Voltage-dependent open-channel block of the acetylcholine-activated K+ channel by Ba2+

Inward rectifying K channels are essential for maintaining resting membrane potential and regulating excitability in many cell types. Previous studies have attributed the rectification properties of strong inward rectifiers such as Kir2.1 to voltage-dependent binding of intracellular polyamines or Mg to the pore (direct open channel block), thereby preventing outward passage of K ions. We have studied interactions between polyamines and the polyamine toxins philanthotoxin and argiotoxin on inward rectification in Kir2.1. We present evidence that high affinity polyamine block is not consistent with direct open channel block, but instead involves polyamines binding to another region of the channel (intrinsic gate) to form a blocking complex that occludes the pore. This interaction defines a novel mechanism of ion channel closure.

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Open Channel Block by Ca2+ Underlies the Voltage Dependence of Drosophila TRPL Channel

The Journal of General Physiology ◽

10.1085/jgp.200609659 ◽

2006 ◽

Vol 129 (1) ◽

pp. 17-28 ◽

Cited By ~ 22

Author(s):

Moshe Parnas ◽

Ben Katz ◽

Baruch Minke

Keyword(s):

Open Channel ◽

Transient Receptor Potential ◽

Voltage Dependence ◽

Trp Channels ◽

Divalent Cations ◽

Dependent Manner ◽

Channel Block ◽

Open Channel Block ◽

Gating Mechanism ◽

Voltage Dependent

The light-activated channels of Drosophila photoreceptors transient receptor potential (TRP) and TRP-like (TRPL) show voltage-dependent conductance during illumination. Recent studies implied that mammalian members of the TRP family, which belong to the TRPV and TRPM subfamilies, are intrinsically voltage-gated channels. However, it is unclear whether the Drosophila TRPs, which belong to the TRPC subfamily, share the same voltage-dependent gating mechanism. Exploring the voltage dependence of Drosophila TRPL expressed in S2 cells, we found that the voltage dependence of this channel is not an intrinsic property since it became linear upon removal of divalent cations. We further found that Ca2+ blocked TRPL in a voltage-dependent manner by an open channel block mechanism, which determines the frequency of channel openings and constitutes the sole parameter that underlies its voltage dependence. Whole cell recordings from a Drosophila mutant expressing only TRPL indicated that Ca2+ block also accounts for the voltage dependence of the native TRPL channels. The open channel block by Ca2+ that we characterized is a useful mechanism to improve the signal to noise ratio of the response to intense light when virtually all the large conductance TRPL channels are blocked and only the low conductance TRP channels with lower Ca2+ affinity are active.

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Y3+ Block Demonstrates an Intracellular Activation Gate for the α1G T-type Ca2+ Channel

The Journal of General Physiology ◽

10.1085/jgp.200409167 ◽

2004 ◽

Vol 124 (6) ◽

pp. 631-640 ◽

Cited By ~ 20

Author(s):

Carlos A. Obejero-Paz ◽

I. Patrick Gray ◽

Stephen W. Jones

Keyword(s):

Open Channel ◽

Voltage Dependence ◽

Channel Block ◽

Open Channel Block ◽

Ion Interactions ◽

Dependent Rate ◽

Activation Gate ◽

Voltage Dependent ◽

Primary Activation ◽

Closed Pore

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular “pore gate” has also been proposed. We have used the voltage dependence of block by extracellular Y3+ as a tool to locate the activation gate of the α1G (CaV3.1) T-type calcium channel. Y3+ block exhibited no clear voltage dependence from −40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y3+ at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca2+ was replaced with Ba2+, the rate of open block by Y3+ was unaffected, but closed block was threefold faster than in Ca2+, suggesting the slower closed-block rate reflects ion–ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.

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Voltage-dependent block of the cystic fibrosis transmembrane conductance regulator Cl- channel by two closely related arylaminobenzoates.

The Journal of General Physiology ◽

10.1085/jgp.102.1.1 ◽

1993 ◽

Vol 102 (1) ◽

pp. 1-23 ◽

Cited By ~ 110

Author(s):

N A McCarty ◽

S McDonough ◽

B N Cohen ◽

J R Riordan ◽

N Davidson ◽

...

Keyword(s):

Cystic Fibrosis ◽

Binding Site ◽

Open Channel ◽

Transmembrane Conductance Regulator ◽

Channel Block ◽

Transmembrane Conductance ◽

Open Channel Block ◽

Whole Cell ◽

Voltage Dependent ◽

Cystic Fibrosis Transmembrane

The gene defective in cystic fibrosis encodes a Cl- channel, the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is blocked by diphenylamine-2-carboxylate (DPC) when applied extracellularly at millimolar concentrations. We studied the block of CFTR expressed in Xenopus oocytes by DPC or by a closely related molecule, flufenamic acid (FFA). Block of whole-cell CFTR currents by bath-applied DPC or by FFA, both at 200 microM, requires several minutes to reach full effect. Blockade is voltage dependent, suggesting open-channel block: currents at positive potentials are not affected but currents at negative potentials are reduced. The binding site for both drugs senses approximately 40% of the electric field across the membrane, measured from the inside. In single-channel recordings from excised patches without blockers, the conductance was 8.0 +/- 0.4 pS in symmetric 150 mM Cl-. A subconductance state, measuring approximately 60% of the main conductance, was often observed. Bursts to the full open state lasting up to tens of seconds were uninterrupted at depolarizing membrane voltages. At hyperpolarizing voltages, bursts were interrupted by brief closures. Either DPC or FFA (50 microM) applied to the cytoplasmic or extracellular face of the channel led to an increase in flicker at Vm = -100 mV and not at Vm = +100 mV, in agreement with whole-cell experiments. DPC induced a higher frequency of flickers from the cytoplasmic side than the extracellular side. FFA produced longer closures than DPC; the FFA closed time was roughly equal (approximately 1.2 ms) at -100 mV with application from either side. In cell-attached patch recordings with DPC or FFA applied to the bath, there was flickery block at Vm = -100 mV, confirming that the drugs permeate through the membrane to reach the binding site. The data are consistent with the presence of a single binding site for both drugs, reached from either end of the channel. Open-channel block by DPC or FFA may offer tools for use with site-directed mutagenesis to describe the permeation pathway.

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Voltage-dependent open-channel block of G protein-gated inward-rectifying K+ (GIRK) current in rat atrial myocytes by tamoxifen

Naunyn-Schmiedeberg s Archives of Pharmacology ◽

10.1007/s00210-012-0801-8 ◽

2012 ◽

Vol 385 (12) ◽

pp. 1149-1160 ◽

Cited By ~ 4

Author(s):

Svenja Vanheiden ◽

Lutz Pott ◽

Marie-Cécile Kienitz

Keyword(s):

G Protein ◽

Open Channel ◽

Channel Block ◽

Atrial Myocytes ◽

Open Channel Block ◽

Voltage Dependent

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W-7 modulates Kv4.3: pore block and Ca2+-calmodulin inhibition

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00409.2005 ◽

2007 ◽

Vol 292 (5) ◽

pp. H2364-H2377 ◽

Cited By ~ 10

Author(s):

Yu-Jie Qu ◽

Vladimir E. Bondarenko ◽

Chang Xie ◽

Shimin Wang ◽

Mouhamed S. Awayda ◽

...

Keyword(s):

Open Channel ◽

Direct Interaction ◽

Xenopus Laevis Oocytes ◽

Channel Block ◽

Wild Type ◽

Open Channel Block ◽

Peak Current ◽

Voltage Dependent ◽

In The Wild ◽

Dependent Protein

Ca+-calmodulin (Ca2+-CaM)-dependent protein kinase II (Ca2+/CaMKII) is an important regulator of cardiac ion channels, and its inhibition may be an approach for treatment of ventricular arrhythmias. Using the two-electrode voltage-clamp technique, we investigated the role of W-7, an inhibitor of Ca2+-occupied CaM, and KN-93, an inhibitor of Ca2+/CaMKII, on the Kv4.3 channel in Xenopus laevis oocytes. W-7 caused a voltage- and concentration-dependent decrease in peak current, with IC50 of 92.4 μM. The block was voltage dependent, with an effective electrical distance of 0.18 ± 0.05, and use dependence was observed, suggesting that a component of W-7 inhibition of Kv4.3 current was due to open-channel block. W-7 made recovery from open-state inactivation a biexponential process, also suggesting open-channel block. We compared the effects of W-7 with those of KN-93 after washout of 500 μM BAPTA-AM. KN-93 reduced peak current without evidence of voltage or use dependence. Both W-7 and KN-93 accelerated all components of inactivation. We used wild-type and mutated Kv4.3 channels with mutant CaMKII consensus phosphorylation sites to examine the effects of W-7 and KN-93. In contrast to W-7, KN-93 at 35 μM selectively accelerated open-state inactivation in the wild-type vs. the mutant channel. W-7 had a significantly greater effect on recovery from inactivation in wild-type than in mutant channels. We conclude that, at certain concentrations, KN-93 selectively inhibits Ca2+/CaMKII activity in Xenopus oocytes and that the effects of W-7 are mediated by direct interaction with the channel pore and inhibition of Ca2+-CaM, as well as a change in activity of Ca2+-CaM-dependent enzymes, including Ca2+/CaMKII.

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Open-channel-block induced by the neurotransmitter glycine in homomeric alpha1-GlyR and heteromeric alpha1/beta1- GlyR

Aktuelle Neurologie ◽

10.1055/s-0028-1086720 ◽

2008 ◽

Vol 35 (S 01) ◽

Author(s):

Y.P Song ◽

F Schlesinger ◽

S Petri ◽

R Dengler ◽

K Krampfl

Keyword(s):

Open Channel ◽

Channel Block ◽

Open Channel Block

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Interactions among DIV voltage-sensor movement, fast inactivation, and resurgent Na current induced by the NaVβ4 open-channel blocking peptide

The Journal of General Physiology ◽

10.1085/jgp.201310984 ◽

2013 ◽

Vol 142 (3) ◽

pp. 191-206 ◽

Cited By ~ 16

Author(s):

Amanda H. Lewis ◽

Indira M. Raman

Keyword(s):

Open Channel ◽

Voltage Sensor ◽

Channel Block ◽

Na Channels ◽

Fast Inactivation ◽

Na Current ◽

Open Channel Block ◽

Channel Blocking ◽

Open States ◽

Resurgent Currents

Resurgent Na current flows as voltage-gated Na channels recover through open states from block by an endogenous open-channel blocking protein, such as the NaVβ4 subunit. The open-channel blocker and fast-inactivation gate apparently compete directly, as slowing the onset of fast inactivation increases resurgent currents by favoring binding of the blocker. Here, we tested whether open-channel block is also sensitive to deployment of the DIV voltage sensor, which facilitates fast inactivation. We expressed NaV1.4 channels in HEK293t cells and assessed block by a free peptide replicating the cytoplasmic tail of NaVβ4 (the “β4 peptide”). Macroscopic fast inactivation was disrupted by mutations of DIS6 (L443C/A444W; “CW” channels), which reduce fast-inactivation gate binding, and/or by the site-3 toxin ATX-II, which interferes with DIV movement. In wild-type channels, the β4 peptide competed poorly with fast inactivation, but block was enhanced by ATX. With the CW mutation, large peptide-induced resurgent currents were present even without ATX, consistent with increased open-channel block upon depolarization and slower deactivation after blocker unbinding upon repolarization. The addition of ATX greatly increased transient current amplitudes and further enlarged resurgent currents, suggesting that pore access by the blocker is actually decreased by full deployment of the DIV voltage sensor. ATX accelerated recovery from block at hyperpolarized potentials, however, suggesting that the peptide unbinds more readily when DIV voltage-sensor deployment is disrupted. These results are consistent with two open states in Na channels, dependent on the DIV voltage-sensor position, which differ in affinity for the blocking protein.

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