at large depolarizing potentials. The results are consistent with the concept that A3-subtype adenosine agonists and swelling activate a common population of Cl− channels.
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Fischer, Horst, Jonathan H. Widdicombe, and Beate Illek. "Acid secretion and proton conductance in human airway epithelium." American Journal of Physiology-Cell Physiology 282, no. 4 (April 1, 2002): C736—C743. http://dx.doi.org/10.1152/ajpcell.00369.2001.
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Acid secretion and proton conductive pathways across primary human airway surface epithelial cultures were investigated with the pH stat method in Ussing chambers and by single cell patch clamping. Cultures showed a basal proton secretion of 0.17 ± 0.04 μmol · h−1 · cm−2, and mucosal pH equilibrated at 6.85 ± 0.26. Addition of histamine or ATP to the mucosal medium increased proton secretion by 0.27 ± 0.09 and 0.24 ± 0.09 μmol · h−1 · cm−2, respectively. Addition of mast cells to the mucosal medium of airway cultures similarly activated proton secretion. Stimulated proton secretion was similar in cultures bathed mucosally with either NaCl Ringer or ion-free mannitol solutions. Proton secretion was potently blocked by mucosal ZnCl2 and was unaffected by mucosal bafilomycin A1, Sch-28080, or ouabain. Mucosal amiloride blocked proton secretion in tissues that showed large amiloride-sensitive potentials. Proton secretion was sensitive to the application of transepithelial current and showed outward rectification. In whole cell patch-clamp recordings a strongly outward-rectifying, zinc-sensitive, depolarization-activated proton conductance was identified with an average chord conductance of 9.2 ± 3.8 pS/pF (at 0 mV and a pH 5.3-to-pH 7.3 gradient). We suggest that inflammatory processes activate proton secretion by the airway epithelium and acidify the airway surface liquid.
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Mitchell, Claire H., Johannes C. Fleischhauer, W. Daniel Stamer, K. Peterson-Yantorno, and Mortimer M. Civan. "Human trabecular meshwork cell volume regulation." American Journal of Physiology-Cell Physiology 283, no. 1 (July 1, 2002): C315—C326. http://dx.doi.org/10.1152/ajpcell.00544.2001.
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The volume of certain subpopulations of trabecular meshwork (TM) cells may modify outflow resistance of aqueous humor, thereby altering intraocular pressure. This study examines the contribution that Na+/H+, Cl−/HCO[Formula: see text]exchange, and K+-Cl− efflux mechanisms have on the volume of TM cells. Volume, Cl− currents, and intracellular Ca2+ activity of cultured human TM cells were studied with calcein fluorescence, whole cell patch clamping, and fura 2 fluorescence, respectively. At physiological bicarbonate concentration, the selective Na+/H+ antiport inhibitor dimethylamiloride reduced isotonic cell volume. Hypotonicity triggered a regulatory volume decrease (RVD), which could be inhibited by the Cl− channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB), the K+channel blockers Ba2+ and tetraethylammonium, and the K+-Cl− symport blocker [(dihydroindenyl)oxy]alkanoic acid. The fluid uptake mechanism in isotonic conditions was dependent on bicarbonate; at physiological levels, the Na+/H+ exchange inhibitor dimethylamiloride reduced cell volume, whereas at low levels the Na+-K+-2Cl− symport inhibitor bumetanide had the predominant effect. Patch-clamp measurements showed that hypotonicity activated an outwardly rectifying, NPPB-sensitive Cl− channel displaying the permeability ranking Cl− > methylsulfonate > aspartate. 2,3-Butanedione 2-monoxime antagonized actomyosin activity and both increased baseline [Ca2+] and abolished swelling-activated increase in [Ca2+], but it did not affect RVD. Results indicate that human TM cells display a Ca2+-independent RVD and that volume is regulated by swelling-activated K+ and Cl− channels, Na+/H+ antiports, and possibly K+-Cl− symports in addition to Na+-K+-2Cl− symports.
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Whalley, D. W., L. C. Hool, R. E. Ten Eick, and H. H. Rasmussen. "Effect of osmotic swelling and shrinkage on Na(+)-K+ pump activity in mammalian cardiac myocytes." American Journal of Physiology-Cell Physiology 265, no. 5 (November 1, 1993): C1201—C1210. http://dx.doi.org/10.1152/ajpcell.1993.265.5.c1201.
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The effect on the sarcolemmal Na(+)-K+ pump of exposure to anisosmolar solutions was examined using whole cell patch clamping and ion-selective microelectrodes. Na(+)-K+ pump currents were measured in single ventricular myocytes by using pipette Na+ concentrations ([Na]pip) of 0-70 mM. The relationship between [Na]pip and pump current was well described by the Hill equation. The [Na]pip for half-maximal pump current (K0.5) was 21.4 mM in isosmolar (310 mosM) solution. K0.5 was 12.8 mM during cell swelling in hyposmolar solution (240 mosM) and 39.0 mM during cell shrinkage in hyperosmolar solution (464 mosM). The maximal pump currents, derived from the best fit of the Hill equation, and the Hill coefficients were similar in isosmolar, hyposmolar, and hyperosmolar solutions. A sustained (> 20 min) decrease in the intracellular Na+ activity developed during exposure of intact papillary muscles to hyposmolar solutions, and a sustained increase developed during exposure to hyperosmolar solutions. We conclude that osmotic myocyte swelling stimulates the sarcolemmal Na(+)-K+ pump at near-physiological levels of intracellular Na+, whereas shrinkage inhibits the pump. These changes are due to increases and decreases, respectively, in the apparent affinity of the pump for Na+.
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Zhang, F., J. L. Ram, P. R. Standley, and J. R. Sowers. "17 beta-Estradiol attenuates voltage-dependent Ca2+ currents in A7r5 vascular smooth muscle cell line." American Journal of Physiology-Cell Physiology 266, no. 4 (April 1, 1994): C975—C980. http://dx.doi.org/10.1152/ajpcell.1994.266.4.c975.
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Previous studies have shown that 17 beta-estradiol (beta-E2) has a direct acute inhibitory effect on vascular smooth muscle (VSM) contraction. To investigate the mechanisms underlying this phenomenon, we utilized whole cell patch-clamping techniques to study effects of beta-E2 on voltage-dependent Ca2+ channels in cultured VSM cells (VSMC). T- and L-type Ca2+ currents were characterized with ramp and pulse protocols in A7r5 cultured VSMC. T-type current, inactivated in < 100 ms, was reduced by Ba2+ and was comparatively little affected by isradipine. L-type current required higher voltages to activate, inactivated slowly, was greatly increased by Ba2+, and could be completely inhibited by 5 microM isradipine. beta-E2 (10 microM) significantly reduced peak L-type Ba2+ current and T-type Ca2+ current within 1-2 min, whereas alpha E2 (a hormonally inactive isomer of estradiol) caused significantly less reduction in both types of current. Vehicle (0.1% ethanol) had no significant effect on either current. The inhibitory effect of beta-E2 on voltage-dependent Ca2+ currents may contribute to previously demonstrated beta-E2 attenuation of VSM contraction.
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Webb, Kevin F., and Paul J. Donaldson. "Whole-Cell Patch Clamping of Isolated Fiber Cells Confirms that Spatially Distinct Cl−Influx and Efflux Pathways Exist in the Cortex of the Rat Lens." Investigative Opthalmology & Visual Science 50, no. 8 (August 1, 2009): 3808. http://dx.doi.org/10.1167/iovs.08-2680.
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Li, H. Y., C. K. Park, S. J. Jung, S. Y. Choi, S. J. Lee, K. Park, J. S. Kim, and S. B. Oh. "Eugenol Inhibits K+ Currents in Trigeminal Ganglion Neurons." Journal of Dental Research 86, no. 9 (September 2007): 898–902. http://dx.doi.org/10.1177/154405910708600918.
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Eugenol, a natural capsaicin congener, is widely used in dentistry. Eugenol inhibits voltage-activated Na+ and Ca2+ channels in a transient receptor potential vanilloid 1 (TRPV1)-independent manner. We hypothesized that eugenol also inhibits voltage-gated K+ currents, and investigated this in rat trigeminal ganglion neurons and in a heterologous system using whole-cell patch clamping. Eugenol inhibited voltage-gated K+ currents, and the inhibitory effects of eugenol were observed in both capsaicin-sensitive and capsaicin-insensitive neurons. Pre-treatment with capsazepine, a well-known antagonist of TRPV1, failed to block the inhibitory effects of eugenol on K+ currents, suggesting no involvement of TRPV1. Eugenol inhibited human Kv1.5 currents stably expressed in Ltk− cells, where TRPV1 is not endogenously expressed. We conclude that eugenol inhibits voltage-gated K+ currents in a TRPV1-independent manner. The inhibition of voltage-gated K+ currents is likely to contribute to the irritable action of eugenol. Abbreviations: human Kv1.5 channel, hKv1.5; transient receptor potential vanilloid 1, TRPV1.
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Chen, Xiao-Wei, Ya-Qin Feng, Chan-Juan Hao, Xiao-Li Guo, Xin He, Zhi-Yong Zhou, Ning Guo, et al. "DTNBP1, a schizophrenia susceptibility gene, affects kinetics of transmitter release." Journal of Cell Biology 181, no. 5 (May 26, 2008): 791–801. http://dx.doi.org/10.1083/jcb.200711021.
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Schizophrenia is one of the most debilitating neuropsychiatric disorders, affecting 0.5–1.0% of the population worldwide. Its pathology, attributed to defects in synaptic transmission, remains elusive. The dystrobrevin-binding protein 1 (DTNBP1) gene, which encodes a coiled-coil protein, dysbindin, is a major susceptibility gene for schizophrenia. Our previous results have demonstrated that the sandy (sdy) mouse harbors a spontaneously occurring deletion in the DTNBP1 gene and expresses no dysbindin protein (Li, W., Q. Zhang, N. Oiso, E.K. Novak, R. Gautam, E.P. O'Brien, C.L. Tinsley, D.J. Blake, R.A. Spritz, N.G. Copeland, et al. 2003. Nat. Genet. 35:84–89). Here, using amperometry, whole-cell patch clamping, and electron microscopy techniques, we discovered specific defects in neurosecretion and vesicular morphology in neuroendocrine cells and hippocampal synapses at the single vesicle level in sdy mice. These defects include larger vesicle size, slower quantal vesicle release, lower release probability, and smaller total population of the readily releasable vesicle pool. These findings suggest that dysbindin functions to regulate exocytosis and vesicle biogenesis in endocrine cells and neurons. Our work also suggests a possible mechanism in the pathogenesis of schizophrenia at the synaptic level.
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Wang, Zhao, Chi Wai Do, Virginijus Valiunas, Chi Ting Leung, Angela K. W. Cheng, Abbott F. Clark, Martin B. Wax, Jon E. Chatterton, and Mortimer M. Civan. "Regulation of gap junction coupling in bovine ciliary epithelium." American Journal of Physiology-Cell Physiology 298, no. 4 (April 2010): C798—C806. http://dx.doi.org/10.1152/ajpcell.00406.2009.
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Aqueous humor is formed by fluid transfer from the ciliary stroma sequentially across the pigmented ciliary epithelial (PE) cells, gap junctions, and nonpigmented ciliary epithelial (NPE) cells. Which connexins (Cx) contribute to PE-NPE gap junctional formation appears species specific. We tested whether small interfering RNA (siRNA) against Cx43 (siCx43) affects bovine PE-NPE communication and whether cAMP affects communication. Native bovine ciliary epithelial cells were studied by dual-cell patch clamping, Lucifer Yellow (LY) transfer, quantitative polymerase chain reaction with reverse transcription (qRT-PCR), and Western immunoblot. qRT-PCR revealed at least 100-fold greater expression for Cx43 than Cx40. siCx43 knocked down target mRNA expression by 55 ± 7% after 24 h, compared with nontargeting control siRNA (NTC1) transfection. After 48 h, siCx43 reduced Cx43 protein expression and LY transfer. The ratio of fluorescence intensity (Rf) in recipient to donor cell was 0.47 ± 0.09 ( n = 11) 10 min after whole cell patch formation in couplets transfected with NTC1. siCx43 decreased Rf by ∼60% to 0.20 ± 0.07 ( n = 13, P < 0.02). Dibutyryl-cAMP (500 μM) also reduced LY dye transfer by ∼60%, reducing Rf from 0.41 ± 0.05 ( n = 15) to 0.17 ± 0.05 ( n = 20) after 10 min. Junctional currents were lowered by ∼50% ( n = 6) after 10-min perfusion with 500 μM dibutyryl-cAMP ( n = 6); thereafter, heptanol abolished the currents ( n = 5). Preincubation with the PKA inhibitor H-89 (2 μM) prevented cAMP-triggered current reduction ( n = 6). We conclude that 1) Cx43, but not Cx40, is a major functional component of bovine PE-NPE gap junctions; and 2) under certain conditions, cAMP may act through PKA to inhibit bovine PE-NPE gap junctional communication.
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Chang, Wenhan, Tsui-Hua Chen, Stacy Pratt, and Dolores Shoback. "Regulation of extracellular calcium-activated cation currents by cAMP in parathyroid cells." American Journal of Physiology-Endocrinology and Metabolism 275, no. 2 (August 1, 1998): E213—E221. http://dx.doi.org/10.1152/ajpendo.1998.275.2.e213.
Full textAbstract:
Parathyroid cells express Ca2+-sensing receptors that couple changes in the extracellular Ca2+ concentration ([Ca2+]o) to increases in the intracellular free Ca2+ concentration ([Ca2+]i) and to the suppression of parathyroid hormone secretion. Using whole cell patch clamping, we previously identified voltage-independent Ca2+-conducting currents in bovine parathyroid cells that increased with rising [Ca2+]oand were blocked by Cd2+ and nifedipine. Because cAMP-dependent phosphorylation regulates dihydropyridine-sensitive Ca2+channels in other systems, we tested whether cAMP modulates these currents. At 0.7 mM Ca2+, nonselective Ca2+-conducting currents were suppressed by 30–50% when the recording pipette was perfused with cAMP. High-[Ca2+]o-induced increases in membrane currents were also abrogated. The effects of cAMP were reversible and dose dependent (3 × 10−9 to 3 × 10−3 M) and required ATP in the pipette solution. Perfusion of the cell interior with the catalytic subunit of protein kinase A mimicked the effects of cAMP, as did perfusion of the bath with the adenylate cyclase activator forskolin. These findings support the idea that cAMP-dependent phosphorylation suppresses high-[Ca2+]o-induced cation currents and may play a role in regulating ion fluxes in parathyroid cells.
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Wu, Delin, Linqing Jiang, Hongjin Wu, Shengqi Wang, Sidao Zheng, Jiyuan Yang, Yuna Liu, Jianxun Ren, and Xianbing Chen. "Inhibitory Effects of Glycyrrhetinic Acid on the Delayed Rectifier Potassium Current in Guinea Pig Ventricular Myocytes and HERG Channel." Evidence-Based Complementary and Alternative Medicine 2013 (2013): 1–11. http://dx.doi.org/10.1155/2013/481830.
Full textAbstract:
Background. Licorice has long been used to treat many ailments including cardiovascular disorders in China. Recent studies have shown that the cardiac actions of licorice can be attributed to its active component, glycyrrhetinic acid (GA). However, the mechanism of action remains poorly understood.Aim. The effects of GA on the delayed rectifier potassium current (IK), the rapidly activating (IKr) and slowly activating (IKs) components ofIK, and the HERG K+channel expressed in HEK-293 cells were investigated.Materials and Methods. Single ventricular myocytes were isolated from guinea pig myocardium using enzymolysis. The wild type HERG gene was stably expressed in HEK293 cells. Whole-cell patch clamping was used to recordIK(IKr,IKs) and the HERG K+current.Results. GA (1, 5, and 10 μM) inhibitedIK(IKr,IKs) and the HERG K+current in a concentration-dependent manner.Conclusion. GA significantly inhibited the potassium currents in a dose- and voltage-dependent manner, suggesting that it exerts its antiarrhythmic action through the prolongation of APD and ERP owing to the inhibition ofIK(IKr,IKs) and HERG K+channel.
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Wong, Kwoon Y., Ethan D. Cohen, and John E. Dowling. "Retinal Bipolar Cell Input Mechanisms in Giant Danio. II. Patch-Clamp Analysis of on Bipolar Cells." Journal of Neurophysiology 93, no. 1 (January 2005): 94–107. http://dx.doi.org/10.1152/jn.00270.2004.
Full textAbstract:
Glutamate receptors on giant danio retinal on bipolar cells were studied with whole cell patch clamping using a slice preparation. Cone-driven on bipolars (Cbs) and mixed-input on bipolars (Mbs) were identified morphologically. Most Cbs responded to the excitatory amino acid transporter (EAAT) substrate d-aspartate but not to the group III metabotropic glutamate receptor (mGluR) agonist l-(+)-2-amino-4-phosphonobutyric acid (l-AP4) or the AMPA/kainate receptor agonist kainate, suggesting EAATs are the primary glutamate receptors on Cbs. The EAAT inhibitor dl- threo-β-benzyloxyasparate (TBOA) blocked all light-evoked responses of Cbs, suggesting these responses are mediated exclusively by EAATs. Conversely, all Mbs responded to d-aspartate and l-AP4 but not to kainate, indicating they have both EAATs and group III mGluRs (presumably mGluR6). The light responses of Mbs involve both receptors because they could be blocked by TBOA plus (RS)-α-cyclopropyl-4-phosphonophenylglycine (CPPG, a group III mGluR antagonist) but not by either alone. Under dark-adapted conditions, the responses of Mbs to green (rod-selective) stimuli were reduced by CPPG but enhanced by TBOA. In contrast, both antagonists reduced the responses to red (cone-selective) stimuli, although TBOA was more effective. Furthermore, under photopic conditions, TBOA failed to eliminate light-evoked responses of Mbs. Thus on Mbs, rod inputs are mediated predominantly by mGluR6, whereas cone inputs are mediated mainly by EAATs but also by mGluR6 to some extent. Finally, we explored the interactions between EAATs and mGluR6 in Mbs. Responses to d-aspartate were reduced by l-AP4 and vice versa. Therefore mGluR6 and EAATs suppress each other, and this might underlie mutual suppression between rod and cone signals in Mbs.
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Baker, Mark D., and Hugh Bostock. "Inactivation of Macroscopic Late Na+ Current and Characteristics of Unitary Late Na+ Currents in Sensory Neurons." Journal of Neurophysiology 80, no. 5 (November 1, 1998): 2538–49. http://dx.doi.org/10.1152/jn.1998.80.5.2538.
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Baker, Mark D. and Hugh Bostock. Inactivation of macroscopic late Na+ current and characteristics of unitary late Na+ currents in sensory neurons. J. Neurophysiol. 80: 2538–2549, 1998. Na+ currents in adult rat large dorsal root ganglion neurons were recorded during long duration voltage-clamp steps by patch clamping whole cells and outside-out membrane patches. Na+ current present >60 ms after the onset of a depolarizing pulse (late Na+ current) underwent partial inactivation; it behaved as the sum of three kinetically distinct components, each of which was blocked by nanomolar concentrations of tetrodotoxin. Inactivation of one component (late-1) of the whole cell current reached equilibrium during the first 60 ms; repolarizing to −40 or −50 mV from potentials of −30 mV or more positive gave rise to a characteristic increase in current (τ ≥ 5 ms), attributed to removal of inactivation. A second component (late-2) underwent slower inactivation (τ > 80 ms) at potentials more positive than −80 mV, and steady-state inactivation appeared complete at −30 mV. In small membrane patches, bursts of brief openings (γ = 13–18 pS) were usually recorded. The distribution of burst durations indicated that two populations of channel were present with inactivation rates corresponding to late-1 and late-2 macroscopic currents. The persistent Na+ current in the whole cell that extended to potentials more positive than −30 mV appeared to correspond to sporadic, brief openings that were recorded in patches (mean open time ∼0.1 ms) over a wide potential range. None of the three types of gating described corresponded to activation/inactivation gating overlap of fast transient currents.
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Bertrand, C. A., H. Danahay, C. T. Poll, C. Laboisse, U. Hopfer, and R. J. Bridges. "Niflumic acid inhibits ATP-stimulated exocytosis in a mucin-secreting epithelial cell line." American Journal of Physiology-Cell Physiology 286, no. 2 (February 2004): C247—C255. http://dx.doi.org/10.1152/ajpcell.00593.2002.
Full textAbstract:
ATP is an efficacious secretagogue for mucin and chloride in the epithelial cell line HT29-Cl.16E. Mucin release has been measured as [3H]glucosamine-labeled product in extracellular medium and as single-cell membrane capacitance increases indicative of exocytosis-related increases in membrane area. The calcium-activated chloride channel blocker niflumic acid, also reported to modulate secretion, was used to probe for divergence in the purinergic signaling of mucin exocytosis and channel activation. With the use of whole cell patch clamping, ATP stimulated a transient capacitance increase of 15 ± 4%. Inclusion of niflumic acid significantly reduced the ATP-stimulated capacitance change to 3 ± 1%, although normalized peak currents were not significantly different. Ratiometric imaging was used to assess intracellular calcium ([Formula: see text]) dynamics during stimulation. In the presence of niflumic acid, the ATP-stimulated peak change in [Formula: see text] was unaffected, but the initial response and overall time to [Formula: see text] peak were significantly affected. Excluding external calcium before ATP stimulation or including the capacitative calcium entry blocker LaCl3 during stimulation muted the initial calcium transient similar to that observed with niflumic acid and significantly reduced peak capacitance change, suggesting that a substantial portion of the ATP-stimulated mucin exocytosis in HT29-Cl.16E depends on a rapid, brief calcium influx through the plasma membrane. Niflumic acid interferes with this influx independent of a chloride channel blockade effect.
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Klenke, Ulrike, Carol Taylor-Burds, and Susan Wray. "Metabolic Influences on Reproduction: Adiponectin Attenuates GnRH Neuronal Activity in Female Mice." Endocrinology 155, no. 5 (May 1, 2014): 1851–63. http://dx.doi.org/10.1210/en.2013-1677.
Full textAbstract:
Metabolic dysfunctions are often linked to reproductive abnormalities. Adiponectin (ADP), a peripheral hormone secreted by white adipose tissue, is important in energy homeostasis and appetite regulation. GnRH neurons are integral components of the reproductive axis, controlling synthesis, and release of gonadotropins. This report examined whether ADP can directly act on GnRH neurons. Double-label immunofluorescence on brain sections from adult female revealed that a subpopulation of GnRH neurons express ADP receptor (AdipoR)2. GnRH/AdipoR2+ cells were distributed throughout the forebrain. To determine the influence of ADP on GnRH neuronal activity and the signal transduction pathway of AdipoR2, GnRH neurons maintained in explants were assayed using whole-cell patch clamping and calcium imaging. This mouse model system circumvents the dispersed distribution of GnRH neurons within the forebrain, making analysis of large numbers of GnRH cells possible. Single-cell PCR analysis and immunocytochemistry confirmed the presence of AdipoR2 in GnRH neurons in explants. Functional analysis revealed 20% of the total GnRH population responded to ADP, exhibiting hyperpolarization or decreased calcium oscillations. Perturbation studies revealed that ADP activates AMP kinase via the protein kinase Cζ/liver kinase B1 pathway. The modulation of GnRH neuronal activity by ADP demonstrated in this report directly links energy balance to neurons controlling reproduction.
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Kim, Hee Young, Inhyung Lee, Sang Woo Chun, and Hee Kee Kim. "Reactive Oxygen Species Donors Increase the Responsiveness of Dorsal Horn Neurons and Induce Mechanical Hyperalgesia in Rats." Neural Plasticity 2015 (2015): 1–10. http://dx.doi.org/10.1155/2015/293423.
Full textAbstract:
Our previous studies suggest that reactive oxygen species (ROS) scavengers have analgesic effect on neuropathic pain through spinal mechanisms in the rat. The studies suggest that superoxide in spinal cord is one of important mediators of persistent pain. To test the hypothesis that increase of superoxide-derived intermediates leads to central sensitization and pain, the effects of an intrathecal injection of chemical ROS donors releasing eitherOH∙,OCl-, or H2O2were examined on pain behaviors. Following treatment witht-BOOH (OH∙donor), dorsal horn neuron responses to mechanical stimuli in normal rats and the changes of neuronal excitability were explored on substantia gelatinosa (SG) neurons using whole-cell patch clamping recordings. Intrathecal administration oft-BOOH or NaOCl (OCl-donor), but not H2O2, significantly decreased mechanical thresholds of hind paws. The responses of wide dynamic range neurons to mechanical stimuli increased after a local application oft-BOOH. Thet-BOOH increased the frequency and the amplitude of excitatory postsynaptic potentials, depolarized membrane potential in SG neurons, and increased the frequency of action potentials evoked by depolarizing current pulses. These results suggest that elevated ROS, especiallyOH∙, in the spinal cord sensitized dorsal horn neurons and produced hyperalgesia in normal rats.
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Nobles, Muriel, David Montaigne, Sonia Sebastian, Lutz Birnbaumer, and Andrew Tinker. "Differential effects of inhibitory G protein isoforms on G protein-gated inwardly rectifying K+ currents in adult murine atria." American Journal of Physiology-Cell Physiology 314, no. 5 (May 1, 2018): C616—C626. http://dx.doi.org/10.1152/ajpcell.00271.2016.
Full textAbstract:
G protein-gated inwardly rectifying K+ (GIRK) channels are the major inwardly rectifying K+ currents in cardiac atrial myocytes and an important determinant of atrial electrophysiology. Inhibitory G protein α-subunits can both mediate activation via acetylcholine but can also suppress basal currents in the absence of agonist. We studied this phenomenon using whole cell patch clamping in murine atria from mice with global genetic deletion of Gαi2, combined deletion of Gαi1/Gαi3, and littermate controls. We found that mice with deletion of Gαi2 had increased basal and agonist-activated currents, particularly in the right atria while in contrast those with Gαi1/Gαi3 deletion had reduced currents. Mice with global genetic deletion of Gαi2 had decreased action potential duration. Tissue preparations of the left atria studied with a multielectrode array from Gαi2 knockout mice showed a shorter effective refractory period, with no change in conduction velocity, than littermate controls. Transcriptional studies revealed increased expression of GIRK channel subunit genes in Gαi2 knockout mice. Thus different G protein isoforms have differential effects on GIRK channel behavior and paradoxically Gαi2 act to increase basal and agonist-activated GIRK currents. Deletion of Gαi2 is potentially proarrhythmic in the atria.
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He, Jianying, Margaret E. Kargacin, Gary J. Kargacin, and Christopher A. Ward. "Tamoxifen inhibits Na+ and K+ currents in rat ventricular myocytes." American Journal of Physiology-Heart and Circulatory Physiology 285, no. 2 (August 2003): H661—H668. http://dx.doi.org/10.1152/ajpheart.00686.2002.
Full textAbstract:
Tamoxifen is an estrogen receptor antagonist used in the treatment of breast cancer. However, tamoxifen has been shown to induce QT prolongation of the electrocardiogram, thereby potentially causing life-threatening polymorphic ventricular arrhythmias. The purpose of the present study was to elucidate the electrophysiological mechanism(s) that underlie the arrhythmogenic effects of tamoxifen. We used standard ruptured whole cell and perforated patch-clamping techniques on rat ventricular myocytes to investigate the effects of tamoxifen on cardiac action potential (AP) waveforms and the underlying K+ currents. Tamoxifen (3 μmol/l) markedly prolonged AP duration, decreased maximal rate of depolarization, and decreased resting membrane potential. At this concentration, tamoxifen significantly depressed the Ca2+-independent transient outward K+ current ( Ito), sustained outward delayed rectifier K+ current ( Isus), inward rectifier K+ current ( IK1), and Na+ current ( INa) in the myocytes. Lower concentrations of tamoxifen (1 μmol/l) also decreased the resting membrane potential and significantly depressed IK1 to 79 ± 5% ( n = 5; at –120 mV) of pretreatment values. The results of this study indicate that inhibition of Ito, Isus, and IK1 by tamoxifen may underlie AP prolongation in cardiac myocytes and thereby contribute to prolonged QT interval observed in patients.
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Oliet, S. H., and C. W. Bourque. "Steady-state osmotic modulation of cationic conductance in neurons of rat supraoptic nucleus." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 265, no. 6 (December 1, 1993): R1475—R1479. http://dx.doi.org/10.1152/ajpregu.1993.265.6.r1475.
Full textAbstract:
Whole cell patch-clamp recordings were obtained from isolated rat supraoptic nucleus magnocellular neurosecretory cells (MNCs). Under current clamping, hyperosmolality produced by the addition of 10-30 mM mannitol depolarized each of 25 cells tested. In contrast, reducing fluid osmolality from 295 to 265 mosmol/kgH2O had the reverse effect, hyperpolarizing 18 of 21 MNCs. Voltage-clamp recordings in 43 cells revealed that the effects of hypo- and hyperosmolality, respectively, were caused by decreases and increases in a nonselective cation conductance reversing near -41 mV. Current-voltage analysis in Na(+)-free solution revealed that the reversal potentials of currents elicited by increases and decreases in osmolality both shifted to a value near -90 mV, suggesting that a single ionic conductance is modulated by these stimuli. The relation between cationic conductance and osmolality was specific, sensitive (+2.14%.mosmol-1.kgH2O-1), and well-fit by linear regression (r = 0.96; n = 22 cells) between 275 and 325 mosmol/kgH2O. These results indicate that MNCs express a depolarizing current that is active under steady-state conditions and that the up- or downregulation of this current contributes to the excitation or inhibition of these cells upon acute exposure to hypo- or hyperosmolar conditions.
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Ma, Linlin, Grigori Y. Rychkov, Bernard P. Hughes, and Allan H. Bretag. "Analysis of carboxyl tail function in the skeletal muscle Cl− channel hClC-1." Biochemical Journal 413, no. 1 (June 12, 2008): 61–69. http://dx.doi.org/10.1042/bj20071489.
Full textAbstract:
Human ClC-1 (skeletal muscle Cl− channel) has a long cytoplasmic C-tail (carboxyl tail), containing two CBS (cystathionine β-synthase) domains, which is very important for channel function. We have now investigated its significance further, using deletion and alanine-scanning mutagenesis, split channels, GST (glutathione transferase)-pull-down and whole-cell patch-clamping. In tagged split-channel experiments, we have demonstrated strong binding between an N-terminal membrane-resident fragment (terminating mid-C-tail at Ser720 and containing CBS1) and its complement (containing CBS2). This interaction is not affected by deletion of some sequences, suggested previously to be important, particularly in channel gating. Contact between CBS1 and CBS2, however, may make a major contribution to assembly of functional channels from such co-expressed complements, although the possibility that C-tail fragments could, in addition, bind to other parts of the membrane-resident component has not been eliminated. We now show such an interaction between a membrane-resident component terminating at Ser720 (but with CBS1 deleted) and a complete C-tail beginning at Leu598. Channel function is rescued in patch-clamped HEK-293T (human embryonic kidney) cells co-expressing these same fragments. From our own results and those of others, we conclude that the CBS1–CBS2 interaction is not sufficient, in itself, for channel assembly, but rather that this might normally assist in bringing some part of the CBS2/C-tail region into appropriate proximity with the membrane-resident portion of the protein. Previously conflicting and anomalous results can now be explained by an hypothesis that, for split channels to be functional, at least one membrane-resident component must include a plasma membrane trafficking signal between Leu665 and Lys680.
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Fuller, William, Jacqueline Howie, Linda M. McLatchie, Roberta J. Weber, C. James Hastie, Kerry Burness, Davor Pavlovic, and Michael J. Shattock. "FXYD1 phosphorylation in vitro and in adult rat cardiac myocytes: threonine 69 is a novel substrate for protein kinase C." American Journal of Physiology-Cell Physiology 296, no. 6 (June 2009): C1346—C1355. http://dx.doi.org/10.1152/ajpcell.00523.2008.
Full textAbstract:
FXYD1 (phospholemman), the primary sarcolemmal kinase substrate in the heart, is a regulator of the cardiac sodium pump. We investigated phosphorylation of FXYD1 peptides by purified kinases using HPLC, mass spectrometry, and Edman sequencing, and FXYD1 phosphorylation in cultured adult rat ventricular myocytes treated with PKA and PKC agonists by phosphospecific immunoblotting. PKA phosphorylates serines 63 and 68 (S63 and S68) and PKC phosphorylates S63, S68, and a new site, threonine 69 (T69). In unstimulated myocytes, FXYD1 is ∼30% phosphorylated at S63 and S68, but barely phosphorylated at T69. S63 and S68 are rapidly dephosphorylated following acute inhibition of PKC in unstimulated cells. Receptor-mediated PKC activation causes sustained phosphorylation of S63 and S68, but transient phosphorylation of T69. To characterize the effect of T69 phosphorylation on sodium pump function, we measured pump currents using whole cell voltage clamping of cultured adult rat ventricular myocytes with 50 mM sodium in the patch pipette. Activation of PKA or PKC increased pump currents (from 2.1 ± 0.2 pA/pF in unstimulated cells to 2.9 ± 0.1 pA/pF for PKA and 3.4 ± 0.2 pA/pF for PKC). Following kinase activation, phosphorylated FXYD1 was coimmunoprecipitated with sodium pump α1-subunit. We conclude that T69 is a previously undescribed phosphorylation site in FXYD1. Acute T69 phosphorylation elicits stimulation of the sodium pump additional to that induced by S63 and S68 phosphorylation.
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Boulkroun, Sheerazed, Dorothée Ruffieux-Daidié, Jean-Jacques Vitagliano, Olivier Poirot, Roch-Philippe Charles, Dagmara Lagnaz, Dmitri Firsov, Stephan Kellenberger, and Olivier Staub. "Vasopressin-inducible ubiquitin-specific protease 10 increases ENaC cell surface expression by deubiquitylating and stabilizing sorting nexin 3." American Journal of Physiology-Renal Physiology 295, no. 4 (October 2008): F889—F900. http://dx.doi.org/10.1152/ajprenal.00001.2008.
Full textAbstract:
Adjustment of Na+ balance in extracellular fluids is achieved by regulated Na+ transport involving the amiloride-sensitive epithelial Na+ channel (ENaC) in the distal nephron. In this context, ENaC is controlled by a number of hormones, including vasopressin, which promotes rapid translocation of water and Na+ channels to the plasma membrane and long-term effects on transcription of vasopressin-induced and -reduced transcripts. We have identified a mRNA encoding the deubiquitylating enzyme ubiquitin-specific protease 10 (Usp10), whose expression is increased by vasopressin at both the mRNA and the protein level. Coexpression of Usp10 in ENaC-transfected HEK-293 cells causes a more than fivefold increase in amiloride-sensitive Na+ currents, as measured by whole cell patch clamping. This is accompanied by a three- to fourfold increase in surface expression of α- and γ-ENaC, as shown by cell surface biotinylation experiments. Although ENaC is well known to be regulated by its direct ubiquitylation, Usp10 does not affect the ubiquitylation level of ENaC, suggesting an indirect effect. A two-hybrid screen identified sorting nexin 3 (SNX3) as a novel substrate of Usp10. We show that it is a ubiquitylated protein that is degraded by the proteasome; interaction with Usp10 leads to its deubiquitylation and stabilization. When coexpressed with ENaC, SNX3 increases the channel's cell surface expression, similarly to Usp10. In mCCDcl1 cells, vasopressin increases SNX3 protein but not mRNA, supporting the idea that the vasopressin-induced Usp10 deubiquitylates and stabilizes endogenous SNX3 and consequently promotes cell surface expression of ENaC.
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Oronowicz, Jakub, Jacqueline Reinhard, Peter Sol Reinach, Szymon Ludwiczak, Huan Luo, Marah Hussain Omar Ba Salem, Miriam Monika Kraemer, Heike Biebermann, Vinodh Kakkassery, and Stefan Mergler. "Ascorbate-induced oxidative stress mediates TRP channel activation and cytotoxicity in human etoposide-sensitive and -resistant retinoblastoma cells." Laboratory Investigation 101, no. 1 (September 18, 2020): 70–88. http://dx.doi.org/10.1038/s41374-020-00485-2.
Full textAbstract:
AbstractThere are indications that pharmacological doses of ascorbate (Asc) used as an adjuvant improve the chemotherapeutic management of cancer. This favorable outcome stems from its cytotoxic effects due to prooxidative mechanisms. Since regulation of intracellular Ca2+ levels contributes to the maintenance of cell viability, we hypothesized that one of the effects of Asc includes disrupting regulation of intracellular Ca2+ homeostasis. Accordingly, we determined if Asc induced intracellular Ca2+ influx through activation of pertussis sensitive Gi/o-coupled GPCR which in turn activated transient receptor potential (TRP) channels in both etoposide-resistant and -sensitive retinoblastoma (WERI-Rb1) tumor cells. Ca2+ imaging, whole-cell patch-clamping, and quantitative real-time PCR (qRT-PCR) were performed in parallel with measurements of RB cell survival using Trypan Blue cell dye exclusion. TRPM7 gene expression levels were similar in both cell lines whereas TRPV1, TRPM2, TRPA1, TRPC5, TRPV4, and TRPM8 gene expression levels were downregulated in the etoposide-resistant WERI-Rb1 cells. In the presence of extracellular Ca2+, 1 mM Asc induced larger intracellular Ca2+ transients in the etoposide-resistant WERI-Rb1 than in their etoposide-sensitive counterpart. With either 100 µM CPZ, 500 µM La3+, 10 mM NAC, or 100 µM 2-APB, these Ca2+ transients were markedly diminished. These inhibitors also had corresponding inhibitory effects on Asc-induced rises in whole-cell currents. Pertussis toxin (PTX) preincubation blocked rises in Ca2+ influx. Microscopic analyses showed that after 4 days of exposure to 1 mM Asc cell viability fell by nearly 100% in both RB cell lines. Taken together, one of the effects underlying oxidative mediated Asc-induced WERI-Rb1 cytotoxicity stems from its promotion of Gi/o coupled GPCR mediated increases in intracellular Ca2+ influx through TRP channels. Therefore, designing drugs targeting TRP channel modulation may be a viable approach to increase the efficacy of chemotherapeutic treatment of RB. Furthermore, Asc may be indicated as a possible supportive agent in anti-cancer therapies.
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Chang, W., T. H. Chen, P. Gardner, and D. Shoback. "Regulation of Ca(2+)-conducting currents in parathyroid cells by extracellular Ca(2+) and channel blockers." American Journal of Physiology-Endocrinology and Metabolism 269, no. 5 (November 1, 1995): E864—E877. http://dx.doi.org/10.1152/ajpendo.1995.269.5.e864.
Full textAbstract:
High extracellular Ca2+ concentrations ([Ca2+]o) produce sustained intracellular Ca2+ responses in parathyroid cells that correlate with suppression of parathyroid hormone release. Using whole cell patch clamping, we identified two types of Ca(2+)-conducting currents in these cells. Type 1 currents were enhanced by raising [Ca2+]o and blocked by Cd2+ and nifedipine, whereas type 2 currents were resistant to blockade by these agents. Both types of membrane currents were cation nonselective, voltage independent over a broad range of membrane potentials, and blocked by the trivalent ions La3+ and Gd3+ (> 98%). Cd2+, La3+, and Gd3+ had biphasic effects on membrane conductance (Gm). At submicromolar concentrations, these ions increased Gm, whereas at higher concentrations they reduced Gm. In contrast to ionic channel blockers, nifedipine had only an inhibitory effect on the Ca(2+)-conducting currents that were sensitive to changes in [Ca2+]o (dose inhibiting 50% of maximal response = approximately 3-10 x 10(-8) M). Microfluorimetric ratio-imaging analysis of single parathyroid cells loaded with fura 2 showed that Gd3+ inhibited sustained intracellular Ca2+ responses to high [Ca2+]o. These findings suggest that the Ca(2+)-conducting currents identified in these studies may play a role in regulating intracellular Ca2+ responses in this system.
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Calvo-Flores Guzmán, Beatriz, SooHyun Kim, Bhavya Chawdhary, Katie Peppercorn, Warren P. Tate, Henry J. Waldvogel, Richard LM Faull, Johanna Montgomery, and Andrea Kwakowsky. "Amyloid-Beta1-42 -Induced Increase in GABAergic Tonic Conductance in Mouse Hippocampal CA1 Pyramidal Cells." Molecules 25, no. 3 (February 6, 2020): 693. http://dx.doi.org/10.3390/molecules25030693.
Full textAbstract:
Alzheimer’s disease (AD) is a complex and chronic neurodegenerative disorder that involves a progressive and severe decline in cognition and memory. During the last few decades a considerable amount of research has been done in order to better understand tau-pathology, inflammatory activity and neuronal synapse loss in AD, all of them contributing to cognitive decline. Early hippocampal network dysfunction is one of the main factors associated with cognitive decline in AD. Much has been published about amyloid-beta1-42 (Aβ1-42)-mediated excitotoxicity in AD. However, increasing evidence demonstrates that the remodeling of the inhibitory gamma-aminobutyric acid (GABAergic) system contributes to the excitatory/inhibitory (E/I) disruption in the AD hippocampus, but the underlying mechanisms are not well understood. In the present study, we show that hippocampal injection of Aβ1-42 is sufficient to induce cognitive deficits 7 days post-injection. We demonstrate using in vitro whole-cell patch-clamping an increased inhibitory GABAergic tonic conductance mediated by extrasynaptic type A GABA receptors (GABAARs), recorded in the CA1 region of the mouse hippocampus following Aβ1-42 micro injection. Such alterations in GABA neurotransmission and/or inhibitory GABAARs could have a significant impact on both hippocampal structure and function, causing E/I balance disruption and potentially contributing to cognitive deficits in AD.
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RYCHKOV, Grigori Y., Tom LITJENS, Michael L. ROBERTS, and Greg J. BARRITT. "Arachidonic acid inhibits the store-operated Ca2+ current in rat liver cells." Biochemical Journal 385, no. 2 (January 7, 2005): 551–56. http://dx.doi.org/10.1042/bj20041604.
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Vasopressin and other phospholipase-C-coupled hormones induce oscillations (waves) of [Ca2+]cyt (cytoplasmic Ca2+ concentration) in liver cells. Maintenance of these oscillations requires replenishment of Ca2+ in intracellular stores through Ca2+ inflow across the plasma membrane. While this may be achieved by SOCs (store-operated Ca2+ channels), some studies in other cell types indicate that it is dependent on AA (arachidonic acid)-activated Ca2+ channels. We studied the effects of AA on membrane conductance of rat liver cells using whole-cell patch clamping. We found no evidence that concentrations of AA in the physiological range could activate Ca2+-permeable channels in either H4IIE liver cells or rat hepatocytes. However, AA (1–10 μM) did inhibit (IC50=2.4±0.1 μM) Ca2+ inflow through SOCs (ISOC) initiated by intracellular application of Ins(1,4,5)P3 in H4IIE cells. Pre-incubation with AA did not inhibit ISOC development, but decreased maximal amplitude of the current. Iso-tetrandrine, widely used to inhibit receptor-activation of phospholipase A2, and therefore AA release, inhibited ISOC directly in H4IIE cells. It is concluded that (i) in rat liver cells, AA does not activate an AA-regulated Ca2+-permeable channel, but does inhibit SOCs, and (ii) iso-tetrandrine and tetrandrine are effective blockers of CRAC (Ca2+-release-activated Ca2+) channel-like SOCs. These results indicate that AA-activated Ca2+-permeable channels do not contribute to hormone-induced increases or oscillations in [Ca2+]cyt in liver cells. However, AA may be a physiological modulator of Ca2+ inflow in these cells.
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Pedersen, Nigel P., Christopher W. Vaughan, and MacDonald J. Christie. "Opioid receptor modulation of GABAergic and serotonergic spinally projecting neurons of the rostral ventromedial medulla in mice." Journal of Neurophysiology 106, no. 2 (August 2011): 731–40. http://dx.doi.org/10.1152/jn.01062.2010.
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The rostral ventromedial medulla (RVM) is an important site of opioid actions and forms part of an analgesic pathway that projects to the spinal cord. The neuronal mechanisms by which opioids act within this brain region remain unclear, particularly in relation to the neurotransmitters GABA and serotonin. In the present study, we examined serotonergic and GABAergic immunoreactivity, identified using immunohistochemistry for tryptophan hydroxylase (TPH) and glutamate decarboxylase (GAD), in combination with in vitro whole cell patch clamping to investigate the role of opioids on the mouse RVM with identified projections to the spinal cord. Tyr-d-Ala-Gly- N-Me-Phe-Gly-ol enkephalin (DAMGO) produced μ-opioid receptor-mediated outward currents in virtually all TPH-immunoreactive projecting neurons and GAD-immunoreactive nonprojecting neurons (87% and 86%). The other groups of RVM neurons displayed mixed responsiveness to DAMGO (40–68%). Deltorphin II and U-69593 produced δ- and κ-opioid receptor-mediated outward currents in smaller subpopulations of RVM neurons, with many of the δ-opioid responders forming a subpopulation of μ-opioid-sensitive GABAergic nonprojecting neurons. These findings are consistent with prior electrophysiological and anatomic studies in the rat RVM and indicate that both serotonergic and GABAergic RVM neurons mediate the actions of μ-opioids. Specifically, μ-opioids have a direct postsynaptic inhibitory influence over both GABAergic and serotonergic neurons, including those that project to the dorsal spinal cord.
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Smith, R. Ya, D. Morgan, L. Sharma, V. V. Cherny, N. Tidswell, M. W. Molo, and T. E. DeCoursey. "Voltage-gated proton channels exist in the plasma membrane of human oocytes." Human Reproduction 34, no. 10 (October 2019): 1974–83. http://dx.doi.org/10.1093/humrep/dez178.
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Abstract STUDY QUESTION Do human oocytes express voltage-gated proton channels? SUMMARY ANSWER Human oocytes exhibit voltage-gated proton currents. WHAT IS KNOWN ALREADY Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. STUDY DESIGN, SIZE, DURATION Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. MAIN RESULTS AND THE ROLE OF CHANCE Proton currents are present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene that codes for the voltage-gated proton channel. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. STUDY FUNDING/COMPETING INTEREST(S) NIH R35GM126902 (TED), Bears Care (DM). No competing interests. TRIAL REGISTRATION NUMBER N/A.
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Lynch, J. W., and P. H. Barry. "Properties of transient K+ currents and underlying single K+ channels in rat olfactory receptor neurons." Journal of General Physiology 97, no. 5 (May 1, 1991): 1043–72. http://dx.doi.org/10.1085/jgp.97.5.1043.
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The transient potassium current, IK(t), of enzymatically dissociated rat olfactory receptor neurons was studied using patch-clamp techniques. Upon depolarization from negative holding potentials, IK(t) activated rapidly and then inactivated with a time course described by the sum of two exponential components with time constants of 22.4 and 143 ms. Single-channel analysis revealed a further small component with a time constant of several seconds. Steady-state inactivation was complete at -20 mV and completely removed at -80 mV (midpoint -45 mV). Activation was significant at -40 mV and appeared to reach a maximum conductance at +40 mV (midpoint -13 mV). Deactivation was described by the sum of two voltage-dependent exponential components. Recovery from inactivation was extraordinarily slow (50 s at -100 mV) and the underlying processes appeared complex. IK(t) was reduced by 4-aminopyridine and tetraethylammonium applied externally. Increasing the external K+ concentration ([K+]o) from 5 to 25 mM partially removed IK(t) inactivation, usually without affecting activation kinetics. The elevated [K+]o also hyperpolarized the steady-state inactivation curve by 9 mV and significantly depolarized the voltage dependence of activation. Single transient K+ channels, with conductances of 17 and 26 pS, were observed in excised patches and often appeared to be localized into large clusters. These channels were similar to IK(t) in their kinetic, pharmacological, and voltage-dependent properties and their inactivation was also subject to modulation by [K+]o. The properties of IK(t) imply a role in action potential repolarization and suggest it may also be important in modulating spike parameters during neuronal burst firing. A simple method is also presented to correct for errors in the measurement of whole-cell resistance (Ro) that can result when patch-clamping very small cells. The analysis revealed a mean corrected Ro of 26 G omega for these cells.
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Guo, X., D. Merlin, R. D. Harvey, C. Laboisse, and U. Hopfer. "Stimulation of Cl- secretion by extracellular ATP does not depend on increased cytosolic Ca2+ in HT-29.cl16E." American Journal of Physiology-Cell Physiology 269, no. 6 (December 1, 1995): C1457—C1463. http://dx.doi.org/10.1152/ajpcell.1995.269.6.c1457.
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Extracellular ATP and elevated cytosolic Ca2+ concentration ([Ca2+]i) are major secretagogues for Cl- in the goblet cell-like clone cl.16E derived from colonic HT-29 cells. The involvement of [Ca2+]i as a messenger for the purinergically stimulated Cl- secretion was investigated using whole cell patch-clamp and Ussing chamber techniques, as well as [Ca2+]i measurements using fura 2-loaded cells. Under voltage-clamp conditions, the whole cell current at +50 mV was 3 +/- 1 pA/pF under unstimulated conditions. Stimulation of purinergic receptors with 200 microM extracellular ATP increased the current at +50 mV to 41 +/- 10 pA/pF, with a half-maximal effective dose (ED50) of approximately 3 microM. The current was transient, usually lasting 1-2 min, and the current-voltage relationship was approximately linear between -70 and +50 mV. Evidence that the ATP-stimulated current was carried by Cl- included 1) the reversal potential of the current closely followed the Cl- equilibrium potential, and 2) the stimulated current was absent when Cl- was removed from both bath and pipette solutions. Exposure to ATP also increased [Ca2+]i, with an ED50 of approximately 1 microM and maximal changes (at 200 microM) from baseline (71 +/- 3 nM) to 459 +/- 50 nM. The ATP-dependent Cl- conductance increase was not diminished when [Ca2+]i was clamped at 100 nM using a Ca(2+)-1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Ca(2+)-ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid buffering system. However, the ATP effect did require some basal level of Ca2+ because clamping [Ca2+]i at < 10 nM abolished activation of the Cl- conductance. The presence of the protein kinase A inhibitor H-89 or the protein kinase C inhibitor staurosprine did not change the ATP-activated Cl-conductance. These data demonstrate that the ATP-stimulated increase in Cl- current does not require an increase in [Ca2+]i, suggesting the involvement of either another signaling pathway or direct activation of Cl- channels by purinergic receptors.
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Ishikawa, Taisuke, Hiroki Kimoto, Hiroyuki Mishima, Kenichiro Yamagata, Soshiro Ogata, Yoshiyasu Aizawa, Kenshi Hayashi, et al. "Functionally validated SCN5A variants allow interpretation of pathogenicity and prediction of lethal events in Brugada syndrome." European Heart Journal 42, no. 29 (June 5, 2021): 2854–63. http://dx.doi.org/10.1093/eurheartj/ehab254.
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Abstract Aims The prognostic value of genetic variants for predicting lethal arrhythmic events (LAEs) in Brugada syndrome (BrS) remains controversial. We investigated whether the functional curation of SCN5A variations improves prognostic predictability. Methods and results Using a heterologous expression system and whole-cell patch clamping, we functionally characterized 22 variants of unknown significance (VUSs) among 55 SCN5A mutations previously curated using in silico prediction algorithms in the Japanese BrS registry (n = 415). According to the loss-of-function (LOF) properties, SCN5A mutation carriers (n = 60) were divided into two groups: LOF-SCN5A mutations and non-LOF SCN5A variations. Functionally proven LOF-SCN5A mutation carriers (n = 45) showed significantly severer electrocardiographic conduction abnormalities and worse prognosis associated with earlier manifestations of LAEs (7.9%/year) than in silico algorithm-predicted SCN5A carriers (5.1%/year) or all BrS probands (2.5%/year). Notably, non-LOF SCN5A variation carriers (n = 15) exhibited no LAEs during the follow-up period. Multivariate analysis demonstrated that only LOF-SCN5A mutations and a history of aborted cardiac arrest were significant predictors of LAEs. Gene-based association studies using whole-exome sequencing data on another independent SCN5A mutation-negative BrS cohort (n = 288) showed no significant enrichment of rare variants in 16 985 genes including 22 non-SCN5A BrS-associated genes as compared with controls (n = 372). Furthermore, rare variations of non-SCN5A BrS-associated genes did not affect LAE-free survival curves. Conclusion In vitro functional validation is key to classifying the pathogenicity of SCN5A VUSs and for risk stratification of genetic predictors of LAEs. Functionally proven LOF-SCN5A mutations are genetic burdens of sudden death in BrS, but evidence for other BrS-associated genes is elusive.
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Miyoshi, Hiroshi, Kaoru Yamaoka, Satoshi Urabe, Miho Kodama, and Yoshiki Kudo. "Functional expression of purinergic P2X7 receptors in pregnant rat myometrium." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 298, no. 4 (April 2010): R1117—R1124. http://dx.doi.org/10.1152/ajpregu.00507.2009.
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ATP has been reported to enhance the membrane conductance of myometrial cells and uterine contractility. Purinergic P2 receptor expression has been reported in the myometrium, using molecular biology, but the functional identity of the receptor subtype has not been determined. In this study, ATP-induced currents were recorded and characterized in single myometrial cells from pregnant rats using whole cell patch clamping. Extracellular ATP was applied in the range of 10 μM-1 mM and induced currents with an EC50 of 74 μM, with no desensitization, time dependency, or voltage dependency. The currents induced carried multiple monovalent cations, with conductances ranked as K+ > Cs+ > Li+ > Na+. They were activated by P2X receptor agonists, with their effectiveness ranked as 2′,3′- O-(4-benzoylbenzoyl)-ATP >> ATP > αβ-methylene-ATP > 2-methylthio ATP ≥ UTP ≥ GTP > ADP. These currents were blocked by the selective P2X7 receptor antagonist 3-[5-(2,3-dichlorophenyl)-1 H-tetrazol-1-yl]methyl pyridine (A-438079). We therefore concluded that ATP-induced currents in rat myometrial cells crossed cell membranes via P2X7 receptors. We further showed that the ATP-induced currents were blocked by extracellular Mg2+ (IC50 = 0.26 mM). Clinically, administering extracellular Mg2+ is known to inhibit uterine contraction. It therefore seems likely that uterine contraction may be induced by raised extracellular ATP and suppressed via Mg2+ inhibiting P2X7 receptors. Further research is needed into the P2X7 receptor as a therapeutic target in abnormal uterine contraction, as a possible treatment for premature labor.
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Cetin, Hakan, Richard Webster, Wei Wei Liu, Akiko Nagaishi, Inga Koneczny, Fritz Zimprich, Susan Maxwell, Judith Cossins, David Beeson, and Angela Vincent. "Myasthenia gravis AChR antibodies inhibit function of rapsyn-clustered AChRs." Journal of Neurology, Neurosurgery & Psychiatry 91, no. 5 (March 12, 2020): 526–32. http://dx.doi.org/10.1136/jnnp-2019-322640.
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ObjectiveDirect inhibition of acetylcholine receptor (AChR) function by autoantibodies (Abs) is considered a rare pathogenic mechanism in myasthenia gravis (MG), but is usually studied on AChRs expressed in cell lines, rather than tightly clustered by the intracellular scaffolding protein, rapsyn, as at the intact neuromuscular junction. We hypothesised that clustered AChRs would provide a better target for investigating the functional effects of AChR-Abs.MethodsAcetylcholine-induced currents were measured using whole-cell patch clamping and a fast perfusion system to assess fast (<2 min) functional effects of the serum samples. The sensitivity, specificity and rapidity of the system were first demonstrated by applying maternal AChR-Ab positive plasmas known to inhibit fetal AChR function in TE671 cells. Eleven previously untested AChR-Ab positive MG sera, 10 AChR-Ab negative MG sera and 5 healthy control sera were then applied to unclustered and rapsyn-clustered human adult AChRs in CN21 cells.ResultsThe maternal AChR-Ab positive plasmas reduced fetal AChR currents, but not adult AChR currents, by >80% within 100 s. Only 2/11 AChR-Ab positive sera inhibited AChR currents in unclustered AChRs, but 6/11 AChR-Ab positive sera compared with none of the 10 AChR-Ab negative sera (p=0.0020) inhibited rapsyn-clustered AChR currents, and current inhibition by the AChR-Ab positive sera was greater when the AChRs were clustered (p=0.0385). None of the sera had detectable effects on desensitisation or recovery from desensitisation.ConclusionThese results show that antibodies can inhibit AChR function rapidly and demonstrate the importance of clustering in exploring pathogenic disease mechanisms of MG Abs.
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Ma, Yi, Lin Zhang, Bradley S. Launikonis, and Chen Chen. "Growth Hormone Secretagogues Preserve the Electrophysiological Properties of Mouse Cardiomyocytes Isolated from in Vitro Ischemia/Reperfusion Heart." Endocrinology 153, no. 11 (November 1, 2012): 5480–90. http://dx.doi.org/10.1210/en.2012-1404.
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Abstract Ischemic heart diseases often induce cardiac arrhythmia with irregular cardiac action potential (AP). This study aims to demonstrate that GH secretagogues (GHS) ghrelin and its synthetic analog hexarelin can preserve the electrophysiological properties of cardiomyocytes experiencing ischemia/reperfusion (I/R). Isolated hearts from adult male mice underwent 20 min global ischemia followed by 30 min reperfusion using a Langendorff apparatus. Ghrelin (10 nm) or hexarelin (1 nm) was administered in the perfusion solution either 10 min before or after ischemia, termed pre- or posttreatments. Cardiomyocytes isolated from these hearts were used for whole-cell patch clamping to measure AP, voltage-gated L-type calcium current (ICaL), transient outward potassium current (Ito), and sodium current (INa). AP amplitude and duration were significantly decreased by I/R, but GHS treatments maintained their normality. GHS treatments prevented the decrease in ICaL and INa after I/R, thereby maintaining AP amplitude. Although the significant increase in Ito after I/R partially explained the shortened AP duration, the normalization of it by GHS treatments might contribute to the preservation of AP duration. Phosphorylated p38 and c-Jun NH2-terminal kinase and the downstream active caspase-9 in the cellular apoptosis pathway were significantly increased after I/R but not when GHS treatments were included, whereas phosphorylation of ERK1/2 associated with cell survival showed increase after I/R and a further increase after GHS treatments by binding to its receptor GHS receptor type 1a. These results suggest GHS can not only preserve the electrophysiological properties of cardiomyocytes after I/R but also inhibit cardiomyocyte apoptosis and promote cell survival by modification of MAPK pathways through activating GHS receptor type 1a.
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Rotordam, Maria Giustina, Elisa Fermo, Nadine Becker, Wilma Barcellini, Andrea Brüggemann, Niels Fertig, Stéphane Egée, Markus Rapedius, Paola Bianchi, and Lars Kaestner. "A Yoda1-Based Approach to Investigate Piezo1 Channels in Red Blood Cells Using Automated Patch Clamp Technology." Blood 132, Supplement 1 (November 29, 2018): 1031. http://dx.doi.org/10.1182/blood-2018-99-118043.
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Abstract Piezo1 is a mechanosensitive ion channel supposed to regulate the volume and maintain the structural integrity in Red Blood Cells (RBCs), as gain-of-function mutations in this channel are associated to the RBC disease Hereditary Xerocytosis (Zarychanski et al. Blood 2012; Bae et al. Proceedings of the National Academy of Sciences 2013). Piezo1 is activated by several mechanical forces, including stretching, poking and shear stress and allows Ca2+ and other cations to enter the cell generating an electrical response. In 2015, it has been discovered that Piezo1 is sensitive to a small molecule, Yoda1 (Syeda et al. Elife 2015), which keeps the channel open and affects its inactivation kinetics. This finding has created new possibilities to elucidate Piezo1 gating mechanism and explore its functional significance in physiological and pathophysiological conditions. Here, we present a patient with a novel PIEZO1 mutation (R2110W) and a patch clamp-based high-throughput screening assay for Piezo1 activity. We established a protocol to detect functional Piezo1 mutations upon chemical stimulation by Yoda1, yet were not able to stimulate the channel via mechanical force, i.e. pressure steps and shear-stress. The assay was first developed on Neuro2A (N2A), a neuroblastoma cell-line endogenously expressing Piezo1 channels (kindly provided by Max-Delbrück Center, Berlin), due to larger abundance of Piezo1 channels in these cells. Initial experiments were performed on the Patchliner (Nanion Technologies GmbH, Munich), a medium-throughput automated patch clamp system able to record up to 8 cells at a time. Currents were elicited using a voltage ramp ranging from -100 to +80 mV for 300 ms, the holding potential was set to -60 mV. A significantly increased whole-cell current was observed upon 10 µM Yoda1 application in half of the recorded cells and the resulting Yoda1-induced currents were inhibited by 30 µM gadolinium chloride, a non-specific blocker of stretch-activated channels. The assay was then implemented on the SyncroPatch 384PE (Nanion Technologies GmbH, Munich), capable of recording up to 384 cells in parallel under identical experimental conditions, thus allowing for reliable statistical analysis. Yoda1 responding cells were selected based on strict quality control (QC) criteria, i.e. the seal resistance stability over time. In one example NPC-384 chip 140 out of 384 N2A cells (37%) passed the QC criteria and 85 cells (60% of the valid cells) were considered as Yoda1 responders. Finally, we investigated Piezo1 electrophysiological properties in healthy and patient RBCs carrying the novel PIEZO1 R2110W mutation. Similar to N2A cells, RBCs currents were analyzed and divided into Yoda1 responders and non-responders according to our QC criteria. The increase in whole-cell currents induced by Yoda1 application was significantly higher in patient compared to control RBCs, which was also reflected by a higher number of Yoda1 responders compared to control. Residue R2110W is structurally located in a gating sensitive area of the channel protein suggesting a gain-of-function. This would be in line with previously described mutations in PIEZO1 (Albuisson et al. Nature Communications 2013) and the mild form of anaemia observed in the patient. Furthermore, we excluded any involvement of Gardos channels in the Yoda1-induced currents by comparing measurements in the presence and absence of the specific Gardos channel inhibitor TRAM-34. Altogether, our work demonstrates that high-throughput patch clamping can provide a robust assay to study functional Piezo1 impairments in primary RBCs without expressing the mutated channel protein in a heterologous expression system. Our approach may be used to detect other channelopathies not only in RBCs and may serve as routine screening assay for diseases related to ion channel dysfunctions in general, complementary to gene sequencing. Disclosures No relevant conflicts of interest to declare.