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.

scholarly journals Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

2013 ◽  
Vol 142 (4) ◽  
pp. 367-380 ◽  
Author(s):  
David J. Combs ◽  
Hyeon-Gyu Shin ◽  
Yanping Xu ◽  
Yajamana Ramu ◽  
Zhe Lu
Keyword(s):  
Membrane Potential ◽  
Mammalian Cells ◽  
Resting Membrane Potential ◽  
Xenopus Laevis Oocytes ◽  
Channel Activity ◽  
Excitable Cells ◽  
Voltage Sensors ◽  
Voltage Gated ◽  
Activated State ◽  
Gated Channel

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K+ (KV) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a KV channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na+ (NaV) and Ca2+ channels but also markedly slows NaV channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native KV1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native NaV channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.

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 High Ca2+ permeability of a peptide-gated DEG/ENaC from Hydra

2012 ◽  
Vol 140 (4) ◽  
pp. 391-402 ◽  
Author(s):  
Stefan Dürrnagel ◽  
Björn H. Falkenburger ◽  
Stefan Gründer
Keyword(s):  
Xenopus Oocytes ◽  
Divalent Cations ◽  
Na Channel ◽  
Low Permeability ◽  
Xenopus Laevis Oocytes ◽  
Na Channels ◽  
Transient Component ◽  
Epithelial Na Channels ◽  
Hydra Magnipapillata ◽  
Secondary Activation

Degenerin/epithelial Na+ channels (DEG/ENaCs) are Na+ channels that are blocked by the diuretic amiloride. In general, they are impermeable for Ca2+ or have a very low permeability for Ca2+. We describe here, however, that a DEG/ENaC from the cnidarian Hydra magnipapillata, the Hydra Na+ channel (HyNaC), is highly permeable for Ca2+ (PCa/PNa = 3.8). HyNaC is directly gated by Hydra neuropeptides, and in Xenopus laevis oocytes expressing HyNaCs, RFamides elicit currents with biphasic kinetics, with a fast transient component and a slower sustained component. Although it was previously reported that the sustained component is unselective for monovalent cations, the selectivity of the transient component had remained unknown. Here, we show that the transient current component arises from secondary activation of the Ca2+-activated Cl− channel (CaCC) of Xenopus oocytes. Inhibiting the activation of the CaCC leads to a simple on–off response of peptide-activated currents with no apparent desensitization. In addition, we identify a conserved ring of negative charges at the outer entrance of the HyNaC pore that is crucial for the high Ca2+ permeability, presumably by attracting divalent cations to the pore. At more positive membrane potentials, the binding of Ca2+ to the ring of negative charges increasingly blocks HyNaC currents. Thus, HyNaC is the first member of the DEG/ENaC gene family with a high Ca2+ permeability.

scholarly journals A Xenopus oocyte model system to study action potentials

2018 ◽  
Vol 150 (11) ◽  
pp. 1583-1593 ◽  
Author(s):  
Aaron Corbin-Leftwich ◽  
Hannah E. Small ◽  
Helen H. Robinson ◽  
Carlos A. Villalba-Galea ◽  
Linda M. Boland
Keyword(s):  
Action Potentials ◽  
Subcellular Location ◽  
Model System ◽  
K Channel ◽  
Functional Coupling ◽  
Xenopus Laevis Oocytes ◽  
Excitable Cells ◽  
Voltage Gated ◽  
Channel Expression ◽  
Electrical Signaling

Action potentials (APs) are the functional units of fast electrical signaling in excitable cells. The upstroke and downstroke of an AP is generated by the competing and asynchronous action of Na+- and K+-selective voltage-gated conductances. Although a mixture of voltage-gated channels has been long recognized to contribute to the generation and temporal characteristics of the AP, understanding how each of these proteins function and are regulated during electrical signaling remains the subject of intense research. AP properties vary among different cellular types because of the expression diversity, subcellular location, and modulation of ion channels. These complexities, in addition to the functional coupling of these proteins by membrane potential, make it challenging to understand the roles of different channels in initiating and “temporally shaping” the AP. Here, to address this problem, we focus our efforts on finding conditions that allow reliable AP recordings from Xenopus laevis oocytes coexpressing Na+ and K+ channels. As a proof of principle, we show how the expression of a variety of K+ channel subtypes can modulate excitability in this minimal model system. This approach raises the prospect of studies on the modulation of APs by pharmacological or biological means with a controlled background of Na+ and K+ channel expression.

Ionic Basis of the Resting Membrane Potential and Action Potential in the Pharyngeal Muscle of Caenorhabditis elegans

2002 ◽  
Vol 87 (2) ◽  
pp. 954-961 ◽  
Author(s):  
Christopher J. Franks ◽  
Darrel Pemberton ◽  
Irina Vinogradova ◽  
Alan Cook ◽  
Robert J. Walker ◽  
...  
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Action Potentials ◽  
Resting Membrane Potential ◽  
Channel Blockers ◽  
Voltage Gated ◽  
C Elegans ◽  
Current Voltage ◽  
Prolongation Of Action Potential ◽  
Ionic Basis

The pharynx of C. elegans is a rhythmically active muscle that pumps bacteria into the gut of the nematode. This activity is maintained by action potentials, which qualitatively bear a resemblance to vertebrate cardiac action potentials. Here, the ionic basis of the resting membrane potential and pharyngeal action potential has been characterized using intracellular recording techniques. The resting membrane potential is largely determined by a K+permeability, and a ouabain-sensitive, electrogenic pump. As previously suggested, the action potential is at least partly dependent on voltage-gated Ca2+ channels, as the amplitude was increased as extracellular Ca2+ was increased, and decreased by L-type Ca2+ channel blockers verapamil and nifedipine. Barium caused a marked prolongation of action potential duration, suggesting that a calcium-activated K+ current may contribute to repolarization. Most notably, however, we found that action potentials were abolished in the absence of external Na+. This may be due, at least in part, to a Na+-dependent pacemaker potential. In addition, the persistence of action potentials in nominally free Ca2+, the inhibition by Na+ channel blockers procaine and quinidine, and the increase in action potential frequency caused by veratridine, a toxin that alters activation of voltage-gated Na+channels, point to the involvement of a voltage-gated Na+ current. Voltage-clamp analysis is required for detailed characterization of this current, and this is in progress. Nonetheless, these observations are quite surprising in view of the lack of any obvious candidate genes for voltage-gated Na+ channels in the C. elegans genome. It would therefore be informative to re-evaluate the data from these homology searches, with the aim of identifying the gene(s) conferring this Na+, quinidine, and veratridine sensitivity to the pharynx.

scholarly journals Statistical Approach to Incorporating Experimental Variability into a Mathematical Model of the Voltage-Gated Na+ Channel and Human Atrial Action Potential

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1516
Author(s):  
Daniel Gratz ◽  
Alexander J Winkle ◽  
Seth H Weinberg ◽  
Thomas J Hund
Keyword(s):  
Action Potential ◽  
Mathematical Models ◽  
Cardiac Myocyte ◽  
Population Level ◽  
Na Channel ◽  
Model Parameters ◽  
Healthy Population ◽  
Experimental Measurements ◽  
Voltage Gated ◽  
Experimental Variability

The voltage-gated Na+ channel Nav1.5 is critical for normal cardiac myocyte excitability. Mathematical models have been widely used to study Nav1.5 function and link to a range of cardiac arrhythmias. There is growing appreciation for the importance of incorporating physiological heterogeneity observed even in a healthy population into mathematical models of the cardiac action potential. Here, we apply methods from Bayesian statistics to capture the variability in experimental measurements on human atrial Nav1.5 across experimental protocols and labs. This variability was used to define a physiological distribution for model parameters in a novel model formulation of Nav1.5, which was then incorporated into an existing human atrial action potential model. Model validation was performed by comparing the simulated distribution of action potential upstroke velocity measurements to experimental measurements from several different sources. Going forward, we hope to apply this approach to other major atrial ion channels to create a comprehensive model of the human atrial AP. We anticipate that such a model will be useful for understanding excitability at the population level, including variable drug response and penetrance of variants linked to inherited cardiac arrhythmia syndromes.

Basis for tetrodotoxin and lidocaine effects on action potentials in dog ventricular myocytes

1988 ◽  
Vol 254 (6) ◽  
pp. H1157-H1166 ◽  
Author(s):  
J. A. Wasserstrom ◽  
J. J. Salata
Keyword(s):  
Action Potential ◽  
Action Potentials ◽  
Ventricular Myocytes ◽  
Ionic Currents ◽  
Detectable Effect ◽  
Na Channel ◽  
Na Current ◽  
Voltage Range ◽  
Purkinje Fibers ◽  
Ventricular Cells

We studied the effects of tetrodotoxin (TTX) and lidocaine on transmembrane action potentials and ionic currents in dog isolated ventricular myocytes. TTX (0.1-1 x 10(-5) M) and lidocaine (0.5-2 x 10(-5) M) decreased action potential duration, but only TTX decreased the maximum rate of depolarization (Vmax). Both TTX (1-2 x 10(-5) M) and lidocaine (2-5 x 10(-5) M) blocked a slowly inactivating toward current in the plateau voltage range. The voltage- and time-dependent characteristics of this current are virtually identical to those described in Purkinje fibers for the slowly inactivating inward Na+ current. In addition, TTX abolished the outward shift in net current at plateau potentials caused by lidocaine alone. Lidocaine had no detectable effect on the slow inward Ca2+ current and the inward K+ current rectifier, Ia. Our results indicate that 1) there is a slowly inactivating inward Na+ current in ventricular cells similar in time, voltage, and TTX sensitivity to that described in Purkinje fibers; 2) both TTX and lidocaine shorten ventricular action potentials by reducing this slowly inactivating Na+ current; 3) lidocaine has no additional actions on other ionic currents that contribute to its ability to abbreviate ventricular action potentials; and 4) although both agents shorten the action potential by the same mechanism, only TTX reduces Vmax. This last point suggests that TTX produces tonic block of Na+ current, whereas lidocaine may produce state-dependent Na+ channel block, namely, blockade of Na+ current only after Na+ channels have already been opened (inactivated-state block).

Exposure to low temperature prepares the turtle brain to withstand anoxic environments during overwintering

2021 ◽  
Author(s):  
Ebrahim Lari ◽  
Leslie T. Buck
Keyword(s):  
Membrane Potential ◽  
Low Temperature ◽  
Action Potential ◽  
Pyramidal Neurons ◽  
Cellular Damage ◽  
Severe Hypoxia ◽  
Painted Turtle ◽  
Whole Cell ◽  
Voltage Gated ◽  
The Impact

In most vertebrates, anoxia drastically reduces the production of the essential adenosine triphosphate (ATP) to power its many necessary functions, and consequently, cell death occurs within minutes. However, some vertebrates, such as the painted turtle (Chrysemys picta bellii), have evolved the ability to survive months without oxygen by simultaneously decreasing ATP supply and demand, surviving the anoxic period without any apparent cellular damage. The impact of anoxia on the metabolic function of painted turtles has received a lot of attention. Still, the impact of low temperature has received less attention and the interactive effect of anoxia and temperature even less. In the present study, we investigated the interactive impacts of reduced temperature and severe hypoxia on the electrophysiological properties of pyramidal neurons in painted turtle cerebral cortex. Our results show that an acute reduction in temperature from 20 to 5°C decreases membrane potential, action potential width and amplitude, and whole-cell conductance. Importantly, acute exposure to 5°C considerably slows membrane repolarization by voltage-gated K+ channels. Exposing pyramidal cells to severe hypoxia in addition to an acute temperature change slightly depolarized membrane potential but did not alter action potential amplitude or width and whole-cell conductance. These results suggest that acclimation to low temperatures, preceding severe environmental hypoxia, induces cellular responses in pyramidal neurons that facilitate survival under low oxygen concentration. In particular, our results show that temperature acclimation invokes a change in voltage-gated K+ channel kinetics that overcomes the acute inhibition of the channel.

Hormonal Signaling Actions on Kv7.1 (KCNQ1) Channels

2021 ◽  
Vol 61 (1) ◽  
pp. 381-400
Author(s):  
Emely Thompson ◽  
Jodene Eldstrom ◽  
David Fedida
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Cardiac Action Potential ◽  
Resting Membrane Potential ◽  
Voltage Gated ◽  
M Current ◽  
Kv7 Channels ◽  
Cardiac Action ◽  
Repolarization Phase ◽  
And Control

Kv7 channels (Kv7.1–7.5) are voltage-gated K+ channels that can be modulated by five β-subunits (KCNE1–5). Kv7.1-KCNE1 channels produce the slow-delayed rectifying K+ current, IKs, which is important during the repolarization phase of the cardiac action potential. Kv7.2–7.5 are predominantly neuronally expressed and constitute the muscarinic M-current and control the resting membrane potential in neurons. Kv7.1 produces drastically different currents as a result of modulation by KCNE subunits. This flexibility allows the Kv7.1 channel to have many roles depending on location and assembly partners. The pharmacological sensitivity of Kv7.1 channels differs from that of Kv7.2–7.5 and is largely dependent upon the number of β-subunits present in the channel complex. As a result, the development of pharmaceuticals targeting Kv7.1 is problematic. This review discusses the roles and the mechanisms by which different signaling pathways affect Kv7.1 and KCNE channels and could potentially provide different ways of targeting the channel.

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