scholarly journals Cyclic GMP–gated Channels in a Sympathetic Neuron Cell Line

1997 ◽  
Vol 110 (2) ◽  
pp. 155-164 ◽  
Author(s):  
Stuart H. Thompson
Keyword(s):  
Cell Line ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Neuroblastoma Cell ◽  
Sympathetic Neuron ◽  
Sympathetic Ganglia ◽  
Guanosine Monophosphate ◽  
Hill Coefficient ◽  
Muscarinic Agonists ◽  
Single Channel Currents

The stimulation of IP3 production by muscarinic agonists causes both intracellular Ca2+ release and activation of a voltage-independent cation current in differentiated N1E-115 cells, a neuroblastoma cell line derived from mouse sympathetic ganglia. Earlier work showed that the membrane current requires an increase in 3′,5′-cyclic guanosine monophosphate (cGMP) produced through the NO-synthase/guanylyl cyclase cascade and suggested that the cells may express cyclic nucleotide–gated ion channels. This was tested using patch clamp methods. The membrane permeable cGMP analogue, 8-br-cGMP, activates Na+ permeable channels in cell attached patches. Single channel currents were recorded in excised patches bathed in symmetrical Na+ solutions. cGMP-dependent single channel activity consists of prolonged bursts of rapid openings and closings that continue without desensitization. The rate of occurrence of bursts as well as the burst length increase with cGMP concentration. The unitary conductance in symmetrical 160 mM Na+ is 47 pS and is independent of voltage in the range −50 to +50 mV. There is no apparent effect of voltage on opening probability. The dose response curve relating cGMP concentration to channel opening probability is fit by the Hill equation assuming an apparent KD of 10 μm and a Hill coefficient of 2. In contrast, cAMP failed to activate the channel at concentrations as high as 100 μm. Cyclic nucleotide gated (CNG) channels in N1E-115 cells share a number of properties with CNG channels in sensory receptors. Their presence in neuronal cells provides a mechanism by which activation of the NO/cGMP pathway by G-protein–coupled neurotransmitter receptors can directly modify Ca2+ influx and electrical excitability. In N1E-115 cells, Ca2+ entry by this pathway is necessary to refill the IP3-sensitive intracellular Ca2+ pool during repeated stimulation and CNG channels may play a similar role in other neurons.

Divalent cations block the cyclic nucleotide-gated channel of olfactory receptor neurons

1993 ◽  
Vol 69 (5) ◽  
pp. 1758-1768 ◽  
Author(s):  
F. Zufall ◽  
S. Firestein
Keyword(s):  
Cyclic Amp ◽  
Calcium Channels ◽  
Cyclic Nucleotide ◽  
Olfactory Receptor ◽  
Single Channel ◽  
Divalent Cations ◽  
Olfactory Receptor Neurons ◽  
Gated Channel ◽  
Receptor Neurons ◽  
Single Channel Currents

1. The effects of external divalent cations on odor-dependent, cyclic AMP-activated single-channel currents from olfactory receptor neurons of the tiger salamander (Ambystoma tigrinum) were studied in inside-out membrane patches taken from dendritic regions of freshly isolated sensory cells. 2. Channels were reversibly activated by 100 microM cyclic AMP. In the absence of divalent cations, the channel had a linear current-voltage relation giving a conductance of 45 pS. With increasing concentrations of either Ca2+ or Mg2+ in the external solution, the channel displayed a rapid flickering behavior. At higher concentrations of divalent cations, the transitions were too rapid to be fully resolved and appeared as a reduction in mean unitary single-channel current amplitude. 3. This effect was voltage dependent, and on analysis was shown to be due to an open channel block by divalent ions. In the case of Mg2+, the block increased steadily with hyperpolarization. In contrast, for Ca2+ the block first increased with hyperpolarization and then decreased with further hyperpolarization beyond -70 mV, providing evidence for Ca2+ permeation of this channel. 4. This block is similar to that seen in voltage-gated calcium channels. Additionally, the cyclic nucleotide-gated channel shows some pharmacological similarities with L-type calcium channels, including a novel block of the cyclic nucleotide channel by nifedipine (50 microM). 5. Our results indicate that the sensory generator current simultaneously depends on the presence of the second messenger and on the membrane potential of the olfactory neuron.

scholarly journals Is Oxytocin Proper for Cancer Adjuvant Therapy?

Proceedings ◽  
2018 ◽  
Vol 2 (25) ◽  
pp. 1582
Author(s):  
Ali Taghizadehghalehjoughi ◽  
Betul Cicek ◽  
Ahmet Hacimuftuoglu ◽  
Mustaf Gul
Keyword(s):  
Cell Culture ◽  
Cell Viability ◽  
Cell Line ◽  
Culture Media ◽  
Neuroblastoma Cell ◽  
Sympathetic Ganglia ◽  
Control Group ◽  
Human Neuroblastoma ◽  
Lactation Stage ◽  
Cortex Cell

Neuroblastomas are solid tumors and mostly seen in the adrenal medulla and sympathetic ganglia. It is known that neuroblastoma cell proliferation is inhibited by cisplatin and vincristine. The aim of this study was to investigate the effect of oxytocin on cell viability in human neuroblastoma SH-SY5Y cell line and primary cerebral cortex cell culture exposed to cisplatin and vincristine. In this direction, SH-SY5Y cell line and cortex neurons were obtained from the medical pharmacology department, Ataturk University. Both cells were grown in the appropriate cell culture media. Cisplatin (5, 10, 15 μg), vincristine (0.5, 1 and 2 ng) and oxytocin (1 μM) were applied to SH-SY5Y cell line and primary cortex cell culture for 24 h. MTT and TAC-TOS tests were performed 24 h after the application. As a result of the MTT assay, the combination of cisplatin and vincristine reduced cell viability in both cultures approximately 25% and 22%, respectively, compared to the control group. It appears that oxytocin increases neuroblastoma and cortex neuron viability, 112% and 95%, respectively. In this relation, we need to investigate why oxytocin increases cell viability and what are the possible implications in women in lactation stage.

Modulation of ATP-sensitive K+ channels by internal acidification in insulin-secreting cells

1994 ◽  
Vol 267 (4) ◽  
pp. C1036-C1044 ◽  
Author(s):  
Z. Fan ◽  
Y. Tokuyama ◽  
J. C. Makielski
Keyword(s):  
Insulin Secretion ◽  
Cell Line ◽  
Single Channel ◽  
Inhibitory Effect ◽  
Channel Activity ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Activation Effect ◽  
Insulin Secreting Cells ◽  
Single Channel Currents

The effect of intracellular acidification (low pHi) on open probability of the ATP-sensitive K+ (KATP) channel was examined in insulin-secretion cells using an inside-out configuration of the patch-clamp technique. In an insulin-secreting cell line beta-TC3, KATP single-channel currents (IKATP) were readily recorded in the absence of internal ATP. ATP (50 microM and 0.5 mM) dramatically decreased the channel activity. A step decrease of intracellular pH (pHi) from 7.4 to 6.7 or 6.3 in the presence of ATP gradually increased the channel activity. In addition, low pHi in the presence of ATP could partially restore channel activity lost in a process called "rundown." Kinetic analysis revealed a change in channel gating at low pHi with ATP. The bursting durations of IKATP at pHi 6.3 in the presence of ATP were significantly longer than those at pHi 7.4 in the absence of ATP. These results suggest that the increased channel activity at low pHi might have resulted from a mechanism involving an alteration of channel conformation. We also observed an inhibitory effect of low pHi on channel activity. However, the inhibitory effect was much more apparent at pHi 5.7 and was only partially reversible. The activation effect of low pHi on IKATP in the presence of ATP was also observed in acutely isolated rat islet cells and in another insulin-secretion cell line RINm5F, although the effect was weaker and was variable among experiments. We conclude that, as in frog skeletal muscle and cardiac muscle, an increase in channel activity at low pHi is one of the mechanisms underlying proton modulation of IKATP in insulin-secreting cells.

Electrophysiological Properties of Mouse Horizontal Cell GABAA Receptors

2004 ◽  
Vol 92 (5) ◽  
pp. 2789-2801 ◽  
Author(s):  
Andreas Feigenspan ◽  
Reto Weiler
Keyword(s):  
Single Channel ◽  
Horizontal Cell ◽  
Chloride Ions ◽  
Horizontal Cells ◽  
Hill Coefficient ◽  
Mouse Retina ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Steady State Current ◽  
Single Channel Currents

GABA-induced currents have been characterized in isolated horizontal cells from lower vertebrates but not in mammalian horizontal cells. Therefore horizontal cells were isolated after enzymatical and mechanical dissociation of the adult mouse retina and visually identified. We recorded from horizontal cell bodies using the whole cell and outside-out configuration of the patch-clamp technique. Extracellular application of GABA induced inward currents carried by chloride ions. GABA-evoked currents were completely and reversibly blocked by the competitive GABAA receptor antagonist bicuculline (IC50 = 1.7 μM), indicating expression of GABAA but not GABAC receptors. Their affinity for GABA was moderate (EC50 = 30 μM), and the Hill coefficient was 1.3, corresponding to two GABA binding sites. GABA responses were partially reduced by picrotoxin with differential effects on peak and steady-state current values. Zinc blocked the GABA response with an IC50 value of 7.3 μM in a noncompetitive manner. Furthermore, GABA receptors of horizontal cells were modulated by extracellular application of diazepam, zolpidem, methyl 6,7-dimethoxy-4-ethyl-β-carboxylate, pentobarbital, and alphaxalone, thus showing typical pharmacological properties of CNS GABAA receptors. GABA-evoked single-channel currents were characterized by a main conductance state of 29.8 pS and two subconductance states (20.2 and 10.8 pS, respectively). Kinetic analysis of single-channel events within bursts revealed similar mean open and closed times for the main conductance and the 20.2-pS subconductance state, resulting in open probabilities of 44.6 and 42.7%, respectively. The ratio of open to closed times, however, was significantly different for the 10.8-pS subconductance state with an open probability of 57.2%.

scholarly journals Single Channel Properties of P2X2 Purinoceptors

1999 ◽  
Vol 113 (5) ◽  
pp. 695-720 ◽  
Author(s):  
Shinghua Ding ◽  
Frederick Sachs
Keyword(s):  
Conformational Changes ◽  
Single Channel ◽  
Excess Noise ◽  
Hill Coefficient ◽  
Inward Rectification ◽  
Channel Current ◽  
Single Channel Current ◽  
The Mean ◽  
Single Channel Currents ◽  
Channel Properties

The single channel properties of cloned P2X2 purinoceptors expressed in human embryonic kidney (HEK) 293 cells and Xenopus oocytes were studied in outside-out patches. The mean single channel current–voltage relationship exhibited inward rectification in symmetric solutions with a chord conductance of ∼30 pS at −100 mV in 145 mM NaCl. The channel open state exhibited fast flickering with significant power beyond 10 kHz. Conformational changes, not ionic blockade, appeared responsible for the flickering. The equilibrium constant of Na+ binding in the pore was ∼150 mM at 0 mV and voltage dependent. The binding site appeared to be ∼0.2 of the electrical distance from the extracellular surface. The mean channel current and the excess noise had the selectivity: K+ > Rb+ > Cs+ > Na+ > Li+. ATP increased the probability of being open (Po) to a maximum of 0.6 with an EC50 of 11.2 μM and a Hill coefficient of 2.3. Lowering extracellular pH enhanced the apparent affinity of the channel for ATP with a pKa of ∼7.9, but did not cause a proton block of the open channel. High pH slowed the rise time to steps of ATP without affecting the fall time. The mean single channel amplitude was independent of pH, but the excess noise increased with decreasing pH. Kinetic analysis showed that ATP shortened the mean closed time but did not affect the mean open time. Maximum likelihood kinetic fitting of idealized single channel currents at different ATP concentrations produced a model with four sequential closed states (three binding steps) branching to two open states that converged on a final closed state. The ATP association rates increased with the sequential binding of ATP showing that the binding sites are not independent, but positively cooperative. Partially liganded channels do not appear to open. The predicted Po vs. ATP concentration closely matches the single channel current dose–response curve.

scholarly journals Ligand binding and activation properties of the purified bacterial cyclic nucleotide–gated channel SthK

2018 ◽  
Vol 150 (6) ◽  
pp. 821-834 ◽  
Author(s):  
Philipp A.M. Schmidpeter ◽  
Xiaolong Gao ◽  
Vikrant Uphadyay ◽  
Jan Rheinberger ◽  
Crina M. Nimigean
Keyword(s):  
Lipid Bilayers ◽  
Conformational Changes ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Sequence Similarity ◽  
Guanosine Monophosphate ◽  
Particle Analysis ◽  
Xenopus Laevis Oocytes ◽  
High Sequence Similarity ◽  
Protein Expression And Purification

Cyclic nucleotide–modulated ion channels play several essential physiological roles. They are involved in signal transduction in photoreceptors and olfactory sensory neurons as well as pacemaking activity in the heart and brain. Investigations of the molecular mechanism of their actions, including structural and electrophysiological characterization, are restricted by the availability of stable, purified protein obtained from accessible systems. Here, we establish that SthK, a cyclic nucleotide–gated (CNG) channel from Spirochaeta thermophila, is an excellent model for investigating the gating of eukaryotic CNG channels at the molecular level. The channel has high sequence similarity with its eukaryotic counterparts and was previously reported to be activated by cyclic nucleotides in patch-clamp experiments with Xenopus laevis oocytes. We optimized protein expression and purification to obtain large quantities of pure, homogeneous, and active recombinant SthK protein from Escherichia coli. A negative-stain electron microscopy (EM) single-particle analysis indicated that this channel is a promising candidate for structural studies with cryo-EM. Using radioactivity and fluorescence flux assays, as well as single-channel recordings in lipid bilayers, we show that the protein is partially activated by micromolar concentrations of cyclic adenosine monophosphate (cAMP) and that channel activity is increased by depolarization. Unlike previous studies, we find that cyclic guanosine monophosphate (cGMP) is also able to activate SthK, but with much lower efficiency than cAMP. The distinct sensitivities to different ligands resemble eukaryotic CNG and hyperpolarization-activated and cyclic nucleotide–modulated channels. Using a fluorescence binding assay, we show that cGMP and cAMP bind to SthK with similar apparent affinities, suggesting that the large difference in channel activation by cAMP or cGMP is caused by the efficacy with which each ligand promotes the conformational changes toward the open state. We conclude that the functional characteristics of SthK reported here will permit future studies to analyze ligand gating and discrimination in CNG channels.

scholarly journals PIP2 Mediated Inhibition of TREK Potassium Currents by Bradykinin in Mouse Sympathetic Neurons

2020 ◽  
Vol 21 (2) ◽  
pp. 389 ◽  
Author(s):  
Paula Rivas-Ramírez ◽  
Antonio Reboreda ◽  
Lola Rueda-Ruzafa ◽  
Salvador Herrera-Pérez ◽  
J. Antonio Lamas
Keyword(s):  
Single Channel ◽  
Sympathetic Neurons ◽  
K Channel ◽  
Muscarinic Agonists ◽  
Open Probability ◽  
M Current ◽  
Voltage Dependent ◽  
Cervical Ganglion ◽  
Patch Technique ◽  
Single Channel Currents

Bradykinin (BK), a hormone inducing pain and inflammation, is known to inhibit potassium M-currents (IM) and to increase the excitability of the superior cervical ganglion (SCG) neurons by activating the Ca2+-calmodulin pathway. M-current is also reduced by muscarinic agonists through the depletion of membrane phosphatidylinositol 4,5-biphosphate (PIP2). Similarly, the activation of muscarinic receptors inhibits the current through two-pore domain potassium channels (K2P) of the “Tandem of pore-domains in a Weakly Inward rectifying K+ channel (TWIK)-related channels” (TREK) subfamily by reducing PIP2 in mouse SCG neurons (mSCG). The aim of this work was to test and characterize the modulation of TREK channels by bradykinin. We used the perforated-patch technique to investigate riluzole (RIL) activated currents in voltage- and current-clamp experiments. RIL is a drug used in the palliative treatment of amyotrophic lateral sclerosis and, in addition to blocking voltage-dependent sodium channels, it also selectively activates the K2P channels of the TREK subfamily. A cell-attached patch-clamp was also used to investigate TREK-2 single channel currents. We report here that BK reduces spike frequency adaptation (SFA), inhibits the riluzole-activated current (IRIL), which flows mainly through TREK-2 channels, by about 45%, and reduces the open probability of identified single TREK-2 channels in cultured mSCG cells. The effect of BK on IRIL was precluded by the bradykinin receptor (B2R) antagonist HOE-140 (d-Arg-[Hyp3, Thi5, d-Tic7, Oic8]BK) but also by diC8PIP2 which prevents PIP2 depletion when phospholipase C (PLC) is activated. On the contrary, antagonizing inositol triphosphate receptors (IP3R) using 2-aminoethoxydiphenylborane (2-APB) or inhibiting protein kinase C (PKC) with bisindolylmaleimide did not affect the inhibition of IRIL by BK. In conclusion, bradykinin inhibits TREK-2 channels through the activation of B2Rs resulting in PIP2 depletion, much like we have demonstrated for muscarinic agonists. This mechanism implies that TREK channels must be relevant for the capture of information about pain and visceral inflammation.

Glucocorticoid-stimulated lung epithelial Na+ transport is associated with regulated ENaC andsgk1 expression

2002 ◽  
Vol 282 (4) ◽  
pp. L631-L641 ◽  
Author(s):  
Omar A. Itani ◽  
Scott D. Auerbach ◽  
Russell F. Husted ◽  
Kenneth A. Volk ◽  
Shana Ageloff ◽  
...  
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
A549 Cells ◽  
Epithelial Cell Line ◽  
Alveolar Type ◽  
Xenopus Laevis Oocytes ◽  
Biophysical Properties ◽  
Transport Pathway ◽  
Slow Kinetics ◽  
Single Channel Currents

H441 cells, a bronchiolar epithelial cell line, develop a glucocorticoid-regulated amiloride-sensitive Na+ transport pathway on permeable supports (R. Sayegh, S. D. Auerbach, X. Li, R. Loftus, R. Husted, J. B. Stokes, and C. P. Thomas. J Biol Chem 274: 12431–12437, 1999). To understand its molecular basis, we examined the effect of glucocorticoids (GC) on epithelial Na+ channel (ENaC)-α, -β, and -γ and sgk1 expression and determined the biophysical properties of Na+ channels in these cells. GC stimulated the expression of ENac-α, -β, and -γ and sgk1 mRNA, with the first effect seen by 1 h. These effects were abolished by actinomycin D, but not by cycloheximide, indicating a direct stimulatory effect on ENaC and sgk1 mRNA synthesis. The GC effect on transcription of ENaC-α mRNA was accompanied by a significant increase in ENaC-α protein levels. GC also stimulated ENaC-α, -β, and -γ and sgk1 mRNA expression in A549 cells, an alveolar type II cell line. To determine the biophysical properties of the Na+channel, single-channel currents were recorded from cell-attached H441 membranes. An Na+-selective channel with slow kinetics and a slope conductance of 10.8 pS was noted, properties similar to ENaC-α, -β, and -γ expressed in Xenopus laevis oocytes. These experiments indicate that amiloride-sensitive Na+ transport is mediated through classic ENaC channels in human lung epithelia and that GC-regulated Na+ transport is accompanied by increased transcription of each of the component subunits and sgk1.

scholarly journals Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane.

1988 ◽  
Vol 92 (1) ◽  
pp. 67-86 ◽  
Author(s):  
A Oberhauser ◽  
O Alvarez ◽  
R Latorre
Keyword(s):  
Lipid Bilayers ◽  
Single Channel ◽  
Divalent Cations ◽  
Hill Coefficient ◽  
Muscle Membrane ◽  
K Channel ◽  
Single Channel Currents ◽  
The Hill ◽  
Channel Currents ◽  
Cytoplasmic Side

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.

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