scholarly journals Mechanism of inhibition of connexin channels by the quinine derivative N-benzylquininium

2011 ◽  
Vol 139 (1) ◽  
pp. 69-82 ◽  
Author(s):  
Clio Rubinos ◽  
Helmuth A. Sánchez ◽  
Vytas K. Verselis ◽  
Miduturu Srinivas
Keyword(s):  
Single Channel ◽  
Hill Coefficient ◽  
Open Probability ◽  
Quaternary Derivative ◽  
Mechanism Of Inhibition ◽  
Open Time ◽  
Voltage Dependent ◽  
Extracellular Side ◽  
Cytoplasmic Side ◽  
Closing Rate

The anti-malarial drug quinine and its quaternary derivative N-benzylquininium (BQ+) have been shown to inhibit gap junction (GJ) channels with specificity for Cx50 over its closely related homologue Cx46. Here, we examined the mechanism of BQ+ action using undocked Cx46 and Cx50 hemichannels, which are more amenable to analyses at the single-channel level. We found that BQ+ (300 µM–1 mM) robustly inhibited Cx50, but not Cx46, hemichannel currents, indicating that the Cx selectivity of BQ+ is preserved in both hemichannel and GJ channel configurations. BQ+ reduced Cx50 hemichannel open probability (Po) without appreciably altering unitary conductance of the fully open state and was effective when added from either extracellular or cytoplasmic sides. The reductions in Po were dependent on BQ+ concentration with a Hill coefficient of 1.8, suggesting binding of at least two BQ+ molecules. Inhibition by BQ+ was voltage dependent, promoted by hyperpolarization from the extracellular side and conversely by depolarization from the cytoplasmic side. These results are consistent with binding of BQ+ in the pore. Substitution of the N-terminal (NT) domain of Cx46 into Cx50 significantly impaired inhibition by BQ+. The NT domain contributes to the formation of the wide cytoplasmic vestibule of the pore and, thus, may contribute to the binding of BQ+. Single-channel analyses showed that BQ+ induced transitions that did not resemble pore block, but rather transitions indistinguishable from the intrinsic gating events ascribed to loop gating, one of two mechanisms that gate Cx channels. Moreover, BQ+ decreased mean open time and increased mean closed time, indicating that inhibition consists of an increase in hemichannel closing rate as well as a stabilization of the closed state. Collectively, these data suggest a mechanism of action for BQ+ that involves modulation loop gating rather than channel block as a result of binding in the NT domain.

scholarly journals Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

1992 ◽  
Vol 100 (5) ◽  
pp. 783-801 ◽  
Author(s):  
L W Haynes
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Single Channel ◽  
Inward Rectification ◽  
Pipette Solution ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Extracellular Side ◽  
Current Voltage Relation ◽  
Cytoplasmic Side

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

scholarly journals A Quantitative Description of KcsA Gating I: Macroscopic Currents

2007 ◽  
Vol 130 (5) ◽  
pp. 465-478 ◽  
Author(s):  
Sudha Chakrapani ◽  
Julio F Cordero-Morales ◽  
Eduardo Perozo
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Ph Dependence ◽  
Quantitative Description ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Transmembrane Voltage ◽  
Voltage Dependent ◽  
Inactivation Process

The prokaryotic K+ channel KcsA is activated by intracellular protons and its gating is modulated by transmembrane voltage. Typically, KcsA functions have been studied under steady-state conditions, using macroscopic Rb+-flux experiments and single-channel current measurements. These studies have provided limited insights into the gating kinetics of KcsA due to its low open probability, uncertainties in the number of channels in the patch, and a very strong intrinsic kinetic variability. In this work, we have carried out a detailed analysis of KcsA gating under nonstationary conditions by examining the influence of pH and voltage on the activation, deactivation, and slow-inactivation gating events. We find that activation and deactivation gating of KcsA are predominantly modulated by pH without a significant effect of voltage. Activation gating showed sigmoidal pH dependence with a pKa of ∼4.2 and a Hill coefficient of ∼2. In the sustained presence of proton, KcsA undergoes a time-dependent decay of conductance. This inactivation process is pH independent but is modulated by voltage and the nature of permeant ion. Recovery from inactivation occurs via deactivation and also appears to be voltage dependent. We further find that inactivation in KcsA is not entirely a property of the open-conducting channel but can also occur from partially “activated” closed states. The time course of onset and recovery of the inactivation process from these pre-open closed states appears to be different from the open-state inactivation, suggesting the presence of multiple inactivated states with diverse kinetic pathways. This information has been analyzed together with a detailed study of KcsA single-channel behavior (in the accompanying paper) in the framework of a kinetic model. Taken together our data constitutes the first quantitative description of KcsA gating.

Chloride channels in the apical membrane of a distal nephron A6 cell line

1990 ◽  
Vol 258 (2) ◽  
pp. C352-C368 ◽  
Author(s):  
Y. Marunaka ◽  
D. C. Eaton
Keyword(s):  
Chloride Channels ◽  
Single Channel ◽  
Apical Membrane ◽  
The Other ◽  
Distal Nephron ◽  
Open Probability ◽  
Cl Channel ◽  
High Calcium ◽  
Voltage Dependent ◽  
Closing Rate

In this report, single-channel recording methods were used to determine whether there are Cl- conductive pathways in the apical membrane of cultured renal distal nephron cells (A6). Two different types of single Cl- channels were observed. In cell-attached patches, one had a unit conductance of 3 pS, whereas the unit conductance of the other was 8 pS. In cell-attached patches, the currents associated with the 3-pS Cl- channel outwardly rectified, whereas the current voltage relationship for the 8-pS Cl-channel was linear. The 3-pS Cl- channel has one open and one closed state; the 8-pS Cl- channel has one open and two closed states. The open probability of the 3-pS Cl- channel was voltage dependent (increasing with depolarization of the membrane) but even at very depolarized potentials (+140 mV) remained small (always less than 0.1). On the other hand, the open probability of the 8-pS Cl- channel was large (approximately 0.8) and voltage independent. The closing rate of the 3-pS Cl- channel was decreased when the patch membrane was depolarized, whereas the opening rate was increased. In contrast, the closing rate of the 8-pS Cl- channel decreased with depolarization, but the opening rates were voltage independent. The outward rectification of the 3-pS channel was markedly reduced in inside-out patches when high calcium concentrations (10-800 microM) were present on the intracellular surface. The open probability of the 3-pS Cl- channel is increased by membrane permeable analogues of adenosine 3',5'-cyclic monophosphate primarily by decreasing the mean closed time.

scholarly journals Gating and Inward Rectifying Properties of the MthK K+ Channel with and without the Gating Ring

2007 ◽  
Vol 129 (2) ◽  
pp. 109-120 ◽  
Author(s):  
Yang Li ◽  
Ian Berke ◽  
Liping Chen ◽  
Youxing Jiang
Keyword(s):  
Single Channel ◽  
Hill Coefficient ◽  
K Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Ion Conduction ◽  
Voltage Dependent ◽  
Inwardly Rectifying ◽  
Kinetics Of ◽  
Gating Process

In MthK, a Ca2+-gated K+ channel from Methanobacterium thermoautotrophicum, eight cytoplasmic RCK domains form an octameric gating ring that controls the intracellular gate of the ion conduction pore. The binding of Ca2+ ions to the RCK domains alters the conformation of the gating ring, thereby opening the gate. In the present study, we examined the Ca2+- and pH-regulated gating and the rectifying conduction properties of MthK at the single-channel level. The open probability (Po) of MthK exhibits a sigmoidal relationship with intracellular [Ca2+], and a Hill coefficient >1 is required to describe the dependence of Po on [Ca2+], suggesting cooperative Ca2+ activation of the channel. Additionally, intracellular Ca2+ also blocks the MthK pore in a voltage-dependent manner, rendering an apparently inwardly rectifying I-V relation. Intracellular pH has a dual effect on MthK gating. Below pH 7.5, the channel becomes insensitive to Ca2+. This occurs because the gating ring is structurally unstable at this pH and tends to disassemble (Ye, S., Y. Li, L. Chen, and Y. Jiang. 2006. Cell. 126:1161–1173). In contrast, above pH 7.5, a further increase in pH shifts the Po-[Ca2+] relation towards a lower Ca2+ concentration, augments Po at saturating [Ca2+], and activates the channel even in the absence of Ca2+. Channel activity is marked by bursts of rapid openings and closings separated by relatively longer interburst closings. The duration of interburst closing and the burst length are highly Ca2+ and pH dependent, whereas the kinetics of intraburst events is Ca2+ and pH independent. The rapid intraburst openings and closings are also observed with the isolated MthK pore lacking the attached intracellular gating ring. The fast kinetic events, independent of both Ca2+ and pH, therefore appear to be determined by processes occurring within the ion conduction pore, whereas the slow events reflect the gating process controlled by Ca2+ and pH through the gating ring.

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).

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

2009 ◽  
Vol 101 (3) ◽  
pp. 1151-1159 ◽  
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.

Effect of divalent heavy metals on epithelial Na+ channels in A6 cells

2007 ◽  
Vol 293 (1) ◽  
pp. F236-F244 ◽  
Author(s):  
Ling Yu ◽  
Douglas C. Eaton ◽  
My N. Helms
Keyword(s):  
Heavy Metal ◽  
Voltage Dependence ◽  
Transmembrane Domain ◽  
Single Channel ◽  
Epithelial Cell Line ◽  
Open Probability ◽  
Renal Epithelial Cell ◽  
Histidine Residues ◽  
Voltage Dependent ◽  
Renal Epithelial Cell Line

To better understand how renal Na+ reabsorption is altered by heavy metal poisoning, we examined the effects of several divalent heavy metal ions (Zn2+, Ni2+, Cu2+, Pb2+, Cd2+, and Hg2+) on the activity of single epithelial Na+ channels (ENaC) in a renal epithelial cell line (A6). None of the cations changed the single-channel conductance. However, ENaC activity [measured as the number of channels ( N) × open probability ( Po)] was decreased by Cd2+ and Hg2+ and increased by Cu2+, Zn2+, and Ni2+ but was not changed by Pb2+. Of the cations that induced an increase in Na+ channel function, Zn2+ increased N, Ni2+ increased Po, and Cu2+ increased both. The cysteine modification reagent [2-(trimethylammonium)ethyl]methanethiosulfonate bromide also increased N, whereas diethylpyrocarbonate, which covalently modifies histidine residues, affected neither Po nor N. Cu2+ increased N and stimulated Po by reducing Na+ self-inhibition. Furthermore, we observed that ENaC activity is slightly voltage dependent and that the voltage dependence of ENaC is insensitive to extracellular Na+ concentration; however, apical application of Ni2+ or diethylpyrocarbonate reduced the channel voltage dependence. Thus the voltage sensor of Xenopus ENaC is different from that of typical voltage-gated channels, since voltage appears to be sensed by histidine residues in the extracellular loops of ENaC, rather than by charged amino acids in a transmembrane domain.

Abstract 950: Direct Evidence for an Important Role of Agonist-independent Constitutive I K,ACh Channel Activity in Atrial Tachycardia Remodeling

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Niels Voigt ◽  
Ange Maguy ◽  
Yung-Hsin Yeh ◽  
Xiao-Yan Qi ◽  
Ursula Ravens ◽  
...  
Keyword(s):  
Atrial Tachycardia ◽  
Single Channel ◽  
Channel Activity ◽  
Open Probability ◽  
Direct Demonstration ◽  
Open Time ◽  
Therapy Target ◽  
Channel Properties ◽  
Constitutively Active

Background: Although atrial tachycardia (AT) appears to promote agonist-independent constitutively active I K,ACh that increases susceptibility to AF, direct demonstration of dysregulated I K,ACh channel function is lacking. We studied AT effects on single I K,ACh channel activity in dog atria. Methods: I K,ACh channel activity was recorded with cell-attached patch clamp in isolated atrial myocytes of control (CTL) and AT (7 days, 400 min −1 ) dogs. Results : AT prolonged inducible AF duration from 44±22 to 413±167 s; N=9 dogs/gp, P<0.001. In the absence of cholinergic stimulation, single-channel openings with typical I K,ACh conductance and rectification were observed in CTL and AT (Figure ). AT produced prominent agonist-independent I K,ACh activity due to 7-fold increased opening frequency (f o ) and 10-fold increased open probability (P o ) vs CTL (P<0.01 for each), but unaltered open time and single channel conductance. With maximum I K,ACh activation (10 μm carbachol, CCh), f o was 38% lower, open time constant 25% higher, and P o and unitary conductance unchanged for AT vs CTL. The selective Kir3 blocker tertiapin (100 nM) reduced f o and P o by 48% and 51% (P<0.05 each) without altering other channel properties, confirming the identity of I K,ACh. Conclusions : AT produces prominent agonist-independent constitutive single-channel I K,ACh activity, providing a molecular basis for previously-observed AT-enhanced macroscopic I K,ACh , as well as associated AP-shortening and tertiapin-suppressible AF promotion. These results suggest an important role for constitutively active I K,ACh channels in AT-remodeling and support their interest as a potential novel AF-therapy target.

Potassium channels activated by GABAB agonists and serotonin in cultured hippocampal neurons

1994 ◽  
Vol 71 (6) ◽  
pp. 2570-2575 ◽  
Author(s):  
L. S. Premkumar ◽  
P. W. Gage
Keyword(s):  
Potassium Channels ◽  
Hippocampal Neurons ◽  
Single Channel ◽  
Gamma Aminobutyric Acid ◽  
Kinetic Behavior ◽  
Open Time ◽  
Voltage Dependent ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Cultured Hippocampal Neurons

1. Single-channel currents were recorded in cell-attached patches on cultured hippocampal neurons in response to gamma-aminobutyric acid-B (GABAB) agonists or serotonin applied to the cell surface outside the patch area. 2. The channels activated by GABAB agonists and serotonin were potassium selective but had a different conductance and kinetic behavior. Channels activated by GABAB agonists had a higher conductance, longer open-time, and longer burst-length than channels activated by serotonin. 3. The kinetic behavior of channels activated by GABAB agonists varied with potential whereas channels activated by serotonin did not show voltage-dependent changes in kinetics. 4. In a few cell-attached patches, both types of channel were activated when the cell was exposed to GABA together with serotonin. 5. It was concluded that GABAB agonists and serotonin activate different potassium channels in the soma of cultured hippocampal neurons.

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.

Sign in / Sign up

Export Citation Format

Share Document