scholarly journals Characterization of Voltage-Dependent Ca2+ Currents in Identified Drosophila Motoneurons In Situ

2008 ◽  
Vol 100 (2) ◽  
pp. 868-878 ◽  
Author(s):  
Jason W. Worrell ◽  
Richard B. Levine
Keyword(s):  
Neurotransmitter Release ◽  
Cell Voltage ◽  
Drosophila Genome ◽  
Whole Cell ◽  
Spike Shape ◽  
Voltage Dependent ◽  
Targeted Expression ◽  
Firing Properties

Voltage-dependent Ca2+ channels contribute to neurotransmitter release, integration of synaptic information, and gene regulation within neurons. Thus understanding where diverse Ca2+ channels are expressed is an important step toward understanding neuronal function within a network. Drosophila provides a useful model for exploring the function of voltage-dependent Ca2+ channels in an intact system, but Ca2+ currents within the central processes of Drosophila neurons in situ have not been well described. The aim of this study was to characterize voltage-dependent Ca2+ currents in situ from identified larval motoneurons. Whole cell recordings from the somata of identified motoneurons revealed a significant influence of extracellular Ca2+ on spike shape and firing rate. Using whole cell voltage clamp, along with blockers of Na+ and K+ channels, a Ca2+-dependent inward current was isolated. The Drosophila genome contains three genes with homology to vertebrate voltage-dependent Ca2+ channels: Dmca1A, Dmca1D, and Dmα1G. We used mutants of Dmca1A and Dmca1D as well as targeted expression of an RNAi transgene to Dmca1D to determine the genes responsible for the voltage-dependent Ca2+ current recorded from two identified motoneurons. Our results implicate Dmca1D as the major contributor to the voltage-dependent Ca2+ current recorded from the somatodendritic processes of motoneurons, whereas Dmca1A has previously been localized to the presynaptic terminal where it is essential for neurotransmitter release. Altered firing properties in cells from both Dmca1D and Dmca1A mutants indicate a role for both genes in shaping firing properties.

Protein kinase C stimulates Ca2+ current in pregnant rat myometrial cells

1994 ◽  
Vol 72 (11) ◽  
pp. 1304-1307 ◽  
Author(s):  
Keiichi Shimamura ◽  
Masumi Kusaka ◽  
Nicholas Sperelakis
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Pregnant Rat ◽  
Whole Cell ◽  
Myometrial Cells ◽  
Kinase C ◽  
Whole Cell Voltage Clamp ◽  
Voltage Dependent

The factors that regulate the voltage-dependent Ca2+ channels in pregnant uterine smooth muscle cells have not been elucidated, including any roles for protein kinase C (PKC). Therefore, the role of PKC in the regulation of the slow (L type) Ca2+ channels was examined in myometrial cells isolated from late pregnant (18–19 day) rat uterus, using the nystatin-perforated whole-cell voltage clamp. A PKC activator, phorbol 12, 13-dibutyrate (PDB), increased the L-type Ca2+ current (ICa(L)). Bath application of PDB (0.03 and 0.3 μM) increased the peak amplitude of ICa(L) by 21 ± 14% (n = 6) and 37 ± 8% (n = 9, p < 0.01), respectively. PDB did not change the holding current or shift the current–voltage relationship for ICa(L). The PKC inhibitors, H-7 (20 μM) or staurosporine (10 nM), reversed the effect of PDB. These results indicate that PKC may play a role in regulating Ca2+ channel function in pregnant rat myometrial cells and, therefore, may be involved in control of uterine contraction.Key words: protein kinase C, phorbol ester, calcium current, myometrial cell, nystatin-perforated patch, whole-cell voltage clamp.

Oxytocin induces an inward current in pregnant rat myometrial cells

1994 ◽  
Vol 72 (7) ◽  
pp. 759-763 ◽  
Author(s):  
Keiichi Shimamura ◽  
Masumi Kusaka ◽  
Nicholas Sperelakis
Keyword(s):  
Smooth Muscle ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Induced Current ◽  
Pregnant Rat ◽  
Whole Cell ◽  
Inward Current ◽  
Uterine Smooth Muscle ◽  
Whole Cell Voltage Clamp ◽  
Voltage Dependent

The effects of oxytocin (OT) on holding current were studied in uterine smooth muscle cells freshly isolated from the longitudinal layer of 18–20 day pregnant rats, using the nystatin method of whole-cell voltage clamp. As we previously reported, the voltage-dependent Ca2+ current (L type) was partially inhibited by OT (about 30% inhibition at 1 μM). When the cells were held at the holding potential (HP) of −60 mV and the holding current was monitored, OT induced an inward current. The amplitude of this OT-induced current was 72 ± 26 pA (n = 27). When the cell was held at more positive potentials (HP 0 or +40 mV), the OT-induced current reversed direction, becoming outward. This current usually was long lasting (74% of cells responding to OT); a transient current was observed in 26% of the cells. In the absence of either Na+ or Ca2+ in the bath solution, OT induced an inward current (at HP −60 mV). However, the OT-induced current was absent when both of these ions were omitted from the bath. These results suggest that OT induces an inward current through receptor-activated nonselective cation channels. The resulting increase of intracellular Ca2+ may contribute to the inhibition of voltage-dependent Ca2+ current produced by OT. This OT-induced current may also play an important role for membrane depolarization and accompanying contraction produced by OT in pregnant rat myometrium.Key words: oxytocin receptor activated channel, uterine smooth muscle, pregnant rat myometrium, holding current, whole-cell voltage clamp.

scholarly journals Identification and Modulation of Voltage-Gated Ca2+ Currents in Zebrafish Rohon-Beard Neurons

2011 ◽  
Vol 105 (1) ◽  
pp. 442-453 ◽  
Author(s):  
Yu-Jin Won ◽  
Fumihito Ono ◽  
Stephen R. Ikeda
Keyword(s):  
Neuronal Excitability ◽  
Fluorescent Protein ◽  
Low Voltage ◽  
Cell Voltage ◽  
Transgenic Zebrafish ◽  
Opioid Agonist ◽  
Zebrafish Model ◽  
Whole Cell ◽  
Voltage Gated ◽  
Voltage Dependent

Electrically excitable cells have voltage-dependent ion channels on the plasma membrane that regulate membrane permeability to specific ions. Voltage-gated Ca2+ channels (VGCCs) are especially important as Ca2+ serves as both a charge carrier and second messenger. Zebrafish ( Danio rerio) are an important model vertebrate for studies of neuronal excitability, circuits, and behavior. However, electrophysiological properties of zebrafish VGCCs remain largely unexplored because a suitable preparation for whole cell voltage-clamp studies is lacking. Rohon-Beard (R-B) sensory neurons represent an attractive candidate for this purpose because of their relatively large somata and functional homology to mammalian dorsal root ganglia (DRG) neurons. Transgenic zebrafish expressing green fluorescent protein in R-B neurons, ( Isl2b:EGFP)ZC7, were used to identify dissociated neurons suitable for whole cell patch-clamp experiments. Based on biophysical and pharmacological properties, zebrafish R-B neurons express both high- and low-voltage-gated Ca2+ current (HVA- and LVA- ICa, respectively). Ni+-sensitive LVA- ICa occur in the minority of R-B neurons (30%) and ω-conotoxin GVIA-sensitive CaV2.2 (N-type) Ca2+ channels underlie the vast majority (90%) of HVA- ICa. To identify G protein coupled receptors (GPCRs) that modulate HVA- ICa, a panel of neurotransmitters was screened. Application of GABA/baclofen or serotonin produced a voltage-dependent inhibition while application of the mu-opioid agonist DAMGO resulted in a voltage-independent inhibition. Unlike in mammalian neurons, GPCR-mediated voltage-dependent modulation of ICa appears to be transduced primarily via a cholera toxin-sensitive Gα subunit. These results provide the basis for using the zebrafish model system to understanding Ca2+ channel function, and in turn, how Ca2+ channels contribute to mechanosensory function.

A model of the action potential and underlying membrane currents in a rabbit atrial cell

1996 ◽  
Vol 271 (4) ◽  
pp. H1666-H1696 ◽  
Author(s):  
D. S. Lindblad ◽  
C. R. Murphey ◽  
J. W. Clark ◽  
W. R. Giles
Keyword(s):  
Action Potential ◽  
Voltage Clamp ◽  
Cell Voltage ◽  
Ionic Currents ◽  
Membrane Currents ◽  
Whole Cell ◽  
Whole Cell Voltage Clamp ◽  
Voltage Dependent ◽  
Atrial Myocyte ◽  
Atrial Cell

We have developed a mathematical model of the rabbit atrial myocyte and have used it in an examination of the ionic basis of the atrial action potential. Available biophysical data have been incorporated into the model to quantify the specific ultrastructural morphology, intracellular ion buffering, and time- and voltage-dependent currents and transport mechanisms of the rabbit atrial cell. When possible, mathematical expressions describing ionic currents identified in rabbit atrium are based on whole cell voltage-clamp data from enzymatically isolated rabbit atrial myocytes. This membrane model is coupled to equations describing Na+, K+, and Ca2+ homeostasis, including the uptake and release of Ca2+ by the sarcoplasmic reticulum and Ca2+ buffering. The resulting formulation can accurately simulate the whole cell voltage-clamp data on which it is based and provides fits to a family of rabbit atrial cell action potentials obtained at 35 degrees C over a range of stimulus rates (0.2–3.0 Hz). The model is utilized to provide a qualitative prediction of the intracellular Ca2+ concentration transient during the action potential and to illustrate the interactions between membrane currents that underlie repolarization in the rabbit atrial myocyte.

scholarly journals Synaptotagmins are trafficked to distinct subcellular domains including the postsynaptic compartment

2004 ◽  
Vol 166 (2) ◽  
pp. 249-260 ◽  
Author(s):  
Bill Adolfsen ◽  
Sudipta Saraswati ◽  
Motojiro Yoshihara ◽  
J. Troy Littleton
Keyword(s):  
Membrane Trafficking ◽  
Neurotransmitter Release ◽  
Neurosecretory Cells ◽  
Drosophila Genome ◽  
Calcium Dependent ◽  
Synaptotagmin 1 ◽  
Subcellular Domains ◽  
Low Levels ◽  
Synaptic Vesicle Fusion

The synaptotagmin family has been implicated in calcium-dependent neurotransmitter release, although Synaptotagmin 1 is the only isoform demonstrated to control synaptic vesicle fusion. Here, we report the characterization of the six remaining synaptotagmin isoforms encoded in the Drosophila genome, including homologues of mammalian Synaptotagmins 4, 7, 12, and 14. Like Synaptotagmin 1, Synaptotagmin 4 is ubiquitously present at synapses, but localizes to the postsynaptic compartment. The remaining isoforms were not found at synapses (Synaptotagmin 7), expressed at very low levels (Synaptotagmins 12 and 14), or in subsets of putative neurosecretory cells (Synaptotagmins α and β). Consistent with their distinct localizations, overexpression of Synaptotagmin 4 or 7 cannot functionally substitute for the loss of Synaptotagmin 1 in synaptic transmission. Our results indicate that synaptotagmins are differentially distributed to unique subcellular compartments. In addition, the identification of a postsynaptic synaptotagmin suggests calcium-dependent membrane-trafficking functions on both sides of the synapse.

scholarly journals Characterization of ion channels and O2 sensitivity in gill neuroepithelial cells of the anoxia-tolerant goldfish (Carassius auratus)

2017 ◽  
Vol 118 (6) ◽  
pp. 3014-3023 ◽  
Author(s):  
Peter C. Zachar ◽  
Wen Pan ◽  
Michael G. Jonz
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Synaptic Vesicle ◽  
Resting Membrane Potential ◽  
Patch Clamp Recording ◽  
Whole Cell ◽  
Neuroepithelial Cells ◽  
Voltage Dependent ◽  
Intracellular Ca

The neuroepithelial cell (NEC) of the fish gill is an important model for O2 sensing in vertebrates; however, a complete picture of the chemosensory mechanisms in NECs is lacking, and O2 chemoreception in vertebrates that are tolerant to anoxia has not yet been explored. Using whole cell patch-clamp recording, we characterized four types of ion channels in NECs isolated from the anoxia-tolerant goldfish. A Ca2+-dependent K+ current ( IKCa) peaked at ~20 mV, was potentiated by increased intracellular Ca2+, and was reduced by 100 μM Cd2+. A voltage-dependent inward current in Ba2+ solution, with peak at 0 mV, confirmed the presence of Ca2+ channels. A voltage-dependent K+ current ( IKV) was inhibited by 20 mM tetraethylammonium and 5 mM 4-aminopyridine, revealing a background K+ current ( IKB) with open rectification. Mean resting membrane potential of −45.2 ± 11.6 mV did not change upon administration of hypoxia (Po2 = 11 mmHg), nor were any of the K+ currents sensitive to changes in Po2 during whole cell recording. By contrast, when the membrane and cytosol were left undisturbed during fura-2 or FM 1-43 imaging experiments, hypoxia increased intracellular Ca2+ concentration and initiated synaptic vesicle activity. 100 μM Cd2+ and 50 μM nifedipine eliminated uptake of FM 1-43. We conclude that Ca2+ influx via L-type Ca2+ channels is correlated with vesicular activity during hypoxic stimulation. In addition, we suggest that expression of IKCa in gill NECs is species specific and, in goldfish, may contribute to an attenuated response to acute hypoxia. NEW & NOTEWORTHY This study provides the first physiological characterization of oxygen chemoreceptors from an anoxia-tolerant vertebrate. Neuroepithelial cells (NECs) from the gills of goldfish displayed L-type Ca2+ channels and three types of K+ channels, one of which was dependent upon intracellular Ca2+. Although membrane currents were not inhibited by hypoxia during patch-clamp recording, this study is the first to show that NECs with an undisturbed cytosol responded to hypoxia with increased intracellular Ca2+ and synaptic vesicle activity.

scholarly journals Computer modeling of whole-cell voltage-clamp analyses to delineate guidelines for good practice of manual and automated patch-clamp

2020 ◽  
Author(s):  
Jérôme Montnach ◽  
Maxime Lorenzini ◽  
Adrien Lesage ◽  
Isabelle Simon ◽  
Sébastien Nicolas ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Voltage Clamp ◽  
Good Practice ◽  
Cell Voltage ◽  
Current Amplitude ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Clamp Technique ◽  
Whole Cell Voltage Clamp

ABSTRACTThe patch-clamp technique has contributed to major advances in the characterization of ion channels. The recent development of high throughput patch-clamp provides a new momentum to the field. However, whole-cell voltage-clamp technique presents certain limits that need to be considered for robust data generation. One major caveat is that current amplitude profoundly impacts the precision of the analyzed characteristics of the ion current under study. For voltagegated channels, the higher the current amplitude is, the less precise the characteristics of voltagedependence are. Similarly, in ion channel pharmacology, the characteristics of dose-response curves are hindered by high current amplitudes. In addition, the recent development of high throughput patch-clamp technique is often associated with the generation of stable cell lines demonstrating high current amplitudes. It is therefore critical to set the limits for current amplitude recordings to avoid inaccuracy in the characterization of channel properties or drug actions, such limits being different from one channel to another. In the present study, we use kinetic models of a voltage-gated sodium channel and a voltage-gated potassium channel to edict simple guidelines for good practice of whole-cell voltage-clamp recordings.

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