Voltage-dependent gating and single-channel conductance of adult mammalian atrial gap junctions.

The purpose of this study was to use whole-cell and cell-attached patches of cultured skeletal muscle myotubes to study the macroscopic and unitary behavior of voltage-dependent calcium channels under similar conditions. With 110 mM BaCl2 as the charge carrier, two types of calcium channels with markedly different single-channel and macroscopic properties were found. One class was DHP-insensitive, had a single-channel conductance of approximately 9 pS, yielded ensembles that displayed an activation threshold near -40 mV, and activated and inactivated rapidly in a voltage-dependent manner (T current). The second class could only be well resolved in the presence of the DHP agonist Bay K 8644 (5 microM) and had a single-channel conductance of approximately 14 pS (L current). The 14-pS channel produced ensembles exhibiting a threshold of approximately -10 mV that activated slowly (tau act approximately 20 ms) and displayed little inactivation. Moreover, the DHP antagonist, (+)-PN 200-110 (10 microM), greatly increased the percentage of null sweeps seen with the 14-pS channel. The open probability versus voltage relationship of the 14-pS channel was fitted by a Boltzmann distribution with a VP0.5 = 6.2 mV and kp = 5.3 mV. L current recorded from whole-cell experiments in the presence of 110 mM BaCl2 + 5 microM Bay K 8644 displayed similar time- and voltage-dependent properties as ensembles of the 14-pS channel. Thus, these data are the first comparison under similar conditions of the single-channel and macroscopic properties of T current and L current in native skeletal muscle, and identify the 9- and 14-pS channels as the single-channel correlates of T current and L current, respectively.

Download Full-text

Double whole-cell patch-clamp of gap junctions in insect epidermal cell pairs: single channel conductance, voltage dependence, and spontaneous uncoupling

Gap Junctions ◽

10.1016/b978-0-444-89871-5.50039-0 ◽

1993 ◽

pp. 239-245

Author(s):

DENNIS CHURCHILL ◽

STANLEY CAVENEY

Keyword(s):

Patch Clamp ◽

Gap Junctions ◽

Epidermal Cell ◽

Voltage Dependence ◽

Single Channel ◽

Channel Conductance ◽

Single Channel Conductance ◽

Whole Cell ◽

Cell Patch Clamp ◽

Whole Cell Patch Clamp

Download Full-text

Cation channels from Tetrahymena cilia incorporated into planar lipid bilayers

AJP Cell Physiology ◽

10.1152/ajpcell.1985.249.1.c177 ◽

1985 ◽

Vol 249 (1) ◽

pp. C177-C179 ◽

Cited By ~ 10

Author(s):

Y. Oosawa ◽

M. Sokabe

Keyword(s):

Lipid Bilayers ◽

Single Channel ◽

Cation Channel ◽

Cation Channels ◽

Planar Lipid Bilayers ◽

Channel Conductance ◽

Single Channel Conductance ◽

Ciliary Membrane ◽

Voltage Dependent

A single cation channel from Tetrahymena cilia was incorporated into planar lipid bilayers. This channel selected for K+, Na+, and Li+ over Cl- and gluconate-, and its single channel conductance (at +25 mV) was 211 +/- 8 pS (mean +/- SE) in 100 mM K+-gluconate. The channel was not voltage dependent and may contribute to the resting K+ conductance of ciliary membrane.

Download Full-text

Horizontal cell gap junctions: single-channel conductance and modulation by dopamine.

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.86.19.7639 ◽

1989 ◽

Vol 86 (19) ◽

pp. 7639-7643 ◽

Cited By ~ 89

Author(s):

D. G. McHahon ◽

A. G. Knapp ◽

J. E. Dowling

Keyword(s):

Gap Junctions ◽

Single Channel ◽

Horizontal Cell ◽

Channel Conductance ◽

Single Channel Conductance

Download Full-text

Voltage-dependent aminoglycoside blockade of the sarcoplasmic reticulum K+ channel

AJP Cell Physiology ◽

10.1152/ajpcell.1986.250.3.c361 ◽

1986 ◽

Vol 250 (3) ◽

pp. C361-C364 ◽

Cited By ~ 18

Author(s):

Y. Oosawa ◽

M. Sokabe

Keyword(s):

Sarcoplasmic Reticulum ◽

Voltage Dependence ◽

Single Channel ◽

Phospholipid Bilayer ◽

K Channel ◽

Dependent Manner ◽

Channel Conductance ◽

Single Channel Conductance ◽

Voltage Dependent ◽

The Way

Single-channel conductance of the K+ channel from sarcoplasmic reticulum (SR) was reduced by aminoglycoside antibiotics such as neomycin and ribostamycin and also by n-hexylamine from either side of the membrane in a dose- and voltage-dependent manner. K+ channels were incorporated into an artificial phospholipid bilayer. This inhibition follows a single-site titration curve. The voltage dependence of the inhibition is explained by assuming that these drugs bind to the open state of a single channel on one site located approximately 40% of the way through the membrane from the cis side (the side to which SR vesicles are added) when drugs are added to the cis side and bind on another site located approximately 40% of the way through the membrane from the trans side (the opposite side to the cis side) when drugs are added to the trans side.

Download Full-text

Effects of Ionic Strength on Gating and Permeation of TREK-2 K2P channels

10.1101/2021.07.08.451686 ◽

2021 ◽

Author(s):

Linus J Conrad ◽

Peter Proks ◽

Stephen J Tucker

Keyword(s):

Ionic Strength ◽

Mechanism Of Action ◽

Single Channel ◽

Channel Conductance ◽

Single Channel Conductance ◽

Open Probability ◽

Voltage Sensing ◽

Voltage Dependent ◽

Single Channel Currents ◽

Gating Process

In addition to the classical voltage-dependent behavior mediated by voltage-sensing-domains (VSD), a growing number of voltage-dependent gating behaviors are being described in ion channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel TREK2 responds to membrane voltage through a gating process that is mediated by the interaction of K+ with its selectivity filter. Recently, we have found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found that the channel is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is more complex.

Download Full-text

Sites of antagonist action on N-methyl-D-aspartic acid receptors studied using fluctuation analysis and a rapid perfusion technique

Journal of Neurophysiology ◽

10.1152/jn.1988.60.2.645 ◽

1988 ◽

Vol 60 (2) ◽

pp. 645-663 ◽

Cited By ~ 82

Author(s):

M. L. Mayer ◽

G. L. Westbrook ◽

L. Vyklicky

Keyword(s):

Nmda Receptor ◽

Kynurenic Acid ◽

Single Channel ◽

Nmda Antagonist ◽

Fluctuation Analysis ◽

Channel Conductance ◽

Single Channel Conductance ◽

Open Time ◽

Voltage Dependent ◽

The Mean

1. Mouse hippocampal neurons in dissociated culture were grown at low density on previously plated hippocampal glial cell cultures and voltage clamped using the tight seal whole-cell patch-clamp technique. Flow pipes were used to rapidly exchange the extracellular solution, and to apply N-methyl-D-aspartic acid (NMDA) and some NMDA antagonists. Fluctuation analysis was used to estimate changes in the behavior of NMDA-activated ion channels during application of antagonists. In the presence of NMDA control spectra were well fit by single Lorentzian functions consistent with mean open times of 5-6 ms. 2. Two antagonists thought to act at the NMDA receptor agonist recognition site, 2-amino-5-phosphonovaleric acid (AP5) and kynurenic acid, did not produce changes in the mean open time or single channel conductance, consistent with their action as competitive antagonists. Onset of antagonism and recovery from the action of both AP5 and kynurenic acid was rapid and complete within 1 s. However, raising the extra-cellular glycine concentration, from 1 microM to 1 mM, reduced the potency of 100 microM kynurenic acid as an NMDA antagonist, suggesting that kynurenate has an additional action as a competitive antagonist at the glycine modulatory site on NMDA receptor channels. 3. In the presence of 150 microM magnesium NMDA spectra recorded at -60 mV were fit by double Lorentzian functions, consistent with single-channel events consisting of bursts of openings lasting 3.3 ms in duration, interrupted by blocking and unblocking events of average duration 0.18 ms. The onset and recovery from magnesium antagonism was rapid, and complete within 1 s, but was highly voltage dependent and at +40 mV magnesium (150 microM) failed to produce NMDA antagonism. These results are consistent with a voltage-dependent channel block of NMDA receptor channels produced by binding of magnesium to a site within the ion channel. 4. Zinc (30 microM) was a potent NMDA antagonist at both -60 and +40 mV, and at either potential appeared to reduce the mean open time of NMDA-activated ion channels from about 5 ms to approximately 3 ms. Over the frequency range measured, 1-1,000 Hz, NMDA spectra were well fit by single Lorentzians during zinc antagonism, in contrast to results obtained with magnesium. The mean single channel conductance also decreased in the presence of zinc to approximately 75% of control. Onset of antagonism and recovery from the action of zinc was rapid and complete within 1 s.(ABSTRACT TRUNCATED AT 400 WORDS)

Download Full-text

Effects of ionic strength on gating and permeation of TREK-2 K2P channels

PLoS ONE ◽

10.1371/journal.pone.0258275 ◽

2021 ◽

Vol 16 (10) ◽

pp. e0258275

Author(s):

Linus J. Conrad ◽

Peter Proks ◽

Stephen J. Tucker

Keyword(s):

Ionic Strength ◽

Mechanism Of Action ◽

Single Channel ◽

Channel Conductance ◽

Single Channel Conductance ◽

Open Probability ◽

Voltage Sensing ◽

Voltage Dependent ◽

Single Channel Currents ◽

Gating Process

In addition to the classical voltage-dependent behavior mediated by the voltage-sensing-domains (VSD) of ion channels, a growing number of voltage-dependent gating behaviors are being described in channels that lack canonical VSDs. A common thread in their mechanism of action is the contribution of the permeating ion to this voltage sensing process. The polymodal K2P K+ channel, TREK2 responds to membrane voltage through a gating process mediated by the interaction of K+ with its selectivity filter. Recently, we found that this action can be modulated by small molecule agonists (e.g. BL1249) which appear to have an electrostatic influence on K+ binding within the inner cavity and produce an increase in the single-channel conductance of TREK-2 channels. Here, we directly probed this K+-dependent gating process by recording both macroscopic and single-channel currents of TREK-2 in the presence of high concentrations of internal K+. Surprisingly we found TREK-2 is inhibited by high internal K+ concentrations and that this is mediated by the concomitant increase in ionic-strength. However, we were still able to determine that the increase in single channel conductance in the presence of BL1249 was blunted in high ionic-strength, whilst its activatory effect (on channel open probability) persisted. These effects are consistent with an electrostatic mechanism of action of negatively charged activators such as BL1249 on permeation, but also suggest that their influence on channel gating is complex.

Download Full-text