Electrophysiological analysis of the neurotoxic action of a funnel-web spider toxin, delta-atracotoxin-HV1a, on insect voltage-gated Na+ channels

2001 ◽  
Vol 204 (4) ◽  
pp. 711-721 ◽  
Author(s):  
F. Grolleau ◽  
M. Stankiewicz ◽  
L. Birinyi-Strachan ◽  
X.H. Wang ◽  
G.M. Nicholson ◽  
...  
Keyword(s):  
Voltage Clamp ◽  
Action Potentials ◽  
Periplaneta Americana ◽  
Repetitive Firing ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Voltage Gated ◽  
Web Spider ◽  
Channel Inactivation

The effects of delta-ACTX-Hv1a, purified from the venom of the funnel-web spider Hadronyche versuta, were studied on the isolated giant axon and dorsal unpaired median (DUM) neurones of the cockroach Periplaneta americana under current- and voltage-clamp conditions using the double oil-gap technique for single axons and the patch-clamp technique for neurones. In parallel, the effects of the toxin were investigated on the excitability of rat dorsal root ganglion (DRG) neurones. In both DRG and DUM neurones, delta-ACTX-Hv1a induced spontaneous repetitive firing accompanied by plateau potentials. However, in the case of DUM neurones, plateau action potentials were facilitated when the membrane was artificially hyperpolarized. In cockroach giant axons, delta-ACTX-Hv1a also produced plateau action potentials, but only when the membrane was pre-treated with 3–4 diaminopyridine. Under voltage-clamp conditions, delta-ACTX-Hv1a specifically affected voltage-gated Na+ channels in both axons and DUM neurones. Both the current/voltage and conductance/voltage curves of the delta-ACTX-Hv1a-modified inward current were shifted 10 mV to the left of control curves. In the presence of delta-ACTX-Hv1a, steady-state Na+ channel inactivation became incomplete, causing the appearance of a non-inactivating component at potentials more positive than −40 mV. The amplitude of this non-inactivating component was dependent on the holding potential. From this study, it is concluded that, in insect neurones, delta-ACTX-Hv1a mainly affects Na+ channel inactivation by a mechanism that differs slightly from that of scorpion alpha-toxins.

scholarly journals Action potentials in Xenopus oocytes triggered by blue light

2020 ◽  
Vol 152 (5) ◽  
Author(s):  
Florian Walther ◽  
Dominic Feind ◽  
Christian vom Dahl ◽  
Christoph Emanuel Müller ◽  
Taulant Kukaj ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Blue Light ◽  
Action Potentials ◽  
Xenopus Oocytes ◽  
Na Channel ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Excitable Cells ◽  
Voltage Gated

Voltage-gated sodium (Na+) channels are responsible for the fast upstroke of the action potential of excitable cells. The different α subunits of Na+ channels respond to brief membrane depolarizations above a threshold level by undergoing conformational changes that result in the opening of the pore and a subsequent inward flux of Na+. Physiologically, these initial membrane depolarizations are caused by other ion channels that are activated by a variety of stimuli such as mechanical stretch, temperature changes, and various ligands. In the present study, we developed an optogenetic approach to activate Na+ channels and elicit action potentials in Xenopus laevis oocytes. All recordings were performed by the two-microelectrode technique. We first coupled channelrhodopsin-2 (ChR2), a light-sensitive ion channel of the green alga Chlamydomonas reinhardtii, to the auxiliary β1 subunit of voltage-gated Na+ channels. The resulting fusion construct, β1-ChR2, retained the ability to modulate Na+ channel kinetics and generate photosensitive inward currents. Stimulation of Xenopus oocytes coexpressing the skeletal muscle Na+ channel Nav1.4 and β1-ChR2 with 25-ms lasting blue-light pulses resulted in rapid alterations of the membrane potential strongly resembling typical action potentials of excitable cells. Blocking Nav1.4 with tetrodotoxin prevented the fast upstroke and the reversal of the membrane potential. Coexpression of the voltage-gated K+ channel Kv2.1 facilitated action potential repolarization considerably. Light-induced action potentials were also obtained by coexpressing β1-ChR2 with either the neuronal Na+ channel Nav1.2 or the cardiac-specific isoform Nav1.5. Potential applications of this novel optogenetic tool are discussed.

Actions of insect toxin and other toxins derived from the venom of the scorpion Androctonus australis on isolated giant axons of the cockroach (Periplaneta americana)

1982 ◽  
Vol 97 (1) ◽  
pp. 67-77
Author(s):  
M. Pelhate ◽  
E. Zlotkin
Keyword(s):  
Voltage Clamp ◽  
Action Potentials ◽  
Time Course ◽  
Periplaneta Americana ◽  
Sodium Current ◽  
Giant Axon ◽  
Repetitive Firing ◽  
Current Clamp ◽  
Axonal Membrane ◽  
Insect Toxin

1. Insect toxin, mammal toxins I and II and crustacean toxin were obtained from the venom of the scorpion Androctonus australis. Their effects on the isolated giant axon of the cockroach Periplaneta americana were investigated by current-clamp and voltage-clamp techniques. 2. In current-clamp conditions, mammal toxins and crustacean toxin (1.3-13 microM) induced a large prolongation of the falling phase of the evoked action potentials. Insect toxin (0.13-3.3 microM) induced a progressive slow depolarization of the membrane potential and repetitive firing of action potentials. No changes in the time-course of the action potential were induced by insect toxin. 3. In voltage-clamp conditions, mammal and crustacean toxins induced a slowing of the turn-off of the transient inward sodium current, with either no change or a small increase in the peak sodium current. Insect toxin by contrast induced an increase in the peak sodium current and a slowing of the sodium current turn-off, this effect being greatest at lower values of the clamped membrane voltage. 4. It is concluded that the repetitive activity induced by insect toxin results from a voltage-dependent modulation of sodium inactivation coupled with an increase in both the resting and active sodium permeabilities of the cockroach axonal membrane.

Two Types of Voltage-Gated K+ Currents in Dissociated Heart Ventricular Muscle Cells of the Snail Lymnaea stagnalis

1999 ◽  
Vol 82 (5) ◽  
pp. 2415-2427 ◽  
Author(s):  
M. S. Yeoman ◽  
P. R. Benjamin
Keyword(s):  
Voltage Clamp ◽  
Action Potentials ◽  
Muscle Cells ◽  
Delayed Rectifier ◽  
Voltage Sensitivity ◽  
Ventricular Muscle ◽  
Current Time ◽  
Voltage Gated ◽  
Time To Peak ◽  
Voltage Dependent

We have used a combination of current-clamp and voltage-clamp techniques to characterize the electrophysiological properties of enzymatically dissociated Lymnaea heart ventricle cells. Dissociated ventricular muscle cells had average resting membrane potentials of −55 ± 5 mV. When hyperpolarized to potentials between −70 and −63 mV, ventricle cells were capable of firing repetitive action potentials (8.5 ± 1.2 spikes/min) that failed to overshoot 0 mV. The action potentials were either simple spikes or more complex spike/plateau events. The latter were always accompanied by strong contractions of the muscle cell. The waveform of the action potentials were shown to be dependent on the presence of extracellular Ca2+ and K+ ions. With the use of the single-electrode voltage-clamp technique, two types of voltage-gated K+ currents were identified that could be separated by differences in their voltage sensitivity and time-dependent kinetics. The first current activated between −50 and −40 mV. It was relatively fast to activate (time-to-peak; 13.7 ± 0.7 ms at +40 mV) and inactivated by 53.3 ± 4.9% during a maintained 200-ms depolarization. It was fully available for activation below −80 mV and was completely inactivated by holding potentials more positive than −40 mV. It was completely blocked by 5 mM 4-aminopyridine (4-AP) and by concentrations of tetraethylammonium chloride (TEA) >10 mM. These properties characterize this current as a member of the A-type family of voltage-dependent K+ currents. The second voltage-gated K+ current activated at more depolarized potentials (−30 to −20 mV). It activated slower than the A-type current (time-to-peak; 74.1 ± 3.9 ms at +40 mV) and showed little inactivation (6.2 ± 2.1%) during a maintained 200-ms depolarization. The current was fully available for activation below −80 mV with a proportion of the current still available for activation at potentials as positive as 0 mV. The current was completely blocked by 1–3 mM TEA. These properties characterize this current as a member of the delayed rectifier family of voltage-dependent K+ currents. The slow activation rates and relatively depolarized activation thresholds of the two K+ currents are suggestive that their main role is to contribute to the repolarization phase of the action potential.

Insulin activates single amiloride-blockable Na channels in a distal nephron cell line (A6)

1992 ◽  
Vol 263 (3) ◽  
pp. F392-F400 ◽  
Author(s):  
Y. Marunaka ◽  
N. Hagiwara ◽  
H. Tohda
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Apical Membrane ◽  
Na Channel ◽  
Na Channels ◽  
Distal Nephron ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Patch Clamp Technique ◽  
Open Probability

Using the patch-clamp technique, we studied the effect of insulin on an amiloride-blockable Na channel in the apical membrane of a distal nephron cell line (A6) cultured on permeable collagen films for 10-14 days. NPo (N, number of channels per patch membrane; Po, average value of open probability of individual channels in the patch) under baseline conditions was 0.88 +/- 0.12 (SE)(n = 17). After making cell-attached patches on the apical membrane which contained Na channels, insulin (1 mU/ml) was applied to the serosal bath. While maintaining the cell-attached patch, NPo significantly increased to 1.48 +/- 0.19 (n = 17; P less than 0.001) after 5-10 min of insulin application. The open probability of Na channels was 0.39 +/- 0.01 (n = 38) under baseline condition, and increased to 0.66 +/- 0.03 (n = 38, P less than 0.001) after addition of insulin. The baseline single-channel conductance was 4pS, and neither the single-channel conductance nor the current-voltage relationship was significantly changed by insulin. These results indicate that insulin increases Na absorption in the distal nephron by increasing the open probability of the amiloride-blockable Na channel.

Slowly inactivating outward currents in a cuticular mechanoreceptor neuron of the cockroach (Periplaneta americana)

1995 ◽  
Vol 74 (3) ◽  
pp. 1200-1211 ◽  
Author(s):  
P. H. Torkkeli ◽  
A. S. French
Keyword(s):  
Voltage Clamp ◽  
Time Constant ◽  
Action Potentials ◽  
Periplaneta Americana ◽  
Outward Current ◽  
Equilibrium Potential ◽  
Channel Blockers ◽  
Outward Currents ◽  
Frequency Of Action Potentials ◽  
K Outward Current

1. Although rapid adaptation is a widespread feature of sensory receptors, its ionic basis has not been clearly established in any touch receptor, because their small sizes have severely restricted the range of experiments tat can be performed. In the cockroach tactile spine, intracellular voltage-clamp recordings are now possible. 2. The basic electrophysiological properties of the cockroach femoral tactile spine neuron were studied using discontinuous (switching) single-electrode current- and voltage-clamp recordings. A slowly inactivating voltage-sensitive K+ outward current was detected after the major inward currents were blocked with tetrodotoxin. 3. The total outward current activated in < 1 ms at voltages above 0 mV. At moderate depolarizations it did not inactivate, but at higher depolarizations an inactivation time constant of approximately 260 ms was measured. Some recordings also revealed an additional, slower inactivation time constant of approximately 2.5 s. 4. More than half of the voltage-sensitive K+ outward current could be blocked with the Ca2+ channel blockers Co2+ and Cd2+. Tetraethylammonium chloride (TEA) also reduced the amplitude of the outward current to about half of its original amplitude. The actions of both blockers were reversible and probably reflect overlapping blockades of two separate outward currents. 5. The reversal potentials of the currents that remained after block with Co2+ (-91.7 mV) or TEA (-86.8 mV) were both near the K+ equilibrium potential expected for the tactile spine neuron. The voltage dependencies of activation of the Co(2+)- and TEA-resistant currents were both well fitted by Boltzmann distributions, giving values of half maximal activation (V50) equal to -34.5 mV for the Co(2+)-resistant current and -51.3 mV for the TEA-resistant current. 6. Current-clamp recordings revealed that the TEA-sensitive K+ current was the major component of action potential repolarization but that it did not effect the frequency of action potentials evoked by steady depolarization. On the other hand, blockers of Ca(2+)-sensitive K+ currents (Cd2+, Co2+, or charybdotoxin) reduced adaptation and increased the frequency of action potentials significantly but did not effect the duration or amplitude of individual action potentials.

scholarly journals Block of Neuronal Na+Channels by Antidepressant Duloxetine in a State-dependent Manner

2010 ◽  
Vol 113 (3) ◽  
pp. 655-665 ◽  
Author(s):  
Sho-Ya Wang ◽  
Joanna Calderon ◽  
Ging Kuo Wang
Keyword(s):  
Open Channel ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Dependent Manner ◽  
Channel Block ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Channel Block ◽  
Human Embryonic Kidney Cells ◽  
Na Currents

Background Duloxetine is a mixed serotonin-norepinephrine reuptake inhibitor used for major depressive disorder. Duloxetine is also beneficial for patients with diabetic peripheral neuropathy and with fibromyalgia, but how it works remains unclear. Methods We used the whole cell, patch clamp technique to test whether duloxetine interacts with the neuronal Nav1.7 Na+ channel as a potential target. Resting and inactivated Nav1.7 Na+ channel block by duloxetine were measured by conventional pulse protocols in transfected human embryonic kidney cells. The open-channel block was determined directly using inactivation-deficient mutant Nav1.7 Na+ channels. Results The 50% inhibitory concentration (IC50) of duloxetine for the resting and inactivated wild-type hNav1.7 Na+ channel were 22.1+/-0.4 and 1.79+/-0.10 microM, respectively (mean+/-SE, n=5). The IC50 for the open Na+ channel was 0.25+/-0.02 microM (n=5), as determined by the block of persistent late Nav1.7 Na+ currents. Similar open-channel block by duloxetine was found in the muscle Nav1.4 isoform (IC50=0.51+/-0.05 microM; n=5). Block by duloxetine appeared via the conserved local anesthetic receptor as determined by site-directed mutagenesis. Finally, duloxetine elicited strong use-dependent block of neuronal transient Nav1.7 Na+ currents during repetitive stimulations. Conclusions Duloxetine blocks persistent late Nav1.7 Na+ currents preferentially, which may in part account for its analgesic action.

scholarly journals Kinetics of 9-aminoacridine block of single Na channels.

1984 ◽  
Vol 84 (3) ◽  
pp. 361-377 ◽  
Author(s):  
D Yamamoto ◽  
J Z Yeh
Keyword(s):  
Rate Constant ◽  
Voltage Dependence ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

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