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Patch Clamp (patch + clamp)

Distribution by Scientific Domains
Life Sciences80%

Terms modified by Patch Clamp

  • patch clamp recording
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  • patch clamp technique
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    Selected Abstracts

    Protein kinase A modulates A-type potassium currents of larval zebrafish (Danio rerio) white muscle fibres

    ACTA PHYSIOLOGICA, Issue 2 2009
    C. A. Coutts
    Abstract Aims:, Potassium (K+) channels are involved in regulating cell excitability and action potential shape. To our knowledge, very little is known about the modulation of A-type K+ currents in skeletal muscle fibres. Therefore, we sought to determine whether K+ currents of zebrafish white skeletal muscle were modulated by protein kinase A (PKA). Methods:, Pharmacology and whole-cell patch clamp were used to examine A-type K+ currents and action potentials associated with zebrafish white skeletal muscle fibres. Results:, Activation of PKA by a combination of forskolin + 3-isobutyl-1-methylxanthine (Fsk + IBMX) decreased the peak current density by ,60% and altered the inactivation kinetics of A-type K+ currents. The specific PKA inhibitor H-89 partially blocked the Fsk + IBMX-induced reduction in peak current density, but had no effect on the change in decay kinetics. Fsk + IBMX treatment did not shift the activation curve, but it significantly reduced the slope factor of activation. Activation of PKA by Fsk + IBMX resulted in a negative shift in the V50 of inactivation. H-89 prevented all Fsk + IBMX-induced changes in the steady-state properties of K+ currents. Application of Fsk + IBMX increased action potential amplitude, but had no significant effect on action potential threshold, half width or recovery rate, when fibres were depolarized with single pulses, paired pulses or with high-frequency stimuli. Conclusion:, PKA modulates the A-type K+ current in zebrafish skeletal muscle and affects action potential properties. Our results provide new insights into the role of A-type K+ channels in muscle physiology. [source]

    Calcium and Fos Involvement in Brain-Derived Ca2+ -Binding Protein (S100)-Dependent Apoptosis in Rat Phaeochromocytoma Cells

    EXPERIMENTAL PHYSIOLOGY, Issue 3 2000
    Stefania Fulle
    Brain-derived calcium-binding protein S100 induces apoptosis in a significant fraction of rat phaeochromocytoma (PC12) cells. We used single cell techniques (patch clamp, videomicroscopy and immunocytochemistry) to clarify some of the specific aspects of S100-induced apoptosis, the modality(ies) of early intracellular Ca2+ concentration increase and the expression of some classes of genes (c-fos, c-jun, bax, bcl-x, p-15, p-21) known to be implicated in apoptosis of different cells. The results show that S100: (1) causes an increase of [Ca2+]i due to an increased conductance of L-type Ca2+ channels; (2) induces a sustained increase of the Fos levels which is evident since the first time point tested (3 h) and remains elevated until to the last time point (72 h). All these data suggest that S100-derived apoptosis in PC12 cells may be the consequence of a system involving an increase in L-type Ca2+ channel conductance with consequent [Ca2+]i increase which up-regulates, directly or indirectly, the expression of Fos. [source]

    Chlorotoxin-sensitive Ca2+ -activated Cl, channel in type R2 reactive astrocytes from adult rat brain

    GLIA, Issue 4 2003
    Stanislava Dalton
    Abstract Astrocytes express four types of Cl, or anion channels, but Ca2+ -activated Cl, (ClCa) channels have not been described. We studied Cl, channels in a morphologically distinct subpopulation (, 5% of cells) of small (10,12 ,m, 11.8 ± 0.6 pF), phase-dark, GFAP-positive native reactive astrocytes (NRAs) freshly isolated from injured adult rat brains. Their resting potential, ,57.1 ± 4.0 mV, polarized to ,72.7 ± 4.5 mV with BAPTA-AM, an intracellular Ca2+ chelator, and depolarized to ,30.7 ± 6.1 mV with thapsigargin, which mobilizes Ca2+ from intracellular stores. With nystatin-perforated patch clamp, thapsigargin activated a current that reversed near the Cl, reversal potential, which was blocked by Cl, channel blockers, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) and Zn2+, by I, (10 mM), and by chlorotoxin (EC50 = 47 nM). With conventional whole-cell clamp, NPPB- and Zn2+ -sensitive currents became larger with increasing [Ca2+]i (10, 150, 300 nM). Single-channel recordings of inside-out patches confirmed Ca2+ sensitivity of the channel and showed open-state conductances of 40, 80, 130, and 180 pS, and outside-out patches confirmed sensitivity to chlorotoxin. In primary culture, small phase-dark NRAs developed into small GFAP-positive bipolar cells with chlorotoxin-sensitive ClCa channels. Imaging with biotinylated chlorotoxin confirmed the presence of label in GFAP-positive cells from regions of brain injury, but not from uninjured brain. Chlorotoxin-tagged cells isolated by flow cytometry and cultured up to two passages exhibit positive labeling for GFAP and vimentin, but not for prolyl 4-hydroxylase (fibroblast), A2B5 (O2A progenitor), or OX-42 (microglia). Expression of a novel chlorotoxin-sensitive ClCa channel in a morphologically distinct subpopulation of NRAs distinguishes these cells as a new subtype of reactive astrocyte. GLIA 42:325,339, 2003. © 2003 Wiley-Liss, Inc. [source]

    Dependence of axon initial segment formation on Na+ channel expression

    JOURNAL OF NEUROSCIENCE RESEARCH, Issue 4 2005
    Xiaorong Xu
    Abstract Spinal motor neurons were isolated from embryonic rats, and grown in culture. By 2 days in vitro, the axon initial segment was characterized by colocalization and clustering of Na+ channels and ankyrinG. By 5 days, NrCAM, and neurofascin could also be detected at most initial segments. We sought to determine, as one important aim, whether Na+ channels themselves played an essential role in establishing this specialized axonal region. Small hairpin RNAs (shRNAs) were used to target multiple subtypes of Na+ channels for reduced expression by RNA interference. Transfection resulted in substantial knockdown of these channels within the cell body and also as clusters at initial segments. Furthermore, Na+ currents originating at the initial segment, and recorded under patch clamp, were strongly reduced by shRNA. Control shRNA against a nonmammalian protein was without effect. Most interestingly, targeting Na+ channels also blocked clustering of ankyrinG, NrCAM, and neurofascin at the initial segment, although these proteins were seen in the soma. Thus, both Na+ channels and ankyrinG are required for formation of this essential axonal domain. Knockdown of Na+ channels was somewhat less effective when introduced after the initial segments had formed. Disruption of actin polymerization by cytochalasin D resulted in multiple initial segments, each with clusters of both Na+ channels and ankyrinG. The results indicate that initial segment formation occurs as Na+ channels are transported into the nascent axon membrane, diffuse distally, and link to the cytoskeleton by ankyrinG. Subsequently, other components are added, and stability is increased. A computational model closely reproduced the experimental results. © 2005 Wiley-Liss, Inc. [source]

    Endothelin-1 Modulates the Arrhythmogenic Activity of Pulmonary Veins

    JOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 3 2008
    AMEYA R. UDYAVAR M.D.
    Objective: Endothelin-1 has important cardiovascular effects and is activated during atrial fibrillation. Pulmonary veins (PVs) play a critical role in the pathophysiology of atrial fibrillation. The aim of this study was to evaluate whether endothelin-1 affects PV arrhythmogenic activity. Methods: Conventional microelectrodes were used to record the action potentials (APs) and contractility in isolated rabbit PV tissue specimens before and after the administration of endothelin-1 (0.1, 1, 10 nM). The ionic currents of isolated PV cardiomyocytes were investigated before and after the administration of endothelin-1 (10 nM) through whole-cell patch clamps. Results: In the tissue preparation, endothelin-1 (1, 10 nM) concentration dependently shortened the AP duration and decreased the PV firing rates. Endothelin-1 (10 nM) decreased the resting membrane potential. Endothelin-1 (0.1, 1, 10 nM) decreased the contractility and increased the resting diastolic tension. In single PV cardiomyocytes, endothelin-1 (10 nM) decreased the PV firing rates from 2.7 ± 1.0 Hz to 0.8 ± 0.5 Hz (n = 16). BQ-485 (100 ,M, endothelin-1 type A receptor blocker) reversed and prevented the chrono-inhibitory effects of endothelin-1 (10 nM). Endothelin-1 (10 nM) reduced the L-type calcium currents, transient outward currents, delayed rectifier currents, transient inward currents, and sodium,calcium exchanger currents in the PV cardiomyocytes with and without pacemaker activity. Endothelin-1 (10 nM) increased the inward rectifier potassium current, hyperpolarization-induced pacemaker current, and the sustained outward potassium current in PV cardiomyocytes with and without pacemaker activity. Conclusion: Endothelin-1 may have an antiarrhythmic potential through its direct electrophysiological effects on the PV cardiomyocytes and its action on multiple ionic currents. [source]