Activation of cardiac ryanodine receptors  by cardiac glycosides

This study investigated the effects of cardiac glycosides on single-channel activity of the cardiac sarcoplasmic reticulum (SR) Ca2+ release channels or ryanodine receptor (RyR2) channels and how this action might contribute to their inotropic and/or toxic actions. Heavy SR vesicles isolated from canine left ventricle were fused with artificial planar lipid bilayers to measure single RyR2 channel activity. Digoxin and actodigin increased single-channel activity at low concentrations normally associated with therapeutic plasma levels, yielding a 50% of maximal effect of ∼0.2 nM for each agent. Channel activation by glycosides did not require MgATP and occurred only when digoxin was applied to the cytoplasmic side of the channel. Similar results were obtained in human RyR2 channels; however, neither the crude skeletal nor the purified cardiac channel was activated by glycosides. Channel activation was dependent on [Ca2+] on the luminal side of the bilayer with maximal stimulation occurring between 0.3 and 10 mM. Rat RyR2 channels were activated by digoxin only at 1 μM, consistent with the lower sensitivity to glycosides in rat heart. These results suggest a model in which RyR2 channel activation by digoxin occurs only when luminal [Ca2+] was increased above 300 μM (in the physiological range). Consequently, increasing SR load (by Na+ pump inhibition) serves to amplify SR release by promoting direct RyR2 channel activation via a luminal Ca2+-sensitive mechanism. This high-affinity effect of glycosides could contribute to increased SR Ca2+ release and might play a role in the inotropic and/or toxic actions of glycosides in vivo.

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Disease-Linked Super-Trafficking of a Mutant Potassium Channel

10.1101/2020.07.29.227231 ◽

2020 ◽

Author(s):

Hui Huang ◽

Laura M. Chamness ◽

Carlos G. Vanoye ◽

Georg Kuenze ◽

Jens Meiler ◽

...

Keyword(s):

Plasma Membrane ◽

Cell Surface ◽

Potassium Channel ◽

Single Channel ◽

Channel Activity ◽

Wild Type ◽

Channel Activation ◽

Voltage Gated ◽

Disease Mutations ◽

Single Channel Activity

ABSTRACTGain-of-function (GOF) mutations in the KCNQ1 voltage-gated potassium channel can induce cardiac arrhythmia. We tested whether any of the known GOF disease mutations in KCNQ1 act by increasing the amount of KCNQ1 that reaches the cell surface—“super-trafficking”. We found that levels of R231C KCNQ1 in the plasma membrane are 5-fold higher than wild type KCNQ1. This arises from both enhanced translocon-mediated membrane integration of the S4 voltage-sensor helix and an energetic linkage of C231 with the V129 and F166 side chains. Whole-cell electrophysiology recordings confirmed that R231C KCNQ1 in complex with KCNE1 is constitutively active, but also revealed the single channel activity of this mutant to be only 20% that of WT. The GOF phenotype associated with R231C therefore reflects the net effects of super-trafficking, reduced single channel activity, and constitutive channel activation. These investigations document membrane protein super-trafficking as a contributing mechanism to human disease.

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The heterogeneity of ion channels in chromaffin granule membranes

Cellular & Molecular Biology Letters ◽

10.2478/s11658-006-0027-1 ◽

2006 ◽

Vol 11 (3) ◽

Cited By ~ 3

Author(s):

Renata Hordejuk ◽

Adam Szewczyk ◽

Krzysztof Dołowy

Keyword(s):

Lipid Bilayers ◽

Adrenal Glands ◽

Single Channel ◽

Ion Homeostasis ◽

Planar Lipid Bilayers ◽

Chromaffin Granules ◽

Channel Activity ◽

Chromaffin Granule ◽

Catecholamine Synthesis ◽

Single Channel Activity

AbstractChromaffin granules are involved in catecholamine synthesis and traffic in the adrenal glands. The transporting membrane proteins of chromaffin granules play an important role in the ion homeostasis of these organelles. In this study, we characterized components of the electrogenic 86Rb+ flux observed in isolated chromaffin granules. In order to study single channel activity, chromaffin granules from the bovine adrenal medulla were incorporated into planar lipid bilayers. Four types of cationic channel were found, each with a different conductance. The unitary conductances of the potassium channels are 360 ± 10 pS, 220 ± 8 pS, 152 ± 8 pS and 13 ± 3 pS in a gradient of 450/150 mM KCl, pH 7.0. A multiconductance potassium channel with a conductivity of 110 ± 8 pS and 31 ± 4 pS was also found. With the exception of the 13 pS conductance channel, all are activated by depolarizing voltages. One type of chloride channel was also found. It has a unitary conductance of about 250 pS in a gradient of 500/150 mM KCl, pH 7.0.

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Single cardiac sarcoplasmic reticulum Ca2+-release channel: activation by caffeine

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.1989.256.2.h328 ◽

1989 ◽

Vol 256 (2) ◽

pp. H328-H333 ◽

Cited By ~ 23

Author(s):

E. Rousseau ◽

G. Meissner

Keyword(s):

Steady State ◽

Sarcoplasmic Reticulum ◽

Lipid Bilayers ◽

Single Channel ◽

Ruthenium Red ◽

Planar Lipid Bilayers ◽

Release Channel ◽

Cardiac Sarcoplasmic Reticulum ◽

Channel Activation ◽

Contraction Coupling

Caffeine is thought to affect excitation-contraction coupling in cardiac muscle by activating the sarcoplasmic reticulum (SR) Ca2+-release channel. The effect of caffeine at the single channel level was studied by incorporating canine cardiac SR vesicles into planar lipid bilayers. Cardiac Ca2+-release channels were activated in a steady-state manner by millimolar cis-caffeine and displayed a unitary conductance (77 pS in 50 mM Ca2+ trans) similar to that previously observed for the Ca2+-activated cardiac channel. The caffeine-activated channel was moderately sensitive to the voltage applied across the bilayer, was sensitive to further activation by ATP, and was inhibited by Mg2+ and ruthenium red. Kinetic analysis showed that at low Ca2+ concentration, caffeine activated the channel by increasing the frequency and the duration of open events.

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Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers.

The Journal of General Physiology ◽

10.1085/jgp.97.2.393 ◽

1991 ◽

Vol 97 (2) ◽

pp. 393-412 ◽

Cited By ~ 19

Author(s):

R Mejía-Alvarez ◽

M Fill ◽

E Stefani

Keyword(s):

Calcium Channels ◽

Lipid Bilayers ◽

Single Channel ◽

Channel Activity ◽

T Tube ◽

Dependent Inactivation ◽

Voltage Dependent ◽

Single Channel Activity ◽

Kinetics Of ◽

Channel Properties

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.

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A calcium-activated chloride channel in sarcoplasmic reticulum vesicles from rabbit skeletal muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1996.270.6.c1675 ◽

1996 ◽

Vol 270 (6) ◽

pp. C1675-C1686 ◽

Cited By ~ 23

Author(s):

J. I. Kourie ◽

D. R. Laver ◽

G. P. Ahern ◽

A. F. Dulhunty

Keyword(s):

Skeletal Muscle ◽

Sarcoplasmic Reticulum ◽

Lipid Bilayers ◽

Chloride Channel ◽

Single Channel ◽

Single Channels ◽

Channel Activity ◽

Open Probability ◽

Single Channel Activity ◽

Maximum Conductance

A Ca(2+)-activated Cl- channel is described in sarcoplasmic reticulum (SR) enriched vesicles of skeletal muscle incorporated into lipid bilayers. Small chloride (SCl) channels (n = 20) were rapidly and reversibly activated when cis- (cytoplasmic) [Ca2+] was increased above 10(-7) M, with trans-(luminal) [Ca2+] at either 10(-3) or 10(-7) M. The open probability of single channels increased from zero when cis-[Ca2+] was 10(-7) M to 0.61 +/- 0.12 when [Ca2+] was 10(-4) M. High- and low-conductance levels in single-channel activity were activated at different cis-[Ca2+]. Channel openings to the maximum conductance, 65-75 pS (250/50 mM Cl-, cis/ trans), were active when cis-[Ca2+] was increased above 5 x 10(-6) M. In contrast to the maximum conductance, channel openings to submaximal levels between 5 and 40 pS were activated at a lower cis-[Ca2+] and dominated channel activity between 5 x 10(-7) and 5 x 10(-6) M. Activation of SCl channels was Ca2+ specific and not reproduced by cytoplasmic Mg2+ concentrations of 10(-3) M. We suggest that the SCl channel arises in the SR membrane. The Ca2+ dependence of this channel implies that it is active at [Ca2+] achieved during muscle contraction.

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Activation of cardiac ryanodine receptors by the calcium channel agonist FPL-64176

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00788.2001 ◽

2002 ◽

Vol 283 (1) ◽

pp. H331-H338 ◽

Cited By ~ 10

Author(s):

J. Andrew Wasserstrom ◽

Leslie A. Wasserstrom ◽

Andrew J. Lokuta ◽

James E. Kelly ◽

Sireen T. Reddy ◽

...

Keyword(s):

Single Channel ◽

Ryanodine Receptors ◽

Channel Activity ◽

Open Probability ◽

Release Channel ◽

Cardiac Sarcoplasmic Reticulum ◽

Significant Difference ◽

Single Channel Activity ◽

Control Versus ◽

Channel Agonist

We investigated the possibility that the Ca2+ channel agonist FPL-64176 (FPL) might also activate the cardiac sarcoplasmic reticulum (SR) Ca2+ release channel ryanodine receptor (RyR). The effects of FPL were tested on single channel activity of purified and crude vesicular RyR (RyR2) isolated from human and dog hearts using the planar lipid bilayer technique. FPL (100–200 μM) increased single channel open probability ( P o) when added to the cytoplasmic side of the channel ( P o = 0.070 ± 0.021 in control RyR2; 0.378 ± 0.086 in 150 μM FPL, n = 9, P < 0.01) by prolonging open times and decreasing closed times without changing current magnitude. FPL had no effect on P o when added to the trans (luminal) side of the bilayer ( P o = 0.079 ± 0.036 in control and 0.103 ± 0.066 in FPL, n = 4, no significant difference). The bell-shaped [Ca2+] dependence of [3H]ryanodine binding and of P o was altered by FPL, suggesting that the mechanism by which FPL increases channel activity is by an increase in Ca2+-induced activation at low [Ca2+] (without a change in threshold) and suppression of Ca2+-induced inactivation at high [Ca2+]. However, the fact that inactivation was restored at elevated [Ca2+] suggests a competitive interaction between Ca2+ and FPL on inactivation. FPL had no effect on RyR skeletal channels (RyR1), where P o was 0.039 ± 0.005 in control versus 0.030 ± 0.006 in 150 μM FPL (no significant difference). These results suggest that, in addition to its ability to activate the L-type Ca2+channels, FPL activates cardiac RyR2 primarily by reducing the Ca2+ sensitivity of inactivation.

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