scholarly journals Adenosine and anoxia reduce N-methyl-D-aspartate receptor open probability in turtle cerebrocortex.

1998 ◽  
Vol 201 (2) ◽  
pp. 289-297 ◽  
Author(s):  
L T Buck ◽  
P E Bickler
Keyword(s):  
Membrane Potential ◽  
Nmda Receptor ◽  
Ion Channel ◽  
Single Channel ◽  
Tissue Injury ◽  
Brain Slices ◽  
Current Amplitude ◽  
Mammalian Brain ◽  
Open Probability ◽  
Experimental Conditions

During normoxia, glutamate and the glutamate family of ion channels play a key role in mediating rapid excitatory synaptic transmission in the central nervous system. However, during hypoxia, intracellular [Ca2+] increases to neurotoxic levels, mediated largely by the N-methyl-D-aspartate (NMDA) subfamily of glutamate receptors. Adenosine has been shown to decrease the magnitude of the hypoxia-induced increase in [Ca2+]i in mammalian brain slices, delaying tissue injury. Turtle brain is remarkably tolerant of anoxia, maintaining a pre-anoxic [Ca2+]i while cerebral adenosine levels increase 12-fold. Employing cell-attached single-channel patch-clamp techniques, we studied the effect of adenosine (200 micromol l-1) and anoxia on NMDA receptor open probability (Popen) and current amplitude. After 60 min of anoxic perfusion, channel Popen decreased by 65 % (from 6.8+/-1.6 to 2.4+/-0.8 %) an effect that could also be achieved with a normoxic perfusion of 200 micromol l-1 adenosine (Popen decreased from 5.8+/-1.1 to 2.3+/-1.2 %). The inclusion of 10 micromol l-1 8-phenyltheophylline, an A1 receptor blocker, prevented the adenosine- and anoxia-induced decrease in Popen. Mean single-channel current amplitude remained at approximately 2.7+/-0.23 pA under all experimental conditions. To determine whether a change in the membrane potential could be part of the mechanism by which Popen decreases, membrane and threshold potential were measured following each experiment. Membrane potential did not change significantly under any condition, ranging from -76.8 to -80.6 mV. Therefore, during anoxia, NMDA receptors cannot be regulated by Mg2+ in a manner dependent on membrane potential. Threshold potentials did decrease significantly following 60 min of anoxic or adenosine perfusion (control -33.3+/-1.9 mV, anoxia -28.4+/-1.5 mV, adenosine -23.4+/-2.8 mV). We conclude that anoxia modulates NMDA receptor activity and that adenosine plays a key role in mediating this change. This is the first direct measurement of ion channel activity in anoxic turtle brain and demonstrates that ion channel regulation is part of the naturally evolved anoxic defence mechanism of this species.

AMPA and Kainate Receptors

2020 ◽  
Author(s):  
G. Brent Dawe ◽  
Patricia M. G. E. Brown ◽  
Derek Bowie
Keyword(s):  
Ion Channel ◽  
Glutamate Receptor ◽  
Single Channel ◽  
Channel Gating ◽  
Regulatory Proteins ◽  
Kainate Receptors ◽  
Mammalian Brain ◽  
Receptor Field ◽  
Neuronal Excitation ◽  
Ion Channel Proteins

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate-type glutamate receptors (AMPARs and KARs) are dynamic ion channel proteins that govern neuronal excitation and signal transduction in the mammalian brain. The four AMPAR and five KAR subunits can heteromerize with other subfamily members to create several combinations of tetrameric channels with unique physiological and pharmacological properties. While both receptor classes are noted for their rapid, millisecond-scale channel gating in response to agonist binding, the intricate structural rearrangements underlying their function have only recently been elucidated. This chapter begins with a review of AMPAR and KAR nomenclature, topology, and rules of assembly. Subsequently, receptor gating properties are outlined for both single-channel and synaptic contexts. The structural biology of AMPAR and KAR proteins is also discussed at length, with particular focus on the ligand-binding domain, where allosteric regulation and alternative splicing work together to dictate gating behavior. Toward the end of the chapter there is an overview of several classes of auxiliary subunits, notably transmembrane AMPAR regulatory proteins and Neto proteins, which enhance native AMPAR and KAR expression and channel gating, respectively. Whether bringing an ion channel novice up to speed with glutamate receptor theory and terminology or providing a refresher for more seasoned biophysicists, there is much to appreciate in this summation of work from the glutamate receptor field.

scholarly journals Comparative analysis of spreading depolarizations in brain slices exposed to osmotic or metabolic stress

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Rita Frank ◽  
Ferenc Bari ◽  
Ákos Menyhárt ◽  
Eszter Farkas
Keyword(s):  
Osmotic Stress ◽  
Tissue Injury ◽  
Brain Slices ◽  
Field Potential ◽  
Metabolic Stress ◽  
Glucose Deprivation ◽  
Optical Signal ◽  
Light Illumination ◽  
Glass Capillary ◽  
Experimental Conditions

Abstract Background Recurrent spreading depolarizations (SDs) occur in stroke and traumatic brain injury and are considered as a hallmark of injury progression. The complexity of conditions associated with SD in the living brain encouraged researchers to study SD in live brain slice preparations, yet methodological differences among laboratories complicate integrative data interpretation. Here we provide a comparative evaluation of SD evolution in live brain slices, in response to selected SD triggers and in various media, under otherwise standardized experimental conditions. Methods Rat live coronal brain slices (350 μm) were prepared (n = 51). Hypo-osmotic medium (Na+ content reduced from 130 to 60 mM, HM) or oxygen-glucose deprivation (OGD) were applied to cause osmotic or ischemic challenge. Brain slices superfused with artificial cerebrospinal fluid (aCSF) served as control. SDs were evoked in the control condition with pressure injection of KCl or electric stimulation. Local field potential (LFP) was recorded via an intracortical glass capillary electrode, or intrinsic optical signal imaging was conducted at white light illumination to characterize SDs. TTC and hematoxylin-eosin staining were used to assess tissue damage. Results Severe osmotic stress or OGD provoked a spontaneous SD. In contrast with SDs triggered in aCSF, these spontaneous depolarizations were characterized by incomplete repolarization and prolonged duration. Further, cortical SDs under HM or OGD propagated over the entire cortex and occassionally invaded the striatum, while SDs in aCSF covered a significantly smaller cortical area before coming to a halt, and never spread to the striatum. SDs in HM displayed the greatest amplitude and the most rapid propagation velocity. Finally, spontaneous SD in HM and especially under OGD was followed by tissue injury. Conclusions While the failure of Na+/K+ ATP-ase is thought to impair tissue recovery from OGD-related SD, the tissue swelling-related hyper excitability and the exhaustion of astrocyte buffering capacity are suggested to promote SD evolution under osmotic stress. In contrast with OGD, SD propagating under hypo-osmotic condition is not terminal, yet it is associated with irreversible tissue injury. Further investigation is required to understand the mechanistic similarities or differences between the evolution of SDs spontaneously occurring in HM and under OGD.

Effect of Prozac on whole cell ionic currents in lens and corneal epithelia

1995 ◽  
Vol 269 (1) ◽  
pp. C250-C256 ◽  
Author(s):  
J. L. Rae ◽  
A. Rich ◽  
A. C. Zamudio ◽  
O. A. Candia
Keyword(s):  
Potassium Channels ◽  
Single Channel ◽  
Ionic Currents ◽  
Current Amplitude ◽  
Human Lens ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Ionic Conductances

Prozac (fluoxetine), a compound used therapeutically in humans to combat depression, has substantial effects on ionic conductances in rabbit corneal epithelial cells and in cultured human lens epithelium. In corneal epithelium, it reduces the current due to the large-conductance potassium channels that dominate this preparation. Its effects seem largely to decrease the open probability while leaving the single-channel current amplitude unaltered. In cultured human epithelium, currents from calcium-activated potassium channels and inward rectifiers are unaffected by Prozac. Delayed-rectifier potassium currents are reduced by Prozac in a complicated way that involves both gating and single-channel current amplitude. Fast tetrodotoxin-blockable sodium currents are also decreased by Prozac in this preparation. For all of these ion conductance effects, Prozac concentrations of 10(-5) to 10(-4) M are required. Whereas these levels are 10- to 100-fold higher than the plasma levels achieved in therapeutic use in humans, they are comparable to or less than levels needed for many other blockers of the ionic conductances studied here.

scholarly journals Ion channel activity in lobster skeletal muscle membrane

1993 ◽  
Vol 182 (1) ◽  
pp. 113-130
Author(s):  
M. K. Worden ◽  
R. Rahamimoff ◽  
E. A. Kravitz
Keyword(s):  
Ion Channel ◽  
Single Channel ◽  
Muscle Fibers ◽  
Muscle Membrane ◽  
Channel Activity ◽  
Experimental Conditions ◽  
Sarcolemmal Membrane ◽  
Excised Patches ◽  
Current Flows

Ion channel activity in the sarcolemmal membrane of muscle fibers is critical for regulating the excitability, and therefore the contractility, of muscle. To begin the characterization of the biophysical properties of the sarcolemmal membrane of lobster exoskeletal muscle fibers, recordings were made from excised patches of membrane from enzymatically induced muscle fiber blebs. Blebs formed as evaginations of the muscle sarcolemmal membrane and were sufficiently free of extracellular debris to allow the formation of gigaohm seals. Under simple experimental conditions using bi-ionic symmetrical recording solutions and maintained holding potentials, a variety of single channel types with conductances in the range 32–380 pS were detected. Two of these ion channel species are described in detail, both are cation channels selective for potassium. They can be distinguished from each other on the basis of their single-channel conductance and gating properties. The results suggest that current flows through a large number of ion channels that open spontaneously in bleb membranes in the absence of exogenous metabolites or hormones.

scholarly journals Effects of Bepridil on Stretch-Activated BKca Channels and Stretch-Induced Extrasystoles in Isolated Chick Hearts

2017 ◽  
pp. 459-465 ◽  
Author(s):  
H. JIN ◽  
G. IRIBE ◽  
K. NARUSE
Keyword(s):  
Membrane Potential ◽  
Single Channel ◽  
Ventricular Myocytes ◽  
Heart Rhythm ◽  
Channel Activity ◽  
Open Probability ◽  
Bkca Channels ◽  
Chick Heart ◽  
Lv Volume ◽  
Stretch Activated Channels

Various types of mechanosensitive ion channels, including cationic stretch-activated channels (SACNS) and stretch-activated BKca (SAKca) channels, modulate heart rhythm. Bepridil has been used as an antiarrhythmic drug with multiple pharmacological effects; however, whether it is effective for mechanically induced arrhythmia has not been well investigated. To test the effects of Bepridil on SAKca channels activity, cultured chick embryonic ventricular myocytes were used for single-channel recordings. Bepridil significantly reduced the open probability of the SAKca channel (PO). Next, to test the effects of bepridil on stretch-induced extrasystoles (SIE), we used an isolated 2-week-old Langendorff-perfused chick heart. The left ventricle (LV) volume was rapidly changed, and the probability of SIE was calculated in the presence and absence of bepridil, and the effect of the drug was compared with that of Gadolinium (Gd3+). Bepridil decreased the probability of SIE despite its suppressive effects on SAKca channel activity. The effects of Gd3+, which blocks both SAKca and SACNS, on the probability of SIE were the same as those of bepridil. Our results suggest that bepridil blocks not only SAKca channels but possibly also blocks SACNS, and thus decreases the stretch-induced cation influx (stabilizing membrane potential) to compensate and override the effects of the decrease in outward SAKca current (destabilizing membrane potential).

Hypoxia modulates nitric oxide-induced regulation of NMDA receptor currents and neuronal cell death

1999 ◽  
Vol 277 (4) ◽  
pp. C673-C683 ◽  
Author(s):  
Muyiwa Gbadegesin ◽  
Stefano Vicini ◽  
Sandra J. Hewett ◽  
David A. Wink ◽  
Michael Espey ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Cell Death ◽  
Steady State ◽  
Nmda Receptor ◽  
Single Channel ◽  
Receptor Activation ◽  
Channel Activity ◽  
Open Probability ◽  
Whole Cell ◽  
Nmda Channel

Nitric oxide (NO) released from a new chemical class of donors enhances N-methyl-d-aspartate (NMDA) channel activity. Using whole cell and single-channel patch-clamp techniques, we have shown that ( Z)-1-[ N-(3-ammoniopropyl)- N-( n-propyl)amino]-NO (PAPA-NO) and diethylamine NO, commonly termed NONOates, potentiate the glutamate-mediated response of recombinant rat NMDA receptors (NR1/NR2A) expressed in HEK-293 cells. The overall effect is an increase in both peak and steady-state whole cell currents induced by glutamate. Single-channel studies demonstrate a significant increase in open probability but no change in the mean single-channel open time or mean channel conductance. Reduction in oxygen levels increased and prolonged the PAPA-NO-induced change in both peak and steady-state glutamate currents in transfected HEK cells. PAPA-NO also enhanced cell death in primary cultures of rodent cortical neurons deprived of oxygen and glucose. This potentiation of neuronal injury was blocked by MK-801, indicating a critical involvement of NMDA receptor activation. The NO-induced increase in NMDA channel activity as well as NMDA receptor-mediated cell death provide firm evidence that NO modulates the NMDA channel in a manner consistent with both a physiological role under normoxic conditions and a pathophysiological role under hypoxic conditions.

A voltage-dependent and calcium-permeable ion channel in fused presynaptic terminals of Torpedo

1996 ◽  
Vol 75 (5) ◽  
pp. 1858-1870 ◽  
Author(s):  
A. Meir ◽  
R. Rahamimoff
Keyword(s):  
Ion Channel ◽  
Nerve Terminal ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Voltage Range ◽  
Voltage Dependent ◽  
The Mean ◽  
Presynaptic Nerve Terminals ◽  
Channel Open Time

1. We used a preparation of fused presynaptic nerve terminals of Torpedo electromotor nerve and the patch-clamp technique for characterization of single ion channels. We report here of a large, nonselective ion channel which is highly voltage dependent. 2. The slope conductance of the I-V relation was estimated by either direct measurement of the single-channel current amplitude at different voltages (850 +/- 18 pS (SE); n = 9) or by variance analysis (834 +/- 23 pS; n = 5). 3. The voltage dependence was examined in three ways. At steady-state DC voltage conditions, NPo (the open probability times the number of channels in the patch) was estimated. At potentials < 0 mV, the probability of the channel to open is negligible and increases dramatically, within a very narrow voltage range, to > 50% at +8 mV (n = 8). 4. In pulse experiments, the activation time delay is shorter as the voltage step reaches more positive values. The mean time for half activation (T1/2) decreases from 15 ms at +10 mV to 4 ms at +30 mV (n = 5). 5. Ensemble currents exhibit rectification in response to voltage ramps at negative potentials (n = 10). 6. The channel was found to be nonselective. Its permeability to Na+, K+, Cl-, glutamate, Ba+2, and Ca+2, relative to Na+, was 1.00, 1.00, 1.22, 1.07, 0.85, and 0.62, respectively. 7. Based on the transport number of calcium, the calculated driving force, and the mean channel open time, we estimated the number of calcium ions entering the nerve terminal upon depolarization. This number is not substantially different from the number of ions entering through voltage-dependent, calcium-selective channels in other cells. 8. We speculate that this nonselective ion channel, may serve as a calcium entry route into the nerve terminal and hence be involved in transmitter release.

scholarly journals Agonist-dependent Single Channel Current and Gating in α4β2δ and α1β2γ2S GABAA Receptors

2008 ◽  
Vol 131 (2) ◽  
pp. 163-181 ◽  
Author(s):  
Angelo Keramidas ◽  
Neil L. Harrison
Keyword(s):  
Single Channel ◽  
Reaction Scheme ◽  
Mammalian Brain ◽  
Receptor Subtype ◽  
Kinetic Properties ◽  
Open Probability ◽  
Electrophysiological Recordings ◽  
Acid Type ◽  
Single Channel Activity ◽  
Open States

The family of γ-aminobutyric acid type A receptors (GABAARs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABAARs that are mainly comprised of α1, β2, and γ2 subunits, whereas tonic inhibition is mediated by extrasynaptic GABAARs comprised of α4/6, β2, and δ subunits. We investigated the activation properties of recombinant α4β2δ and α1β2γ2S GABAARs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at α4β2δ GABAARs (β ∼1600 s−1) than at α1β2γ2S GABAARs (β ∼ 460 s−1), whereas GABA activated α1β2γ2S GABAARs more rapidly (β ∼1800 s−1) than α4β2δ GABAARs (β &lt; 440 s−1). Single channel recordings of α1β2γ2S and α4β2δ GABAARs showed that both channels open to a main conductance state of ∼25 pS at −70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited ∼36 pS openings at both channels. Saturating concentrations of GABA elicited brief (&lt;10 ms) openings with low intraburst open probability (PO ∼ 0.3) at α4β2δ GABAARs and at least two “modes” of single channel bursting activity, lasting ∼100 ms at α1β2γ2S GABAARs. The most prevalent bursting mode had a PO of ∼0.7 and was described by a reaction scheme with three open and three shut states, whereas the “high” PO mode (∼0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in α4β2δ and α1β2γ2S GABAARs occurred as a single population of bursts (PO ∼0.4–0.5) of moderate duration (∼33 ms) that could be described by schemes containing two shut and two open states for both GABAARs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.

scholarly journals NMDA receptor channel gating control by the pre-M1 helix

2020 ◽  
Vol 152 (4) ◽  
Author(s):  
Miranda J. McDaniel ◽  
Kevin K. Ogden ◽  
Steven A. Kell ◽  
Pieter B. Burger ◽  
Dennis C. Liotta ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Conformational Changes ◽  
Time Course ◽  
Transmembrane Domain ◽  
Single Channel ◽  
De Novo ◽  
Channel Gating ◽  
Open Probability ◽  
Single Channel Currents

The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

Abstract MP220: Identification And Functional Characterization Of Exosomal Bk Channels

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Shridhar Sanghvi ◽  
Julie A Dougherty ◽  
Parker Evans ◽  
Divya Sridharan ◽  
Devasena Ponnalagu ◽  
...  
Keyword(s):  
Ion Channel ◽  
Reperfusion Injury ◽  
Single Channel ◽  
Ischemia Reperfusion ◽  
Functional Characterization ◽  
Physiological Role ◽  
Bk Channels ◽  
Target Cells ◽  
Open Probability ◽  
Planar Bilayers

Extracellular vesicles (EVs) specifically exosomes are important in mediating intracellular communications, and are capable of transferring genetic information between cells. Exosomes are used as a drug delivery vehicle to carry cargo for targeted therapy and have emerged as one of the most promising candidate for treating cardiovascular diseases. Exosomes get packaged inside the cell, excise out to the extracellular environment, and deliver the cargo to the target cells. However, the precise mechanism of how exosomes handle the differential ionic environment and the physiological role of their ion channels is not determined. Given that potassium (K + ) ions has the largest gradient, we focused on identifying the presence and physiological relevance of K+ channels in exosomes. Using the in silico approach, several ion channel candidates were identified, the most prominent ion channel being large conductance Ca 2+ and voltage-activated potassium channel (BK). To record BK in exosomes, we incorporated planar bilayers and a novel electrophysiology approach called near field electrophysiology (NFE), as the canonical patch-clamp methods are not feasible due to the size of EVs. Our NFE indicates a presence of K + channels in intact exosomes and 45% of them are sensitive to IbTX. Since IbTX specifically blocks, BK channels, we estimated 2 functional channels (single-channel conductance of 300 pS with 50% open probability) in a single exosome. Plasma-derived exosomes from BK +/+ and BK -/- mice subjected to differential K + gradient indicated that functional BK channels exist in exosomes, and help in maintaining their structural integrity. Furthermore, plasma derived exosomes from BK +/+ mice showed cardioprotection from ischemia-reperfusion injury whereas exosomes from BK -/- mice did not. Thus, the presence of BK determines the packaging as well as cardioprotective function of exosomes. Overall, the study for the first time indicate a presence of functional ion channel (BK) in exosomes which plays a role in protecting cells and heart against ischemia and reperfusion injury.

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