Acid-sensing ion channels contribute to the increase in vesicular release from SH-SY5Y cells stimulated by extracellular protons

2012 ◽  
Vol 303 (4) ◽  
pp. C376-C384 ◽  
Author(s):  
Qiu-Ju Xiong ◽  
Zhuang-Li Hu ◽  
Peng-Fei Wu ◽  
Lan Ni ◽  
Zhi-Fang Deng ◽  
...  
Keyword(s):  
Ion Channels ◽  
Neurotransmitter Release ◽  
Neuronal Cell ◽  
Channel Blockers ◽  
Patch Clamp Recording ◽  
Human Neuroblastoma ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
Vesicular Release ◽  
Inward Currents

Acid-sensing ion channels (ASICs) have been reported to play a role in the neuronal dopamine pathway, but the exact role in neurotransmitter release remains elusive. Human neuroblastoma SH-SY5Y is a dopaminergic neuronal cell line, which can release monoamine neurotransmitters. In this study, the expression of ASICs was identified in SH-SY5Y cells to further explore the role of ASICs in vesicular release stimulated by acid. We gathered evidence that ASICs could be detected in SH-SY5Y cells. In whole cell patch-clamp recording, a rapid decrease in extracellular pH evoked inward currents, which were reversibly inhibited by 100 μM amiloride. The currents were pH dependent, with a pH of half-maximal activation (pH0.5) of 6.01 ± 0.04. Furthermore, in calcium imaging and FM 1-43 dye labeling, it was shown that extracellular protons increased intracellular calcium levels and vesicular release in SH-SY5Y cells, which was attenuated by PcTx1 and amiloride. Interestingly, N-type calcium channel blockers inhibited the vesicular release induced by acidification. In conclusion, ASICs are functionally expressed in SH-SY5Y cells and involved in vesicular release stimulated by acidification. N-type calcium channels may be involved in the increase in vesicular release induced by acid. Our results provide a preliminary study on ASICs in SH-SY5Y cells and neurotransmitter release, which helps to further investigate the relationship between ASICs and dopaminergic neurons.

Local Excitatory Circuits in the Intermediate Gray Layer of the Superior Colliculus

1999 ◽  
Vol 81 (3) ◽  
pp. 1424-1427 ◽  
Author(s):  
Diana L. Pettit ◽  
Matthew C. Helms ◽  
Psyche Lee ◽  
George J. Augustine ◽  
William C. Hall
Keyword(s):  
Superior Colliculus ◽  
Intermediate Layer ◽  
Tree Shrew ◽  
Patch Clamp Recording ◽  
Inward Current ◽  
Whole Cell Patch Clamp ◽  
Synaptic Interactions ◽  
Layer Neuron ◽  
Inward Currents ◽  
Synaptic Responses

Local excitatory circuits in the intermediate gray layer of the superior colliculus. We have used photostimulation and whole cell patch-clamp recording techniques to examine local synaptic interactions in slices from the superior colliculus of the tree shrew. Uncaging glutamate 10–75 μm from the somata of neurons in the intermediate gray layer elicited a long-lasting inward current, due to direct activation of glutamate receptors on these neurons, and brief inward currents caused by activation of presynaptic neurons. The synaptic responses occurred as individual currents or as clusters that lasted up to several hundred milliseconds. Excitatory synaptic responses, which reversed at membrane potentials near 0 mV, could be evoked by uncaging glutamate anywhere within 75 μm of an intermediate layer neuron. Our results indicate the presence of extensive local excitatory circuits in the intermediate layer of the superior colliculus and support the hypothesis that such intrinsic circuitry contributes to the development of presaccadic command bursts.

Vasopressin-Induced Currents in Rat Neonatal Spinal Lateral Horn Neurons Are G-Protein Mediated and Involve Two Conductances

1998 ◽  
Vol 80 (4) ◽  
pp. 1900-1910 ◽  
Author(s):  
Miloslav Kolaj ◽  
Leo P. Renaud
Keyword(s):  
Spinal Cord ◽  
G Protein ◽  
Wide Distribution ◽  
Channel Blockers ◽  
Patch Clamp Recording ◽  
Inward Current ◽  
Lateral Column ◽  
Lateral Horn ◽  
Inward Currents ◽  
Induced Currents

Kolaj, Miloslav and Leo P. Renaud. Vasopressin-induced currents in rat neonatal spinal lateral horn neurons are G-protein mediated and involve two conductances . J. Neurophysiol. 80: 1900–1910, 1998. Arginine vasopressin (AVP) receptors are expressed early in the developing spinal cord. To characterize AVP-induced conductances in lower thoracic sympathetic preganglionic (SPN) and other lateral horn neurons, we used patch-clamp recording techniques in neonatal (11–21 days) rat spinal cord slices. Most (90%) of 273 neurons, including all 68 SPNs, responded to AVP with membrane depolarization and/or a V1 receptor-mediated, dose-dependent (0.01–1.0 μM) and tetrodotoxin (TTX)-resistant inward current. A role for G-proteins was indicated by persistence of this inward current after intracellular dialysis with GTP-γ-S or GMP-PNP, its marked reduction with GDP-β-S, and significant reduction, but not abolition, after preincubation with pertussis toxin or in the presence of N-ethylmaleimide. Analysis of individual current-voltage ( I- V) relationships in 57 cells indicated the presence of two different membrane conductances. In 21 cells, net AVP-induced currents reversed around −103 mV, reflecting reduction in one or more barium-sensitive potassium conductances; in 12 cells, net AVP-induced current reversed around −40 mV and was not significantly sensitive to several potassium channel blockers including barium, tetraethylammonium chloride (TEA), 4-aminopyridine (4AP), cesium, or glibenclamide, suggesting increase in a nonselective cationic conductance that was separate from I h; in 24 cells where I- V lines shifted in parallel, AVP-induced inward currents were significantly greater and probably involved both conductances. These data indicate that SPNs and a majority of unidentified neonatal lateral horn neurons possess functional G-protein–coupled V1-type vasopressin receptors. The wide distribution of AVP receptors in neonatal spinal lateral column cells suggests a role that may extend beyond involvement in regulation of autonomic nervous system function.

Activation of subfornical organ neurons in rats through pre- and postsynaptic α-adrenoceptors

2006 ◽  
Vol 290 (6) ◽  
pp. R1646-R1653 ◽  
Author(s):  
Eiko Honda ◽  
Kentaro Ono ◽  
Shinji Kataoka ◽  
Kiyotoshi Inenaga
Keyword(s):  
Gaba Receptor ◽  
Membrane Conductance ◽  
Subfornical Organ ◽  
Patch Clamp Recording ◽  
Pcr Assay ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Current Clamp Mode ◽  
Inward Currents ◽  
Pulse Stimulation

The effects of noradrenaline (NA) and its analogs on subfornical organ (SFO) neurons in rat slice preparations were investigated by using whole cell patch-clamp recording. In the current-clamp mode, the application of NA at 10–100 μM produced membrane depolarization (63%, 17 responsive neurons/27 neurons tested) and hyperpolarization (22%, 6/27 neurons). In the voltage-clamp mode, NA application at 1–100 μM produced inward currents (69%, 42/61 neurons) and outward currents (23%, 14/61 neurons). These currents remained in the presence of TTX or both glutamate and GABA receptor antagonists. In most of the neurons (25/31 neurons) showing inward currents in the presence of NA, the membrane conductance was not changed by voltage ramps or hyperpolarizing pulse stimulation. Similar responses were obtained by the application of the α1-agonist phenylephrine. The phenylephrine-induced inward currents were inhibited by the α1-antagonist prazosin. The α2-agonist clonidine decreased the frequency of spontaneous GABAergic inhibitory postsynaptic currents (4/10 neurons). In addition, RT-PCR assay and immunohistochemical staining showed the existence of α1-adrenoceptors in the SFO. The results suggest that SFO neurons in rats are activated postsynaptically through α1-adrenoceptors and that the activation is enhanced by suppressing GABAergic inhibitory synaptic inputs through presynaptic α2-adrenoceptors.

scholarly journals Large-Conductance Calcium-Activated Potassium Channels and Voltage-Dependent Sodium Channels in Human Cementoblasts

2021 ◽  
Vol 12 ◽  
Author(s):  
Satomi Kamata ◽  
Maki Kimura ◽  
Sadao Ohyama ◽  
Shuichiro Yamashita ◽  
Yoshiyuki Shibukawa
Keyword(s):  
Alveolar Bone ◽  
Attachment Site ◽  
Ionic Currents ◽  
Ionic Channels ◽  
Patch Clamp Recording ◽  
Physiological Processes ◽  
Whole Cell Patch Clamp ◽  
Outward Currents ◽  
Voltage Dependent ◽  
Inward Currents

Cementum, which is excreted by cementoblasts, provides an attachment site for collagen fibers that connect to the alveolar bone and fix the teeth into the alveolar sockets. Transmembrane ionic signaling, associated with ionic transporters, regulate various physiological processes in a wide variety of cells. However, the properties of the signals generated by plasma membrane ionic channels in cementoblasts have not yet been described in detail. We investigated the biophysical and pharmacological properties of ion channels expressed in human cementoblast (HCEM) cell lines by measuring ionic currents using conventional whole-cell patch-clamp recording. The application of depolarizing voltage steps in 10 mV increments from a holding potential (Vh) of −70 mV evoked outwardly rectifying currents at positive potentials. When intracellular K+ was substituted with an equimolar concentration of Cs+, the outward currents almost disappeared. Using tail current analysis, the contributions of both K+ and background Na+ permeabilities were estimated for the outward currents. Extracellular application of tetraethylammonium chloride (TEA) and iberiotoxin (IbTX) reduced the densities of the outward currents significantly and reversibly, whereas apamin and TRAM-34 had no effect. When the Vh was changed to −100 mV, we observed voltage-dependent inward currents in 30% of the recorded cells. These results suggest that HCEM express TEA- and IbTX-sensitive large-conductance Ca2+-activated K+ channels and voltage-dependent Na+ channels.

scholarly journals The Effect of Hydrophobic Monoamines on Acid-Sensing Ion Channels ASIC1B

Acta Naturae ◽  
2015 ◽  
Vol 7 (2) ◽  
pp. 95-101 ◽  
Author(s):  
E. I. Nagaeva ◽  
N. N. Potapieva ◽  
D. B. Tikhonov
Keyword(s):  
Ion Channels ◽  
Patch Clamp ◽  
Inhibitory Effect ◽  
Hill Coefficient ◽  
Patch Clamp Technique ◽  
Recombinant Receptors ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
The Hill ◽  
Cooperative Activation

Acid-sensing ion channels (ASICs) are widely distributed in both the central and peripheral nervous systems of vertebrates. The pharmacology of these receptors remains poorly investigated, while the search for new ASIC modulators is very important. Recently, we found that some monoamines, which are blockers of NMDA receptors, inhibit and/or potentiate acid-sensing ion channels, depending on the subunit composition of the channels. The effect of 9-aminoacridine, IEM-1921, IEM-2117, and memantine both on native receptors and on recombinant ASIC1a, ASIC2a, and ASIC3 homomers was studied. In the present study, we have investigated the effect of these four compounds on homomeric ASIC1b channels. Experiments were performed on recombinant receptors expressed in CHO cells using the whole-cell patch clamp technique. Only two compounds, 9-aminoacridine and memantine, inhibited ASIC1b channels. IEM-1921 and IEM-2117 were inactive even at a 1000 M concentration. In most aspects, the effect of the compounds on ASIC1b was similar to their effect on ASIC1a. The distinguishing feature of homomeric ASIC1b channels is a steep activation-dependence, indicating cooperative activation by protons. In our experiments, the curve of the concentration dependence of ASIC1b inhibition by 9-aminoacridine also had a slope (Hill coefficient) of 3.8, unlike ASIC1a homomers, for which the Hill coefficient was close to 1. This finding indicates that the inhibitory effect of 9-aminoacridine is associated with changes in the activation properties of acid-sensing ion channels.

Stretch-activated ion channels in tissue-cultured chick heart

1993 ◽  
Vol 264 (3) ◽  
pp. H960-H972 ◽  
Author(s):  
A. Ruknudin ◽  
F. Sachs ◽  
J. O. Bustamante
Keyword(s):  
Ion Channels ◽  
Single Channel ◽  
Inward Rectifier ◽  
Channel Noise ◽  
Patch Clamp Recording ◽  
Alkali Cations ◽  
Channel Expression ◽  
Inward Currents ◽  
Extracellular Ions ◽  
Stretch Activated Ion Channels

With use of single-channel patch-clamp recording, we found five distinct types of stretch-activated ion channels (SACs) in tissue-cultured embryonic chick cardiac myocytes. With 140 mM K+ saline in the pipette, four channels had linear conductances of approximately equal to 25, 50, 100, and 200 pS and other channel was an inward rectifier of approximately equal to 25 pS at 0 mV membrane potential. The 100- and 200-pS channels were K+ selective, whereas the others passed alkali cations and Ca2+. From reversal potentials, the permeability ratio of K+/Na+, PK/PNa, was 3–7 for nonselective channels and 7–16 for K(+)-selective channels. Channel density was approximately equal to 0.3/microns2 for linear conductances and approximately equal to 0.1/microns2 for inward rectifier. Open-channel noise was a function of pipette filling solution with root-mean-square (RMS) noise increasing in the order K+ < isosmotic sucrose (plus trace ions) < Na+, probably reflecting short-lived block by extracellular ions. All channels were blocked by 20 microM Gd3+. The 25-pS linear channel was also blocked by 12.5 microM tetrodotoxin and 10 microM diltiazem, but the others were insensitive at these concentrations. Extracellular Cs+ and tetraethylammonium chloride did not block any channels. We saw no SAC activity in cells grown without embryo extract (EE), which demonstrates that channel expression, or some necessary cofactor, is under control of growth factors. Basic fibroblast growth factor (FGF) could replace EE in supporting channel expression. The presence of SACs capable of generating inward currents might explain how stretch increases automaticity in the heart. Because some SACs were permeable to Ca2+, they could contribute to the Starling curve and perhaps to initiating stretch-induced hypertrophy.

scholarly journals Acid-Sensing Ion Channels in Acidosis-Induced Injury of Human Brain Neurons

2010 ◽  
Vol 30 (6) ◽  
pp. 1247-1260 ◽  
Author(s):  
Minghua Li ◽  
Koichi Inoue ◽  
Deborah Branigan ◽  
Eric Kratzer ◽  
Jillian C Hansen ◽  
...  
Keyword(s):  
Ion Channels ◽  
Human Brain ◽  
Cortical Neurons ◽  
Cell Injury ◽  
Acidic Solution ◽  
Brain Neurons ◽  
Acid Sensing Ion Channels ◽  
Intracellular Ca ◽  
Inward Currents ◽  
High Level

Acidosis is a common feature of the human brain during ischemic stroke and is known to cause neuronal injury. However, the mechanism underlying acidosis-mediated injury of the human brain remains elusive. We show that a decrease in the extracellular pH evoked inward currents characteristic of acid-sensing ion channels (ASICs) and increased intracellular Ca2+ in cultured human cortical neurons. Acid-sensing ion channels in human cortical neurons show electrophysiological and pharmacological properties distinct from those in neurons of the rodent brain. Reverse transcriptase-PCR and western blot detected a high level of the ASIC1a subunit with little or no expression of other ASIC subunits. Treatment of human cortical neurons with acidic solution induced substantial cell injury, which was attenuated by the ASIC1a blockade. Thus, functional homomeric ASIC1a channels are predominantly expressed in neurons from the human brain. Activation of these channels has an important role in acidosis-mediated injury of human brain neurons.

scholarly journals Neurodegenerative Disease: What Potential Therapeutic Role of Acid-Sensing Ion Channels?

2021 ◽  
Vol 15 ◽  
Author(s):  
Dalila Mango ◽  
Robert Nisticò
Keyword(s):  
Ion Channels ◽  
Neurodegenerative Diseases ◽  
Ph Sensor ◽  
Channel Blockers ◽  
Neuroprotective Effects ◽  
Ion Channel Blockers ◽  
Brain Physiology ◽  
Acid Sensing Ion Channels ◽  
Complex Process ◽  
Potential Therapeutic Role

Acidic pH shift occurs in many physiological neuronal activities such as synaptic transmission and synaptic plasticity but also represents a characteristic feature of many pathological conditions including inflammation and ischemia. Neuroinflammation is a complex process that occurs in various neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and Huntington’s disease. Acid-sensing ion channels (ASICs) represent a widely expressed pH sensor in the brain that play a key role in neuroinflammation. On this basis, acid-sensing ion channel blockers are able to exert neuroprotective effects in different neurodegenerative diseases. In this review, we discuss the multifaceted roles of ASICs in brain physiology and pathology and highlight ASIC1a as a potential pharmacological target in neurodegenerative diseases.

scholarly journals Acid-sensing ion channels (ASICs) are differentially modulated by anions dependent on their subunit composition

2013 ◽  
Vol 304 (1) ◽  
pp. C89-C101 ◽  
Author(s):  
Nobuyoshi Kusama ◽  
Mamta Gautam ◽  
Anne Marie S. Harding ◽  
Peter M. Snyder ◽  
Christopher J. Benson
Keyword(s):  
Ion Channels ◽  
Ph Dependence ◽  
Extracellular Fluid ◽  
Dorsal Root Ganglion Neurons ◽  
Subunit Composition ◽  
Fluid Composition ◽  
Whole Cell Patch Clamp ◽  
Acid Sensing Ion Channels ◽  
Ganglion Neurons ◽  
Kinetics Of

Acid-sensing ion channels (ASICs) are sodium channels gated by extracellular protons. ASIC1a channels possess intersubunit Cl−-binding sites in the extracellular domain, which are highly conserved between ASIC subunits. We previously found that anions modulate ASIC1a gating via these sites. Here we investigated the effect of anion substitution on native ASICs in rat sensory neurons and heterologously expressed ASIC2a and ASIC3 channels by whole cell patch clamp. Similar to ASIC1a, anions modulated the kinetics of desensitization of other ASIC channels. However, unlike ASIC1a, anions also modulated the pH dependence of activation. Moreover, the order of efficacy of different anions to modulate ASIC2a and -3 was very different from that of ASIC1a. More surprising, mutations of conserved residues that form an intersubunit Cl−-binding site in ASIC1a only partially abrogated the effects of anion modulation of ASIC2a and had no effect on anion modulation of ASIC3. The effects of anions on native ASICs in rat dorsal root ganglion neurons mimicked those in heterologously expressed ASIC1a/3 heteromeric channels. Our data show that anions modulate a variety of ASIC properties and are dependent on the subunit composition, and the mechanism of modulation for ASIC2a and -3 is distinct from that of ASIC1a. We speculate that modulation of ASIC gating by Cl− is a novel mechanism to sense shifts in extracellular fluid composition.

P2X2 receptors are essential for [Ca2+]i increases in response to ATP in cultured rat myenteric neurons

2005 ◽  
Vol 289 (5) ◽  
pp. G935-G948 ◽  
Author(s):  
Toshio Ohta ◽  
Akane Kubota ◽  
Matsuka Murakami ◽  
Ken-ichi Otsuguro ◽  
Shigeo Ito
Keyword(s):  
Rank Order ◽  
Reversal Potential ◽  
Specific Protein ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Patch Clamp Technique ◽  
Myenteric Neurons ◽  
Whole Cell Patch Clamp ◽  
Intracellular Ca ◽  
Inward Currents

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At −60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP ≥ adenosine 5′- O-3-triphosphate ≥ CTP ≥ 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.

Sign in / Sign up

Export Citation Format

Share Document