Extracellular Solution Protocol v3 (protocols.io.uiweufe)

protocols.io ◽  
2018 ◽  
Author(s):  
Anna Kristin
Keyword(s):  
Extracellular Solution

scholarly journals T-type Ca2+channel modulation by otilonium bromide

2010 ◽  
Vol 298 (5) ◽  
pp. G706-G713 ◽  
Author(s):  
Peter R. Strege ◽  
Lei Sha ◽  
Arthur Beyder ◽  
Cheryl E. Bernard ◽  
Edward Perez-Reyes ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Contractile Activity ◽  
Clinical Effectiveness ◽  
Smooth Muscle Contraction ◽  
Hek293 Cells ◽  
Channel Blockers ◽  
Patch Clamp Technique ◽  
Extracellular Solution ◽  
Channel Modulation ◽  
Main Pathway

Antispasmodics are used clinically to treat a variety of gastrointestinal disorders by inhibition of smooth muscle contraction. The main pathway for smooth muscle Ca2+entry is through L-type channels; however, there is increasing evidence that T-type Ca2+channels also play a role in regulating contractility. Otilonium bromide, an antispasmodic, has previously been shown to inhibit L-type Ca2+channels and colonic contractile activity. The objective of this study was to determine whether otilonium bromide also inhibits T-type Ca2+channels. Whole cell currents were recorded by patch-clamp technique from HEK293 cells transfected with cDNAs encoding the T-type Ca2+channels, CaV3.1 (α1G), CaV3.2 (α1H), or CaV3.3 (α1I) alpha subunits. Extracellular solution was exchanged with otilonium bromide (10−8to 10−5M). Otilonium bromide reversibly blocked all T-type Ca2+channels with a significantly greater affinity for CaV3.3 than CaV3.1 or CaV3.2. Additionally, the drug slowed inactivation in CaV3.1 and CaV3.3. Inhibition of T-type Ca2+channels may contribute to inhibition of contractility by otilonium bromide. This may represent a new mechanism of action for antispasmodics and may contribute to the observed increased clinical effectiveness of antispasmodics compared with selective L-type Ca2+channel blockers.

Molecular and Physiological Bases of the K+ Circulation in the Mammalian Inner Ear

Physiology ◽  
2006 ◽  
Vol 21 (5) ◽  
pp. 336-345 ◽  
Author(s):  
Hiroshi Hibino ◽  
Yoshihisa Kurachi
Keyword(s):  
Ion Transport ◽  
Inner Ear ◽  
Lateral Wall ◽  
Extracellular Solution ◽  
Transport Molecules

Endolymph, the extracellular solution in cochlea, contains 150 mM K+ and exhibits a potential of approximately +80 mV relative to neighboring extracellular spaces. This unique situation, essential for hearing, is maintained by K+ circulation from perilymph to endolymph through the cochlear lateral wall. Recent studies have identified ion-transport molecules involved in the K+ circulation and their pathophysiological relevance.

Depletion of intracellular Ca2+ stores activates a Ca(2+)-selective channel in vascular endothelium

1994 ◽  
Vol 267 (4) ◽  
pp. C920-C925 ◽  
Author(s):  
L. Vaca ◽  
D. L. Kunze
Keyword(s):  
Vascular Endothelium ◽  
Reversal Potential ◽  
Inward Rectification ◽  
Ion Permeability ◽  
Channel Activity ◽  
Permeability Ratio ◽  
Extracellular Solution ◽  
Aortic Endothelial Cells ◽  
Bovine Aortic Endothelial Cells ◽  
Selective Channel

The present study was designed to identify the channel responsible for Ca2+ influx after depletion of intracellular Ca2+ stores. Different maneuvers that deplete intracellular Ca2+ stores activated a Ca(2+)-selective channel. Superfusion of single bovine aortic endothelial cells with 50 nmol/l bradykinin, 10 mumol/l ATP, or 10 mumol/l 2,5-di(tert-butyl)-1,4-benzohydroquinone produced activation of channels of the same amplitude in cell-attached patches. Channel activity declined within the first minute after patch excision. The channel showed strong inward rectification and a reversal potential of 0 mV in symmetrical sodium sulfate (Na2SO4) solution. Under these conditions, the conductance was 5 pS in the inward direction. Addition of 10 mmol/l Ca2+ to the extracellular solution shifted the reversal potential to +30 +/- 5 mV, and the conductance for inward current was 11 pS. The reversal potential was used to calculate an ion permeability ratio of Ca2+/Na+ > 10:1.

Adrenergic Facilitation of GABAergic Transmission in Rat Entorhinal Cortex

2007 ◽  
Vol 98 (5) ◽  
pp. 2868-2877 ◽  
Author(s):  
Saobo Lei ◽  
Pan-Yue Deng ◽  
James E. Porter ◽  
Hee-Sup Shin
Keyword(s):  
Entorhinal Cortex ◽  
Adrenergic Receptors ◽  
Gaba Release ◽  
Channel Blockers ◽  
Extracellular Solution ◽  
Gabaergic Transmission ◽  
Voltage Gated ◽  
Postsynaptic Currents ◽  
Kinase C ◽  
Gabaergic Function

Whereas the entorhinal cortex (EC) receives noradrenergic innervations from the locus coeruleus of the pons and expresses adrenergic receptors, the function of norepinephrine (NE) in the EC is still elusive. We examined the effects of NE on GABAA receptor–mediated synaptic transmission in the superficial layers of the EC. Application of NE dose-dependently increased the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) recorded from the principal neurons in layer II/III through activation of α1 adrenergic receptors. NE increased the frequency and not the amplitude of miniature IPSCs (mIPSCs) recorded in the presence of TTX, suggesting that NE increases presynaptic GABA release with no effects on postsynaptic GABAA receptors. Application of Ca2+ channel blockers (Cd2+ and Ni2+), omission of Ca2+ in the extracellular solution, or replacement of extracellular Na+ with N-methyl-d-glucamine (NMDG) failed to alter NE-induced increase in mIPSC frequency, suggesting that Ca2+ influx through voltage-gated Ca2+ or other cationic channels is not required. Application of BAPTA-AM, thapsigargin, and ryanodine did not change NE-induced increase in mIPSC frequency, suggesting that Ca2+ release from intracellular stores is not necessary for NE-induced increase in GABA release. Whereas α1 receptors are coupled to Gq/11 resulting in activation of the phospholipase C (PLC) pathway, NE-mediated facilitation of GABAergic transmission was independent of PLC, protein kinase C, and tyrosine kinase activities. Our results suggest that NE-mediated facilitation of GABAergic function contributes to its antiepileptic effects in the EC.

Ca2+ Enhances U-Type Inactivation of N-Type (CaV2.2) Calcium Current in Rat Sympathetic Neurons

2006 ◽  
Vol 96 (3) ◽  
pp. 1075-1083 ◽  
Author(s):  
Yong Sook Goo ◽  
Wonil Lim ◽  
Keith S. Elmslie
Keyword(s):  
Slow Component ◽  
Divalent Cations ◽  
Sympathetic Neurons ◽  
Slow Inactivation ◽  
Fast Inactivation ◽  
Extracellular Solution ◽  
Channel Inactivation ◽  
Dependent Inactivation ◽  
Preferential Inactivation

Ca2+-dependent inactivation (CDI) has recently been shown in heterologously expressed N-type calcium channels (CaV2.2), but CDI has been inconsistently observed in native N-current. We examined the effect of Ca2+ on N-channel inactivation in rat sympathetic neurons to determine the role of CDI on mammalian N-channels. N-current inactivated with fast (τ ∼ 150 ms) and slow (τ ∼ 3 s) components in Ba2+. Ca2+ differentially affected these components by accelerating the slow component (slow inactivation) and enhancing the amplitude of the fast component (fast inactivation). Lowering intracellular BAPTA concentration from 20 to 0.1 mM accelerated slow inactivation, but only in Ca2+ as expected from CDI. However, low BAPTA accelerated fast inactivation in either Ca2+ or Ba2+, which was unexpected. Fast inactivation was abolished with monovalent cations as the charge carrier, but slow inactivation was similar to that in Ba2+. Increased Ca2+, but not Ba2+, concentration (5–30 mM) enhanced the amplitude of fast inactivation and accelerated slow inactivation. However, the enhancement of fast inactivation was independent of Ca2+ influx, which indicates the relevant site is exposed to the extracellular solution and is inconsistent with CDI. Fast inactivation showed U-shaped voltage dependence in both Ba2+ and Ca2+, which appears to result from preferential inactivation from intermediate closed states (U-type inactivation). Taken together, the data support a role for extracellular divalent cations in modulating U-type inactivation. CDI appears to play a role in N-channel inactivation, but on a slower (sec) time scale.

scholarly journals K+ Occupancy of the N-Methyl-d-Aspartate Receptor Channel Probed by Mg2+ Block

2001 ◽  
Vol 117 (3) ◽  
pp. 287-298 ◽  
Author(s):  
Yongling Zhu ◽  
Anthony Auerbach
Keyword(s):  
Binding Sites ◽  
Voltage Dependence ◽  
Single Channel ◽  
Extracellular Solution ◽  
Aspartate Receptor ◽  
Apparent Decrease ◽  
Internal Site ◽  
Equilibrium Dissociation ◽  
Kinetics Of ◽  
Receptor Channel

The single-channel kinetics of extracellular Mg2+ block was used to probe K+ binding sites in the permeation pathway of rat recombinant NR1/NR2B NMDA receptor channels. K+ binds to three sites: two that are external and one that is internal to the site of Mg2+ block. The internal site is ∼0.84 through the electric field from the extracellular surface. The equilibrium dissociation constant for this site for K+ is 304 mM at 0 mV and with Mg2+ in the pore. The occupancy of any one of the three sites by K+ effectively prevents the association of extracellular Mg2+. Occupancy of the internal site also prevents Mg2+ permeation and increases (by approximately sevenfold) the rate constant for Mg2+ dissociation back to the extracellular solution. Under physiological intracellular ionic conditions and at −60 mV, there is ∼1,400-fold apparent decrease in the affinity of the channel for extracellular Mg2+ and ∼2-fold enhancement of the apparent voltage dependence of Mg2+ block caused by the voltage dependence of K+ occupancy of the external and internal sites.

Visualizing formation and dynamics of vacuoles in living cells using contrasting dextran-bound indicator: endocytic and nonendocytic vacuoles

2007 ◽  
Vol 293 (6) ◽  
pp. G1333-G1338 ◽  
Author(s):  
Svetlana G. Voronina ◽  
Mark W. Sherwood ◽  
Oleg V. Gerasimenko ◽  
Ole H. Petersen ◽  
Alexei V. Tepikin
Keyword(s):  
Endoplasmic Reticulum ◽  
Confocal Microscopy ◽  
Acinar Cells ◽  
Cell Types ◽  
Living Cells ◽  
Pancreatic Acinar Cells ◽  
Extracellular Solution ◽  
Whole Cell Patch Clamp ◽  
Patch Pipette ◽  
Cellular Organelles

Here we describe a technique that allows us to visualize in real time the formation and dynamics (fusion, changes of shape, and translocation) of vacuoles in living cells. The technique involves infusion of a dextran-bound fluorescent probe into the cytosol of the cell via a patch pipette, using the whole-cell patch-clamp configuration. Experiments were conducted on pancreatic acinar cells stimulated with supramaximal concentrations of cholecystokinin (CCK). The vacuoles, forming in the cytoplasm of the cell, were revealed as dark imprints on a bright fluorescence background, produced by the probe and visualized by confocal microscopy. A combination of two dextran-bound probes, one infused into the cytosol and the second added to the extracellular solution, was used to identify endocytic and nonendocytic vacuoles. The cytosolic dextran-bound probe was also used together with a Golgi indicator to illustrate the possibility of combining the probes and identifying the localization of vacuoles with respect to other cellular organelles in pancreatic acinar cells. Combinations of cytosolic dextran-bound probes with endoplasmic reticulum (ER) or mitochondrial probes were also used to simultaneously visualize vacuoles and corresponding organelles. We expect that the new technique will also be applicable and useful for studies of vacuole dynamics in other cell types.

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