Voltage Dependent Conductances: Gating Currents and Single Channel Recordings

AbstractThe activation of voltage-dependent ion channels is associated with the movement gating charges, that give rise to gating currents. Although gating currents originating from a single channel are too small to be detected, analysis of the fluctuations of macroscopic gating currents originating from a population of channels can make a good guess of their magnitude. The analysis of experimental gating current fluctuations, when interpreted in terms of a Markov model of channel activation, are in accordance with the presence of a main step along the activation pathway carrying 2.3-2.4 e0 of charge. To give a physical interpretation to these results and to relate them to the known atomic structure of the voltage sensor domain, we employed a Brownian model of voltage-dependent gating that we recently developed using structural information and applying the laws of electrodynamics. The model was capable to reproduce gating currents and gating current fluctuations essentially similar to those experimentally observed. The detailed study of this model output, also performed by making several simplifications aimed at understanding the basic dependencies of the gating current fluctuations, suggests that in real ion channels the voltage sensor does not move in a fully Markovian regimen due to the relatively low (<5 kT) energy barriers separating successive intermediate states. As a consequence, the simultaneous jump of multiple gating charges through the gating pore becomes frequent, and this occurrence is at the origin of the relatively high single-step charge detected by assuming Markovian behavior.

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Activation and Two Modes of Blockade by Strontium of Ca2+-activated K+ Channels in Goldfish Saccular Hair Cells

The Journal of General Physiology ◽

10.1085/jgp.111.2.363 ◽

1998 ◽

Vol 111 (2) ◽

pp. 363-379 ◽

Cited By ~ 11

Author(s):

Izumi Sugihara

Keyword(s):

Dissociation Constant ◽

Time Constant ◽

Hair Cells ◽

Single Channel ◽

Hill Coefficient ◽

K Channels ◽

Voltage Dependent ◽

Time Courses ◽

The Hill ◽

Inside Out

Effects of internal Sr2+ on the activity of large-conductance Ca2+-activated K+ channels were studied in inside-out membrane patches from goldfish saccular hair cells. Sr2+ was approximately one-fourth as potent as Ca2+ in activating these channels. Although the Hill coefficient for Sr2+ was smaller than that for Ca2+, maximum open-state probability, voltage dependence, steady state gating kinetics, and time courses of activation and deactivation of the channel were very similar under the presence of equipotent concentrations of Ca2+ and Sr2+. This suggests that voltage-dependent activation is partially independent of the ligand. Internal Sr2+ at higher concentrations (>100 μM) produced fast and slow blockade both concentration and voltage dependently. The reduction in single-channel amplitude (fast blockade) could be fitted with a modified Woodhull equation that incorporated the Hill coefficient. The dissociation constant at 0 mV, the Hill coefficient, and zd (a product of the charge of the blocking ion and the fraction of the voltage difference at the binding site from the inside) in this equation were 58–209 mM, 0.69–0.75, 0.45–0.51, respectively (n = 4). Long shut events (slow blockade) produced by Sr2+ lasted ∼10–200 ms and could be fitted with single-exponential curves (time constant, τl−s) in shut-time histograms. Durations of burst events, periods intercalated by long shut events, could also be fitted with single exponentials (time constant, τb). A significant decrease in τb and no large changes in τl−s were observed with increased Sr2+ concentration and voltage. These findings on slow blockade could be approximated by a model in which single Sr2+ ions bind to a blocking site within the channel pore beyond the energy barrier from the inside, as proposed for Ba2+ blockade. The dissociation constant at 0 mV and zd in the Woodhull equation for this model were 36–150 mM and 1–1.8, respectively (n = 3).

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Mechanically Stable Lipid Bilayers in Teflon-Coated Silicon Chips for Single-Channel Recordings

Micro and Nanosystems ◽

10.2174/1876402911204010002 ◽

2012 ◽

Vol 4 (1) ◽

pp. 2-7 ◽

Cited By ~ 10

Author(s):

Azusa Oshima ◽

Ayumi Hirano-Iwata ◽

Tomohiro Nasu ◽

Yasuo Kimura ◽

Michio Niwano

Keyword(s):

Lipid Bilayers ◽

Single Channel ◽

Single Channel Recordings ◽

Silicon Chips

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The Na+/Ca2+ Exchanger Inhibitor, KB-R7943, Blocks a Nonselective Cation Channel Implicated in Chemosensory Transduction

Journal of Neurophysiology ◽

10.1152/jn.90903.2008 ◽

2009 ◽

Vol 101 (3) ◽

pp. 1151-1159 ◽

Cited By ~ 12

Author(s):

A. Pezier ◽

Y. V. Bobkov ◽

B. W. Ache

Keyword(s):

Transient Receptor Potential ◽

Single Channel ◽

Receptor Potential ◽

Trpc Channels ◽

Dependent Manner ◽

Open Probability ◽

Olfactory Transduction ◽

Voltage Dependent ◽

Chemosensory Transduction ◽

Transient Receptor

The mechanism(s) of olfactory transduction in invertebrates remains to be fully understood. In lobster olfactory receptor neurons (ORNs), a nonselective sodium-gated cation (SGC) channel, a presumptive transient receptor potential (TRP)C channel homolog, plays a crucial role in olfactory transduction, at least in part by amplifying the primary transduction current. To better determine the functional role of the channel, it is important to selectively block the channel independently of other elements of the transduction cascade, causing us to search for specific pharmacological blockers of the SGC channel. Given evidence that the Na+/Ca2+ exchange inhibitor, KB-R7943, blocks mammalian TRPC channels, we studied this probe as a potential blocker of the lobster SGC channel. KB-R7943 reversibly blocked the SGC current in both inside- and outside-out patch recordings in a dose- and voltage-dependent manner. KB-R7943 decreased the channel open probability without changing single channel amplitude. KB-R7943 also reversibly and in a dose-dependent manner inhibited both the odorant-evoked discharge of lobster ORNs and the odorant-evoked whole cell current. Our findings strongly imply that KB-R7943 potently blocks the lobster SGC channel and likely does so directly and not through its ability to block the Na+/Ca2+ exchanger.

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Single calcium channels in resistance-sized cerebral arteries from rats

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1993.264.2.h470 ◽

1993 ◽

Vol 264 (2) ◽

pp. H470-H478 ◽

Cited By ~ 5

Author(s):

J. M. Quayle ◽

J. G. McCarron ◽

J. R. Asbury ◽

M. T. Nelson

Keyword(s):

Steady State ◽

Calcium Channels ◽

Single Channel ◽

Cerebral Arteries ◽

Membrane Depolarization ◽

Barium Concentration ◽

Voltage Dependent ◽

Steady State Inactivation ◽

Wistar Kyoto ◽

Single Calcium Channels

Unitary currents through single calcium channels were measured from cell-attached patches on smooth muscle cells isolated from resistance-sized branches of posterior cerebral arteries from Wistar-Kyoto normotensive rats. Barium (80 and 10 mM) was used as the charge carrier, with and without the dihydropyridine calcium channel agonist BAY R 5417. Unitary currents decreased on membrane depolarization, with a slope conductance of 19.4 pS (80 mM barium). Channel open-state probability (Po) was steeply voltage dependent. Peak Po during test pulses from -70 mV increased e-fold per 4.5-mV depolarization. Mean peak Po at potentials positive to +10 mV was 0.44. Po at steady membrane potentials was also steeply voltage dependent, changing e-fold per 4.5 mV in the absence of inactivation. Steady-state Po at positive potentials was substantially lower than peak Po elicited by test pulses, suggesting that steady-state inactivation can reduce Po by as much as 10-fold. Membrane depolarization decreased the longest mean closed time but had little effect on the mean open time of single calcium channels measured during steady-state recordings. Lowering the external barium concentration from 80 to 10 mM reduced the single channel conductance to 12.4 pS and shifted the relationship between steady-state Po and membrane potential by about -30 mV. BAY R 5417 also shifted this relationship by about -15 mV.

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Voltage-dependent block of endothelial volume-regulated anion channels by calix[4]arenes

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.3.c646 ◽

1998 ◽

Vol 275 (3) ◽

pp. C646-C652 ◽

Cited By ~ 34

Author(s):

Guy Droogmans ◽

Jean Prenen ◽

Jan Eggermont ◽

Thomas Voets ◽

Bernd Nilius

Keyword(s):

Open Channel ◽

Single Channel ◽

Anion Channel ◽

Anion Channels ◽

Channel Conductance ◽

Open Channel Block ◽

Maximum Inhibition ◽

Voltage Dependent ◽

A Site ◽

Maximal Block

We have studied the effects of calix[4]arenes on the volume-regulated anion channel (VRAC) currents in cultured calf pulmonary artery endothelial cells. TS- and TS-TM-calix[4]arenes induced a fast inhibition at positive potentials but were ineffective at negative potentials. Maximal block occurred at potentials between 30 and 50 mV. Lowering extracellular pH enhanced the block and shifted the maximum inhibition to more negative potentials. Current inhibition was also accompanied by an increased current noise. From the analysis of the calix[4]arene-induced noise, we obtained a single-channel conductance of 9.3 ± 2.1 pS ( n = 9) at +30 mV. The voltage- and time-dependent block were described using a model in which calix[4]arenes bind to a site at an electrical distance of 0.25 inside the channel with an affinity of 220 μM at 0 mV. Binding occludes VRAC at moderately positive potentials, but calix[4]arenes permeate the channel at more positive potentials. In conclusion, our data suggest an open-channel block of VRAC by calix[4]arenes that also depends on the protonation of the binding site within the pore.

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Ryanodine receptor dysfunction in porcine stunned myocardium

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1997.273.2.h796 ◽

1997 ◽

Vol 273 (2) ◽

pp. H796-H804 ◽

Cited By ~ 3

Author(s):

C. Valdivia ◽

J. O. Hegge ◽

R. D. Lasley ◽

H. H. Valdivia ◽

R. Mentzer

Keyword(s):

Coronary Artery ◽

Lipid Bilayers ◽

Ryanodine Receptor ◽

Myocardial Stunning ◽

Single Channel ◽

Stunned Myocardium ◽

Wall Thickening ◽

Planar Lipid Bilayers ◽

Open Probability ◽

Single Channel Recordings

We investigated the effects of myocardial stunning on the function of the two main Ca2+ transport proteins of the sarcoplasmic reticulum (SR), the Ca(2+)-adenosinetriphosphatase and the Ca(2+)-release channel or ryanodine receptor. Regional myocardial stunning was induced in open-chest pigs (n = 6) by a 10-min occlusion of the left anterior descending coronary artery (LAD) and 2 h reperfusion. SR vesicles isolated from the LAD-perfused region (stunned) and the normal left circumflex coronary artery (LC)-perfused region were used to assess the oxalate-supported 45Ca2+ uptake, [3H]ryanodine binding, and single-channel recordings of ryanodine-sensitive Ca(2+)-release channels in planar lipid bilayers. Myocardial stunning decreased LAD systolic wall thickening to 20% of preischemic values. The rate of SR 45Ca2+ uptake in the stunned LAD bed was reduced by 37% compared with that of the normal LC bed (P < 0.05). Stunning was also associated with a 38% reduction in the maximal density of high-affinity [3H]ryanodine binding sites (P < 0.05 vs. normal LC) but had no effect on the dissociation constant. The open probability of ryanodine-sensitive Ca(2+)-release channels determined by single channel recordings in planar lipid bilayers was 26 +/- 2% for control SR (n = 33 channels from 3 animals) and 14 +/- 2% for stunned SR (n = 21 channels; P < 0.05). This depressed activity of SR function observed in postischemic myocardium could be one of the mechanisms underlying myocardial stunning.

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The Joy of Markov Models—Channel Gating and Transport Cycling Made Easy

The Biophysicist ◽

10.35459/tbp.2019.000125 ◽

2021 ◽

Author(s):

G. Zifarelli ◽

P. Zuccolini ◽

S. Bertelli ◽

M. Pusch

Keyword(s):

Single Molecule ◽

Protein Function ◽

Markov Models ◽

Single Channel ◽

Real Life ◽

Power Spectra ◽

K Channel ◽

Modular Architecture ◽

Gating Currents ◽

Discrete State

ABSTRACT The behavior of ion channels and transporters is often modeled using discrete state continuous-time Markov models. Such models are helpful for the interpretation of experimental data and can guide the design of experiments by testing specific predictions. Here, we describe a computational tool that allows us to create Markov models of chosen complexity and to calculate the predictions on a macroscopic scale, as well on a single-molecule scale. The program calculates steady-state properties (current, state probabilities, and cycle frequencies), deterministic macroscopic and stochastic time courses, gating currents, dwell-time histograms, and power spectra of channels and transporters. In addition, a visual simulation mode allows us to follow the time-dependent stochastic behavior of a single channel or transporter. After a basic introduction into the concept of Markov models, real-life examples are discussed, including a model of a simple K+ channel, a voltage-gated sodium channel, a 3-state ligand-gated channel, and an electrogenic uniporter. In this manner, the article has a modular architecture, progressing from basic to more advanced topics. This illustrates how the MarkovEditor program can serve students to explore Markov models at a basic level but is also suited for research scientists to test and develop models on the mechanisms of protein function.

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