Effects of Propofol on Sodium Channel-dependent Sodium Influx and Glutamate Release in Rat Cerebrocortical Synaptosomes

1997 ◽  
Vol 86 (2) ◽  
pp. 428-439 ◽  
Author(s):  
L. Ratnakumari ◽  
H. C. Hemmings
Keyword(s):  
Glutamate Release ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
General Anesthetic ◽  
Adult Rats ◽  
Sodium Influx ◽  
Voltage Dependent ◽  
Channel Dependent ◽  
Presynaptic Nerve Terminals

Background Previous electrophysiologic studies have implicated voltage-dependent Na+ channels as a molecular site of action for propofol. This study considered the effects of propofol on Na+ channel-mediated Na+ influx and neurotransmitter release in rat brain synaptosomes (isolated presynaptic nerve terminals). Methods Purified cerebrocortical synaptosomes from adult rats were used to determine the effects of propofol on Na+ influx through voltage-dependent Na+ channels (measured using 22Na+) and intracellular [Na+] (measured by ion-specific spectrofluorimetry). For comparison, the effects of propofol on synaptosomal glutamate release evoked by 4-aminopyridine (Na+ channel dependent), veratridine (Na+ channel dependent), KCi (Na+ channel independent) were studied using enzyme-coupled fluorimetry. Results Propofol inhibited veratridine-evoked 22Na+ influx (inhibitory concentration of 50% [IC50] = 46 microM; 8.9 microM free) and changes in intracellular [Na+] (IC50 = 13 microM; 6.3 microM free) in synaptosomes in a dose-dependent manner. Propofol also inhibited 4-aminopyridine-evoked (IC50 = 39 microM; 19 microM free) and veratridine (20 microM)-evoked (IC50 = 30 microM; 14 microM free), but not KCi-evoked (up to 100 microM) glutamate release from synaptosomes. Conclusions Inhibition of Na+ channel-mediated Na+ influx, increased in intracellular [Na+], and glutamate release occurred in synaptosomes at concentrations of propofol achieved clinically. These results support a role for neuronal voltage-dependent Na+ channels as a molecular target for presynaptic general anesthetic effects.

Inhibition of Presynaptic Sodium Channels by Halothane 

1998 ◽  
Vol 88 (4) ◽  
pp. 1043-1054 ◽  
Author(s):  
Lingamaneni Ratnakumari ◽  
Hugh C. Hemmings
Keyword(s):  
Radioligand Binding ◽  
Glutamate Release ◽  
Receptor Site ◽  
Na Channel ◽  
Na Channels ◽  
General Anesthetic ◽  
Adult Rat ◽  
Anesthetic Agents ◽  
Receptor Sites ◽  
Channel Dependent

Background Recent electrophysiologic studies indicate that clinical concentrations of volatile general anesthetic agents inhibit central nervous system sodium (Na+) channels. In this study, the biochemical effects of halothane on Na+ channel function were determined using rat brain synaptosomes (pinched-off nerve terminals) to assess the role of presynaptic Na+ channels in anesthetic effects. Methods Synaptosomes from adult rat cerebral cortex were used to determine the effects of halothane on veratridine-evoked Na+ channel-dependent Na+ influx (using 22Na+), changes in intrasynaptosomal [Na+] (using ion-specific spectrofluorometry), and neurotoxin interactions with specific receptor sites of the Na+ channel (by radioligand binding). The potential physiologic and functional significance of these effects was determined by measuring the effects of halothane on veratridine-evoked Na+ channel-dependent glutamate release (using enzyme-coupled spectrofluorometry). Results Halothane inhibited veratridine-evoked 22Na+ influx (IC50 = 1.1 mM) and changes in intrasynaptosomal [Na+] (concentration for 50% inhibition [IC50] = 0.97 mM), and it specifically antagonized [3H]batrachotoxinin-A 20-alpha-benzoate binding to receptor site two of the Na+ channel (IC50 = 0.53 mM). Scatchard and kinetic analysis revealed an allosteric competitive mechanism for inhibition of toxin binding. Halothane inhibited veratridine-evoked glutamate release from synaptosomes with comparable potency (IC50 = 0.67 mM). Conclusions Halothane significantly inhibited Na+ channel-mediated Na influx, increases in intrasynaptosomal [Na+] and glutamate release, and competed with neurotoxin binding to site two of the Na+ channel in synaptosomes at concentrations within its clinical range (minimum alveolar concentration, 1-2). These findings support a role for presynaptic Na+ channels as a molecular target for general anesthetic effects.

scholarly journals Agonist-specific voltage-dependent gating of lysosomal two-pore Na+ channels

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Xiaoli Zhang ◽  
Wei Chen ◽  
Ping Li ◽  
Raul Calvo ◽  
Noel Southall ◽  
...  
Keyword(s):  
Small Molecule ◽  
Membrane Trafficking ◽  
High Throughput Screening ◽  
Voltage Dependence ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Channels ◽  
Channel Activity ◽  
Ph Homeostasis ◽  
Voltage Dependent

Mammalian two-pore-channels (TPC1, 2; TPCN1, TPCN2) are ubiquitously- expressed, PI(3,5)P2-activated, Na+-selective channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and Ebola viral infection. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of the presumed ‘S4 voltage-sensing’ domains. By performing high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tricyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, referred to as Lysosomal Na+ channel Voltage-dependent Activators (LyNa-VAs). We also identified another compound which, like PI(3,5)P2, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal functions.

scholarly journals Kinetic analysis of the action of Leiurus scorpion alpha-toxin on ionic currents in myelinated nerve.

1985 ◽  
Vol 86 (5) ◽  
pp. 739-762 ◽  
Author(s):  
G K Wang ◽  
G Strichartz
Keyword(s):  
Steady State ◽  
Ionic Currents ◽  
Na Channel ◽  
Dependent Manner ◽  
Na Current ◽  
Toxin Concentration ◽  
Toxin Molecule ◽  
Channel Inactivation ◽  
Na Currents ◽  
Voltage Dependent

The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.

Structure and function of voltage-dependent sodium channels: comparison of brain II and cardiac isoforms

1996 ◽  
Vol 76 (3) ◽  
pp. 887-926 ◽  
Author(s):  
H. A. Fozzard ◽  
D. A. Hanck
Keyword(s):  
Amino Acid ◽  
Three Dimensional ◽  
Point Mutations ◽  
Amino Acid Sequences ◽  
Na Channel ◽  
Na Channels ◽  
Voltage Dependent ◽  
And Function ◽  
Relationship Of ◽  
The Relationship

Cardiac and nerve Na channels have broadly similar functional properties and amino acid sequences, but they demonstrate specific differences in gating, permeation, ionic block, modulation, and pharmacology. Resolution of three-dimensional structures of Na channels is unlikely in the near future, but a number of amino acid sequences from a variety of species and isoforms are known so that channel differences can be exploited to gain insight into the relationship of structure to function. The combination of molecular biology to create chimeras and channels with point mutations and high-resolution electrophysiological techniques to study function encourage the idea that predictions of structure from function are possible. With the goal of understanding the special properties of the cardiac Na channel, this review examines the structural (sequence) similarities between the cardiac and nerve channels and considers what is known about the relationship of structure to function for voltage-dependent Na channels in general and for the cardiac Na channels in particular.

scholarly journals Dynamics of 9-aminoacridine block of sodium channels in squid axons.

1979 ◽  
Vol 73 (1) ◽  
pp. 1-21 ◽  
Author(s):  
J Z Yeh
Keyword(s):  
Ionic Channels ◽  
Na Channel ◽  
Na Channels ◽  
Drug Molecule ◽  
Na Currents ◽  
Voltage Dependent ◽  
A Site ◽  
Kinetics Of ◽  
Block Of Na Channels ◽  
Single Exponential Function

The interactions of 9-aminoacridine with ionic channels were studied in internally perfused squid axons. The kinetics of block of Na channels with 9-aminoacridine varies depending on the voltage-clamp pulses and the state of gating machinery of Na channels. In an axon with intact h gate, the block exhibits frequency- and voltage-dependent characteristics. However, in the pronase-perfused axon, the frequency-dependent block disappears, whereas the voltage-dependent block remains unchanged. A time-dependent decrease in Na currents indicative of direct block of Na channel by drug molecule follows a single exponential function with a time constant of 2.0 +/- 0.18 and 1.0 +/- 0.19 ms (at 10 degrees C and 80 m V) for 30 and 100 microM 9-aminoacridine, respectively. A steady-state block can be achieved during a single 8-ms depolarizing pulse when the h gate has been removed. The block in the h-gate intact axon can be achieved only with multiple conditioning pulses. The voltage-dependent block suggests that 9-aminoacridine binds to a site located halfway across the membrane with a dissociation constant of 62 microM at 0 m V. 9-Aminoacridine also blocks K channels, and the block is time- and voltage-dependent.

scholarly journals Comparative Effects of Halogenated Inhaled Anesthetics on Voltage-gated Na+Channel Function

2009 ◽  
Vol 110 (3) ◽  
pp. 582-590 ◽  
Author(s):  
Wei Ouyang ◽  
Karl F. Herold ◽  
Hugh C. Hemmings
Keyword(s):  
Rank Order ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Na Channel ◽  
Dependent Manner ◽  
Fast Inactivation ◽  
Inhaled Anesthetics ◽  
Voltage Gated ◽  
Channel Inhibition ◽  
Voltage Dependent

Background Inhibition of voltage-gated Na channels (Na(v)) is implicated in the synaptic actions of volatile anesthetics. We studied the effects of the major halogenated inhaled anesthetics (halothane, isoflurane, sevoflurane, enflurane, and desflurane) on Na(v)1.4, a well-characterized pharmacological model for Na(v) effects. Methods Na currents (I(Na)) from rat Na(v)1.4 alpha-subunits heterologously expressed in Chinese hamster ovary cells were analyzed by whole cell voltage-clamp electrophysiological recording. Results Halogenated inhaled anesthetics reversibly inhibited Na(v)1.4 in a concentration- and voltage-dependent manner at clinical concentrations. At equianesthetic concentrations, peak I(Na) was inhibited with a rank order of desflurane > halothane approximately enflurane > isoflurane approximately sevoflurane from a physiologic holding potential (-80 mV). This suggests that the contribution of Na channel block to anesthesia might vary in an agent-specific manner. From a hyperpolarized holding potential that minimizes inactivation (-120 mV), peak I(Na) was inhibited with a rank order of potency for tonic inhibition of peak I(Na) of halothane > isoflurane approximately sevoflurane > enflurane > desflurane. Desflurane produced the largest negative shift in voltage-dependence of fast inactivation consistent with its more prominent voltage-dependent effects. A comparison between isoflurane and halothane showed that halothane produced greater facilitation of current decay, slowing of recovery from fast inactivation, and use-dependent block than isoflurane. Conclusions Five halogenated inhaled anesthetics all inhibit a voltage-gated Na channel by voltage- and use-dependent mechanisms. Agent-specific differences in efficacy for Na channel inhibition due to differential state-dependent mechanisms creates pharmacologic diversity that could underlie subtle differences in anesthetic and nonanesthetic actions.

Calcium Channel Currents in Acutely Dissociated Intracardiac Neurons From Adult Rats

1997 ◽  
Vol 77 (4) ◽  
pp. 1769-1778 ◽  
Author(s):  
Seong-Woo Jeong ◽  
Robert D. Wurster
Keyword(s):  
Calcium Channel ◽  
Fold Change ◽  
Dependent Manner ◽  
High Concentration ◽  
Patch Clamp Technique ◽  
Adult Rats ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Channel Currents ◽  
Intracardiac Neurons

Jeong, Seong-Woo and Robert D. Wurster. Calcium channel currents in acutely dissociated intracardiac neurons from adult rats. J. Neurophysiol. 77: 1769–1778, 1997. With the use of the whole cell patch-clamp technique, multiple subtypes of voltage-activated calcium channels, as indicated by measuring Ba2+ currents, were pharmacologically identified in acutely dissociated intracardiac neurons from adult rats. All tested neurons that were held at −80 mV displayed only high-voltage-activated (HVA) Ca2+ channel currents that were completely blocked by 100 μM CdCl2. The current density of HVA Ca2+ currents was dependent on the external Ca2+ concentration. The Ba2+ (5 mM) currents were half-activated at −16.3 mV with a slope of 5.6 mV per e-fold change. The steady-state inactivation was also voltage dependent with half-inactivation at −33.7 mV and a slope of −12.1 mV per e-fold change. The most effective L-type channel activator, FPL 64176 (2 μM), enhanced the Ba2+ current in a voltage-dependent manner. When cells were held at −80 mV, the saturating concentration (10 μM) of nifedipine blocked ∼11% of the control Ba2+ current. The major component of the Ca2+ channels was N type (63%), which was blocked by a saturating concentration (1 μM) of ω-conotoxin GVIA. Approximately 19% of the control Ba2+ current was sensitive to ω-conotoxin MVIIC (5 μM) but insensitive to low concentrations (30 and 100 nM) of ω-agatoxin IVA (ω-Aga IVA). In addition, a high concentration (1 μM) of ω-Aga IVA occluded the effect of ω-conotoxin MVIIC. Taken together, these results indicate that the ω-conotoxin MVIIC-sensitive current represents only the Q type of Ca2+ channels. The current that was insensitive to nifedipine and various toxins represents the R-type current (7%), which was sensitive to 100 μM NiCl2. In conclusion, the intracardiac neurons from adult rats express at least four different subtypes (L, N, Q, and R) of HVA Ca2+ channels. This information is essential for understanding the regulation of synaptic transmission and excitability of intracardiac neurons by different neurotransmitters and neural regulation of cardiac functions.

scholarly journals RBP2 stabilizes slow Cav1.3 Ca2+ channel inactivation properties of cochlear inner hair cells

2019 ◽  
Vol 472 (1) ◽  
pp. 3-25 ◽  
Author(s):  
Nadine J. Ortner ◽  
Alexandra Pinggera ◽  
Nadja T. Hofer ◽  
Anita Siller ◽  
Niels Brandt ◽  
...  
Keyword(s):  
Hair Cells ◽  
Splice Variant ◽  
Glutamate Release ◽  
Inactivation Kinetics ◽  
Dependent Manner ◽  
Voltage Range ◽  
Inner Hair Cells ◽  
Whole Cell Patch Clamp ◽  
Dependent Inactivation ◽  
Voltage Dependent

AbstractCav1.3 L-type Ca2+ channels (LTCCs) in cochlear inner hair cells (IHCs) are essential for hearing as they convert sound-induced graded receptor potentials into tonic postsynaptic glutamate release. To enable fast and indefatigable presynaptic Ca2+ signaling, IHC Cav1.3 channels exhibit a negative activation voltage range and uniquely slow inactivation kinetics. Interaction with CaM-like Ca2+-binding proteins inhibits Ca2+-dependent inactivation, while the mechanisms underlying slow voltage-dependent inactivation (VDI) are not completely understood. Here we studied if the complex formation of Cav1.3 LTCCs with the presynaptic active zone proteins RIM2α and RIM-binding protein 2 (RBP2) can stabilize slow VDI. We detected both RIM2α and RBP isoforms in adult mouse IHCs, where they co-localized with Cav1.3 and synaptic ribbons. Using whole-cell patch-clamp recordings (tsA-201 cells), we assessed their effect on the VDI of the C-terminal full-length Cav1.3 (Cav1.3L) and a short splice variant (Cav1.342A) that lacks the C-terminal RBP2 interaction site. When co-expressed with the auxiliary β3 subunit, RIM2α alone (Cav1.342A) or RIM2α/RBP2 (Cav1.3L) reduced Cav1.3 VDI to a similar extent as observed in IHCs. Membrane-anchored β2 variants (β2a, β2e) that inhibit inactivation on their own allowed no further modulation of inactivation kinetics by RIM2α/RBP2. Moreover, association with RIM2α and/or RBP2 consolidated the negative Cav1.3 voltage operating range by shifting the channel’s activation threshold toward more hyperpolarized potentials. Taken together, the association with “slow” β subunits (β2a, β2e) or presynaptic scaffolding proteins such as RIM2α and RBP2 stabilizes physiological gating properties of IHC Cav1.3 LTCCs in a splice variant-dependent manner ensuring proper IHC function.

scholarly journals Augmentation of Recovery from Inactivation by Site-3 Na Channel Toxins

1999 ◽  
Vol 113 (2) ◽  
pp. 333-346 ◽  
Author(s):  
G. Richard Benzinger ◽  
Gayle S. Tonkovich ◽  
Dorothy A. Hanck
Keyword(s):  
Time Course ◽  
Genetic Diseases ◽  
Low Frequency ◽  
Periodic Paralysis ◽  
Na Channel ◽  
Na Channels ◽  
Cell Recovery ◽  
Reverse Transcription Pcr ◽  
Recovery From Inactivation ◽  
Voltage Dependent

Site-3 toxins isolated from several species of scorpion and sea anemone bind to voltage-gated Na channels and prolong the time course of INa by interfering with inactivation with little or no effect on activation, effects that have similarities to those produced by genetic diseases in skeletal muscle (myotonias and periodic paralysis) and heart (long QT syndrome). Some published reports have also reported the presence of a noninactivating persistent current in site-3 toxin-treated cells. We have used the high affinity site-3 toxin Anthopleurin B to study the kinetics of this current and to evaluate kinetic differences between cardiac (in RT4-B8 cells) and neuronal (in N1E-115 cells) Na channels. By reverse transcription–PCR from N1E-115 cell RNA multiple Na channel transcripts were detected; most often isolated were sequences homologous to rBrII, although at low frequency sequences homologous to rPN1 and rBrIII were also detected. Toxin treatment induced a voltage-dependent plateau current in both isoforms for which the relative amplitude (plateau current/peak current) approached a constant value with depolarization, although the magnitude was much greater for neuronal (17%) than cardiac (5%) INa. Cell-attached patch recordings revealed distinct quantitative differences in open times and burst durations between isoforms, but for both isoforms the plateau current comprised discrete bursts separated by quiescent periods, consistent with toxin induction of an increase in the rate of recovery from inactivation rather than a modal failure of inactivation. In accord with this hypothesis, toxin increased the rate of whole-cell recovery at all tested voltages. Moreover, experimental data support a model whereby recovery at negative voltages is augmented through closed states rather than through the open state. We conclude that site-3 toxins produce qualitatively similar effects in cardiac and neuronal channels and discuss implications for channel kinetics.

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart

1995 ◽  
Vol 269 (1) ◽  
pp. H203-H214 ◽  
Author(s):  
A. I. Undrovinas ◽  
G. S. Shander ◽  
J. C. Makielski
Keyword(s):  
Membrane Proteins ◽  
Actin Polymerization ◽  
Integral Membrane Proteins ◽  
Na Channel ◽  
Na Channels ◽  
Open Probability ◽  
Whole Cell ◽  
Voltage Dependent ◽  
D 20 ◽  
Inside Out

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

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