scholarly journals Blocker State Dependence and Trapping in Hyperpolarization-Activated Cation Channels

2001 ◽  
Vol 117 (2) ◽  
pp. 91-102 ◽  
Author(s):  
Ki Soon Shin ◽  
Brad S. Rothberg ◽  
Gary Yellen
Keyword(s):  
Molecular Basis ◽  
State Dependence ◽  
Activation Gate ◽  
Voltage Dependent ◽  
The Difference ◽  
Pore Lining ◽  
Selective Blocker ◽  
Inside Out ◽  
Differential Affinity ◽  
Structural Determinant

Hyperpolarization-activated cation currents (Ih) are key determinants of repetitive electrical activity in heart and nerve cells. The bradycardic agent ZD7288 is a selective blocker of these currents. We studied the mechanism for ZD7288 blockade of cloned Ih channels in excised inside-out patches. ZD7288 blockade of the mammalian mHCN1 channel appeared to require opening of the channel, but strong hyperpolarization disfavored blockade. The steepness of this voltage-dependent effect (an apparent valence of ∼4) makes it unlikely to arise solely from a direct effect of voltage on blocker binding. Instead, it probably indicates a differential affinity of the blocker for different channel conformations. Similar properties were seen for ZD7288 blockade of the sea urchin homologue of Ih channels (SPIH), but some of the blockade was irreversible. To explore the molecular basis for the difference in reversibility, we constructed chimeric channels from mHCN1 and SPIH and localized the structural determinant for the reversibility to three residues in the S6 region likely to line the pore. Using a triple point mutant in S6, we also revealed the trapping of ZD7288 by the closing of the channel. Overall, the observations led us to hypothesize that the residues responsible for ZD7288 block of Ih channels are located in the pore lining, and are guarded by an intracellular activation gate of the channel.

scholarly journals Localization of the Activation Gate of a Voltage-gated Ca2+ Channel

2005 ◽  
Vol 126 (3) ◽  
pp. 205-212 ◽  
Author(s):  
Cheng Xie ◽  
Xiao-guang Zhen ◽  
Jian Yang
Keyword(s):  
State Dependence ◽  
K Channels ◽  
Voltage Gated ◽  
Closed State ◽  
State Dependent ◽  
Transmembrane Voltage ◽  
Activation Gate ◽  
Pore Lining ◽  
Ca2 Channels ◽  
Α1 Subunit

Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the α1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting ∼1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the α1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.

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

1998 ◽  
Vol 111 (2) ◽  
pp. 363-379 ◽  
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).

Analysis of voltage-gated and synaptic conductances contributing to network excitability defects in the mutant mouse tottering

1994 ◽  
Vol 71 (1) ◽  
pp. 1-10 ◽  
Author(s):  
S. A. Helekar ◽  
J. L. Noebels
Keyword(s):  
Gabaa Receptor ◽  
Pyramidal Neurons ◽  
Hippocampal Slices ◽  
Channel Blockers ◽  
Gamma Aminobutyric Acid ◽  
Voltage Gated ◽  
Voltage Dependent ◽  
Prolonged Duration ◽  
Ca3 Pyramidal Neurons ◽  
The Difference

1. Intracellular current- and voltage-clamp recordings were carried out in CA3 pyramidal neurons from hippocampal slices of adult tg/tg mice and their coisogenic C57BL/6J (+/+) controls with the use of the single-electrode switch-clamp technique. The principal aim of this study was to investigate the mechanisms responsible for the tg gene-linked prolongation (mean 60%) of a giant synaptic response, the potassium-induced paroxysmal depolarizing shift (PDS) at depolarized membrane potentials (Vm -47 to -54 mV) during synchronous network bursting induced by 10 mM potassium ([K+]o). 2. To examine the role of intrinsic voltage-dependent conductances underlying the mutant PDS prolongation, neurons were voltage clamped by the use of microelectrodes filled with 100 mM QX-314 or QX-222 chloride (voltage-gated sodium channel blockers) and 2 M cesium sulphate (potassium channel blocker). The whole-cell currents active during the PDS showed a significantly prolonged duration (mean 34%) at depolarized Vms in tg/tg compared with +/+ cells, indicating that a defect in voltage-dependent conductances is unlikely to completely account for the mutant phenotype. 3. Bath application of 40 microM (DL)-2-aminophosphonovalerate (DL-APV) produced a 30% reduction in PDS duration in both genotypes but failed to significantly alter the tg gene-linked prolongation compared with the wild type. These data indicate that the mutant PDS abnormality does not result from a selective increase of the N-methyl-D-aspartate (NMDA) receptor-mediated excitatory synaptic component. 4. Blockade of gamma-aminobutyric acid-A (GABAA) transmission with picrotoxin (50 microM) or bicuculline (1–5 microM) completely eliminated the difference in PDS duration between the genotypes. Furthermore, although both GABAA receptor antagonists increased the mean PDS duration in +/+ neurons, they did not significantly alter it in tg/tg neurons. These findings are consistent with a reduction in GABAA receptor-mediated synaptic inhibition during bursting in the tg CA3 hippocampal network. 5. To test this hypothesis, bursting CA3 pyramidal neurons were loaded intracellularly with chloride by the use of KCl-filled microelectrodes to examine the effect of reversing the hyperpolarizing chloride-dependent GABAA receptor-mediated inhibitory postsynaptic component of the PDS. Chloride loading prolonged PDS duration in both genotypes, but the increase was greater in +/+ than in tg/tg neurons, indicating that a smaller GABAA inhibitory postsynaptic potential (IPSP) component was reversed in the mutant.(ABSTRACT TRUNCATED AT 400 WORDS)

Inside-out, outside-in: what's the difference?

Blood ◽  
2007 ◽  
Vol 109 (8) ◽  
pp. 3128-3129 ◽  
Author(s):  
Edward F. Plow ◽  
Yan-Qing Ma
Keyword(s):  
The Difference ◽  
Inside Out

scholarly journals Singlet oxygen modification abolishes voltage-dependent inactivation of the sea urchin spHCN channel

2018 ◽  
Vol 150 (9) ◽  
pp. 1273-1286 ◽  
Author(s):  
Vinay Idikuda ◽  
Weihua Gao ◽  
Khade Grant ◽  
Zhuocheng Su ◽  
Qinglian Liu ◽  
...  
Keyword(s):  
Singlet Oxygen ◽  
Molecular Level ◽  
Basic Research ◽  
Laser Pulses ◽  
Oxygen Species ◽  
Dependent Inactivation ◽  
Activation Gate ◽  
Voltage Dependent ◽  
Short Laser Pulses ◽  
Working Distance

Photochemically or metabolically generated singlet oxygen (1O2) reacts broadly with macromolecules in the cell. Because of its short lifetime and working distance, 1O2 holds potential as an effective and precise nanoscale tool for basic research and clinical practice. Here we investigate the modification of the spHCN channel that results from photochemically and chemically generated 1O2. The spHCN channel shows strong voltage-dependent inactivation in the absence of cAMP. In the presence of photosensitizers, short laser pulses transform the gating properties of spHCN by abolishing inactivation and increasing the macroscopic current amplitude. Alanine replacement of a histidine residue near the activation gate within the channel’s pore abolishes key modification effects. Application of a variety of chemicals including 1O2 scavengers and 1O2 generators supports the involvement of 1O2 and excludes other reactive oxygen species. This study provides new understanding about the photodynamic modification of ion channels by 1O2 at the molecular level.

scholarly journals Spermine Block of the Strong Inward Rectifier Potassium Channel Kir2.1

2002 ◽  
Vol 120 (1) ◽  
pp. 53-66 ◽  
Author(s):  
Lai-Hua Xie ◽  
Scott A. John ◽  
James N. Weiss
Keyword(s):  
Single Channel ◽  
Quantitative Model ◽  
Inward Rectification ◽  
Channel Blockers ◽  
Surface Charges ◽  
Open Probability ◽  
Dependent Component ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Inside Out

Inward rectification in strong inward rectifiers such as Kir2.1 is attributed to voltage-dependent block by intracellular polyamines and Mg2+. Block by the polyamine spermine has a complex voltage dependence with shallow and steep components and complex concentration dependence. To understand the mechanism, we measured macroscopic Kir2.1 currents in excised inside-out giant patches from Xenopus oocytes expressing Kir2.1, and single channel currents in the inside-out patches from COS7 cells transfected with Kir2.1. We found that as spermine concentration or voltage increased, the shallow voltage-dependent component of spermine block at more negative voltages was caused by progressive reduction in the single channel current amplitude, without a decrease in open probability. We attributed this effect to spermine screening negative surface charges involving E224 and E299 near the inner vestibule of the channel, thereby reducing K ion permeation rate. This idea was further supported by experiments in which increasing ionic strength also decreased Kir2.1 single channel amplitude, and by mutagenesis experiments showing that this component of spermine block decreased when E224 and E299, but not D172, were neutralized. The steep voltage-dependent component of block at more depolarized voltages was attributed to spermine migrating deeper into the pore and causing fast open channel block. A quantitative model incorporating both features showed excellent agreement with the steady-state and kinetic data. In addition, this model accounts for previously described substate behavior induced by a variety of Kir2.1 channel blockers.

Two types of voltage-dependent potassium channels in outer hair cells from the guinea pig cochlea

1999 ◽  
Vol 277 (5) ◽  
pp. C913-C925 ◽  
Author(s):  
Thierry van den Abbeele ◽  
Jacques Teulon ◽  
Patrice Tran Ba Huy
Keyword(s):  
Guinea Pig ◽  
Hair Cells ◽  
Basolateral Membrane ◽  
Outer Hair Cells ◽  
Catalytic Subunit ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Guinea Pig Cochlea ◽  
Voltage Dependent ◽  
Inside Out

Cell-attached and cell-free configurations of the patch-clamp technique were used to investigate the conductive properties and regulation of the major K+channels in the basolateral membrane of outer hair cells freshly isolated from the guinea pig cochlea. There were two major voltage-dependent K+ channels. A Ca2+-activated K+ channel with a high conductance (220 pS, P K/ P Na= 8) was found in almost 20% of the patches. The inside-out activity of the channel was increased by depolarizations above 0 mV and increasing the intracellular Ca2+concentration. External ATP or adenosine did not alter the cell-attached activity of the channel. The open probability of the excised channel remained stable for several minutes without rundown and was not altered by the catalytic subunit of protein kinase A (PKA) applied internally. The most frequent K+ channel had a low conductance and a small outward rectification in symmetrical K+ conditions (10 pS for inward currents and 20 pS for outward currents, P K/ P Na= 28). It was found significantly more frequently in cell-attached and inside-out patches when the pipette contained 100 μM acetylcholine. It was not sensitive to internal Ca2+, was inhibited by 4-aminopyridine, was activated by depolarization above −30 mV, and exhibited a rundown after excision. It also had a slow inactivation on ensemble-averaged sweeps in response to depolarizing pulses. The cell-attached activity of the channel was increased when adenosine was superfused outside the pipette. This effect also occurred with permeant analogs of cAMP and internally applied catalytic subunit of PKA. Both channels could control the cell membrane voltage of outer hair cells.

Ifenprodil reduces excitatory synaptic transmission by blocking presynaptic P/Q type calcium channels

2012 ◽  
Vol 107 (6) ◽  
pp. 1571-1575 ◽  
Author(s):  
Andrew J. Delaney ◽  
John M. Power ◽  
Pankaj Sah
Keyword(s):  
Calcium Channels ◽  
Synaptic Transmission ◽  
Nmda Receptors ◽  
Basolateral Amygdala ◽  
Excitatory Transmission ◽  
Voltage Dependent ◽  
Voltage Dependent Calcium Channels ◽  
P Type ◽  
Selective Blocker

Ifenprodil is a selective blocker of NMDA receptors that are heterodimers composed of GluN1/GluN2B subunits. This pharmacological profile has been extensively used to test the role of GluN2B-containing NMDA receptors in learning and memory formation. However, ifenprodil has also been reported to have actions at a number of other receptors, including high voltage-activated calcium channels. Here we show that, in the basolateral amygdala, ifenprodil dose dependently blocks excitatory transmission to principal neurons by a presynaptic mechanism. This action of ifenprodil has an IC50 of ∼10 μM and is fully occluded by the P/Q type calcium channel blocker ω-agatoxin. We conclude that ifenprodil reduces synaptic transmission in the basolateral amygdala by partially blocking P-type voltage-dependent calcium channels.

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