scholarly journals Mechanism of Inhibition of Cyclic Nucleotide–Gated Ion Channels by Diacylglycerol

2000 ◽  
Vol 116 (6) ◽  
pp. 755-768 ◽  
Author(s):  
Jennifer I. Crary ◽  
Dylan M. Dean ◽  
Wang Nguitragool ◽  
Peri T. Kurshan ◽  
Anita L. Zimmerman
Keyword(s):  
Cyclic Nucleotide ◽  
Single Channel ◽  
Open Probability ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Closed State ◽  
Cng Channel ◽  
Channel Interactions ◽  
Cloned Bovine ◽  
Critical Components

Cyclic nucleotide–gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409–417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (α subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (Po), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the Po but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer–channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.

scholarly journals Mechanism of Inhibition of Cyclic Nucleotide–Gated Channel by Protein Tyrosine Kinase Probed with Genistein

2001 ◽  
Vol 117 (3) ◽  
pp. 219-234 ◽  
Author(s):  
Elena Molokanova ◽  
Richard H. Kramer
Keyword(s):  
Tyrosine Phosphorylation ◽  
Cyclic Nucleotide ◽  
Tyrosine Kinases ◽  
Single Channel ◽  
Channel Gating ◽  
Open Probability ◽  
Inhibitory Interaction ◽  
Α Subunit ◽  
Protein Tyrosine ◽  
Cng Channel

Rod cyclic nucleotide–gated (CNG) channels are modulated by changes in tyrosine phosphorylation catalyzed by protein tyrosine kinases (PTKs) and phosphatases (PTPs). We used genistein, a PTK inhibitor, to probe the interaction between the channel and PTKs. Previously, we found that in addition to inhibiting tyrosine phosphorylation of the rod CNG channel α-subunit (RETα), genistein triggers a noncatalytic inhibitory interaction between the PTK and the channel. These studies suggest that PTKs affects RETα channels in two ways: (1) by catalyzing phosphorylation of the channel protein, and (2) by allosterically regulating channel activation. Here, we study the mechanism of noncatalytic inhibition. We find that noncatalytic inhibition follows the same activity dependence pattern as catalytic modulation (phosphorylation): the efficacy and apparent affinity of genistein inhibition are much higher for closed than for fully activated channels. Association rates with the genistein–PTK complex were similar for closed and fully activated channels and independent of genistein concentration. Dissociation rates were 100 times slower for closed channels, which is consistent with a much higher affinity for genistein–PTK. Genistein–PTK affects channel gating, but not single channel conductance or the number of active channels. By analyzing single channel gating during genistein–PTK dissociation, we determined the maximal open probability for normal and genistein–PTK-bound channels. genistein–PTK decreases open probability by increasing the free energy required for opening, making opening dramatically less favorable. Ni2+, which potentiates RETα channel gating, partially relieves genistein inhibition, possibly by disrupting the association between the genistein–PTK and the channel. Studies on chimeric channels containing portions of RETα, which exhibits genistein inhibition, and the rat olfactory CNG channel α-subunit, which does not, reveals that a domain containing S6 and flanking regions is the crucial for genistein inhibition and may constitute the genistein–PTK binding site. Thus, genistein–PTK stabilizes the closed state of the channel by interacting with portions of the channel that participate in gating.

scholarly journals Access of Quaternary Ammonium Blockers to the Internal Pore of Cyclic Nucleotide-gated Channels: Implications for the Location of the Gate

2006 ◽  
Vol 127 (5) ◽  
pp. 481-494 ◽  
Author(s):  
Jorge E. Contreras ◽  
Miguel Holmgren
Keyword(s):  
Binding Site ◽  
Quaternary Ammonium ◽  
Cyclic Nucleotide ◽  
Single Channel ◽  
Dependent Manner ◽  
Receptor Binding Site ◽  
Α Subunit ◽  
Cyclic Nucleotide Gated Channels ◽  
Closed State ◽  
Kv Channels

Cyclic nucleotide-gated (CNG) channels play important roles in the transduction of visual and olfactory information by sensing changes in the intracellular concentration of cyclic nucleotides. We have investigated the interactions between intracellularly applied quaternary ammonium (QA) ions and the α subunit of rod cyclic nucleotide-gated channels. We have used a family of alkyl-triethylammonium derivatives in which the length of one chain is altered. These QA derivatives blocked the permeation pathway of CNG channels in a concentration- and voltage-dependent manner. For QA compounds with tails longer than six methylene groups, increasing the length of the chain resulted in higher apparent affinities of ∼1.2 RT per methylene group added, which is consistent with the presence of a hydrophobic pocket within the intracellular mouth of the channel that serves as part of the receptor binding site. At the single channel level, decyltriethyl ammonium (C10-TEA) ions did not change the unitary conductance but they did reduce the apparent mean open time, suggesting that the blocker binds to open channels. We provide four lines of evidence suggesting that QA ions can also bind to closed channels: (1) the extent of C10-TEA blockade at subsaturating [cGMP] was larger than at saturating agonist concentration, (2) under saturating concentrations of cGMP, cIMP, or cAMP, blockade levels were inversely correlated with the maximal probability of opening achieved by each agonist, (3) in the closed state, MTS reagents of comparable sizes to QA ions were able to modify V391C in the inner vestibule of the channel, and (4) in the closed state, C10-TEA was able to slow the Cd2+ inhibition observed in V391C channels. These results are in stark contrast to the well-established QA blockade mechanism in Kv channels, where these compounds can only access the inner vestibule in the open state because the gate that opens and closes the channel is located cytoplasmically with respect to the binding site of QA ions. Therefore, in the context of Kv channels, our observations suggest that the regions involved in opening and closing the permeation pathways in these two types of channels are different.

scholarly journals The Extracellular Linker of Muscle Acetylcholine Receptor Channels Is a Gating Control Element

2000 ◽  
Vol 116 (3) ◽  
pp. 327-340 ◽  
Author(s):  
Claudio Grosman ◽  
Frank N. Salamone ◽  
Steven M. Sine ◽  
Anthony Auerbach
Keyword(s):  
Acetylcholine Receptors ◽  
Single Channel ◽  
Channel Gating ◽  
Congenital Myasthenic Syndrome ◽  
Control Element ◽  
Dependent Manner ◽  
Α Subunit ◽  
Transmembrane Segments ◽  
Channel Opening ◽  
Myasthenic Syndrome

We describe the functional consequences of mutations in the linker between the second and third transmembrane segments (M2–M3L) of muscle acetylcholine receptors at the single-channel level. Hydrophobic mutations (Ile, Cys, and Phe) placed near the middle of the linker of the α subunit (αS269) prolong apparent openings elicited by low concentrations of acetylcholine (ACh), whereas hydrophilic mutations (Asp, Lys, and Gln) are without effect. Because the gating kinetics of the αS269I receptor (a congenital myasthenic syndrome mutant) in the presence of ACh are too fast, choline was used as the agonist. This revealed an ∼92-fold increased gating equilibrium constant, which is consistent with an ∼10-fold decreased EC50 in the presence of ACh. With choline, this mutation accelerates channel opening ∼28-fold, slows channel closing ∼3-fold, but does not affect agonist binding to the closed state. These ratios suggest that, with ACh, αS269I acetylcholine receptors open at a rate of ∼1.4 × 106 s−1 and close at a rate of ∼760 s−1. These gating rate constants, together with the measured duration of apparent openings at low ACh concentrations, further suggest that ACh dissociates from the diliganded open receptor at a rate of ∼140 s−1. Ile mutations at positions flanking αS269 impair, rather than enhance, channel gating. Inserting or deleting one residue from this linker in the α subunit increased and decreased, respectively, the apparent open time approximately twofold. Contrary to the αS269I mutation, Ile mutations at equivalent positions of the β, ε, and δ subunits do not affect apparent open-channel lifetimes. However, in β and ε, shifting the mutation one residue to the NH2-terminal end enhances channel gating. The overall results indicate that this linker is a control element whose hydrophobicity determines channel gating in a position- and subunit-dependent manner. Characterization of the transition state of the gating reaction suggests that during channel opening the M2–M3L of the α subunit moves before the corresponding linkers of the β and ε subunits.

scholarly journals Mechanism of Allosteric Modulation of Rod Cyclic Nucleotide–gated Channels

1999 ◽  
Vol 113 (5) ◽  
pp. 601-620 ◽  
Author(s):  
Elizabeth R. Sunderman ◽  
William N. Zagotta
Keyword(s):  
Transition State ◽  
Rate Constant ◽  
Rate Constants ◽  
Cyclic Nucleotide ◽  
Equilibrium Constants ◽  
Rod Photoreceptor ◽  
Open Probability ◽  
Allosteric Transition ◽  
Cng Channel ◽  
Single Channel Currents

The cyclic nucleotide–gated (CNG) channel of retinal rod photoreceptor cells is an allosteric protein whose activation is coupled to a conformational change in the ligand-binding site. The bovine rod CNG channel can be activated by a number of different agonists, including cGMP, cIMP, and cAMP. These agonists span three orders of magnitude in their equilibrium constants for the allosteric transition. We recorded single-channel currents at saturating cyclic nucleotide concentrations from the bovine rod CNG channel expressed in Xenopus oocytes as homomultimers of α subunits. The median open probability was 0.93 for cGMP, 0.47 for cIMP, and 0.01 for cAMP. The channels opened to a single conductance level of 26–30 pS at +80 mV. Using signal processing methods based on hidden Markov models, we determined that two closed and one open states are required to explain the gating at saturating ligand concentrations. We determined the maximum likelihood rate constants for two gating schemes containing two closed (denoted C) and one open (denoted O) states. For the C ↔ C ↔ O scheme, all rate constants were dependent on cyclic nucleotide. For the C ↔ O ↔ C scheme, the rate constants for only one of the transitions were cyclic nucleotide dependent. The opening rate constant was fastest for cGMP, intermediate for cIMP, and slowest for cAMP, while the closing rate constant was fastest for cAMP, intermediate for cIMP, and slowest for cGMP. We propose that interactions between the purine ring of the cyclic nucleotide and the binding domain are partially formed at the time of the transition state for the allosteric transition and serve to reduce the transition state energy and stabilize the activated conformation of the channel. When 1 μM Ni2+ was applied in addition to cyclic nucleotide, the open time increased markedly, and the closed time decreased slightly. The interactions between H420 and Ni2+ occur primarily after the transition state for the allosteric transition.

scholarly journals Estimating Binding Affinities of the Nicotinic Receptor for Low-efficacy Ligands Using Mixtures of Agonists and Two-dimensional Concentration–Response Relationships

2006 ◽  
Vol 127 (6) ◽  
pp. 719-735 ◽  
Author(s):  
Yamini Purohit ◽  
Claudio Grosman
Keyword(s):  
Nicotinic Receptor ◽  
Rational Design ◽  
Single Channel ◽  
Specific Binding ◽  
Kinetic Scheme ◽  
Binding Affinities ◽  
Open Probability ◽  
Channel Response ◽  
Closed State ◽  
Dissociation Equilibrium

The phenomenon of ligand-induced ion channel gating hinges upon the ability of a receptor channel to bind ligand molecules with conformation-specific affinities. However, our understanding of this fundamental phenomenon is notably limited, not only because the changes in binding site structure and ligand conformation that occur upon gating are largely unknown but, also, because the strength of these ligand–receptor interactions are experimentally elusive. Both high- and low-efficacy ligands pose a number of analytical and experimental challenges that can render the estimation of their conformation-specific binding affinities impossible. In this paper, we present a novel assay that overcomes some of the hurdles presented by weak agonists of the muscle nicotinic receptor and allows the estimation of their closed-state affinities. The method, which we have termed the “activation-competition” assay, consists of a single-channel concentration–response assay performed in the presence of a binary mixture of ligands of widely different efficacies. By plotting the channel response (i.e., the open probability) as a function of the concentration of each agonist in the mixture, interpreting the observed response in the framework of a plausible kinetic scheme, and fitting the open probability surface with the corresponding function, the affinities of the closed receptor for the two agonists can be simultaneously extracted as free parameters. Here, we applied this methodology to estimate the closed-state affinity of the muscle nicotinic receptor for choline (a very weak agonist) using acetylcholine (ACh) as the partner in the mixture. We estimated the dissociation equilibrium constant of choline (KD) from the wild type's closed state to be 4.1 ± 0.5 mM (and that of ACh to be 106 ± 6 μM). We also discuss the use of accurate estimates of affinities for low-efficacy agonists as a tool to discriminate between binding and gating effects of mutations, and in the context of the rational design of therapeutic drugs.

scholarly journals CFTR displays voltage dependence and two gating modes during stimulation.

1994 ◽  
Vol 104 (3) ◽  
pp. 541-566 ◽  
Author(s):  
H Fischer ◽  
T E Machen
Keyword(s):  
Single Channel ◽  
Noise Analysis ◽  
Current Noise ◽  
Corner Frequency ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Closed State ◽  
Open States ◽  
Multichannel Recordings ◽  
Long Channel

The patch-clamp technique in conjunction with current noise analysis was employed to clarify the events underlying the regulation of the CFTR (cystic fibrosis transmembrane conductance regulator) during cAMP-dependent stimulation. 3T3 fibroblast cells expressing the CFTR were stimulated in cell-attached mode with forskolin. The number (N) of activated channels per patch ranged from 1 to approximately 100. In true single-channel recordings, CFTR's gating was best described by two open states (approximately 5 and approximately 100 ms) and three closed states (< or = 5, approximately 100, and approximately 1,000 ms). Current noise analysis resulted in spectra containing two distinct Lorentzian noise components with corner frequencies of 1.3 Hz and approximately 50 Hz, respectively. Single-channel time constants were dependent on voltage. The fastest closed state increased its contribution from 48% at +100 mV to 87% at -100 mV, and the medium open state reduced its length to one half, resulting in gating dominated by fast events. Similarly, the fast Lorentzian increased its amplitude, and its corner frequency increased from 44 Hz at +100 mV to 91 Hz at -100 mV, while the slow Lorentzian was voltage independent. In multi-channel recordings N.Po (i.e., N times open probability) increased significantly, on average by 52% between -90 and +90 mV. Stimulation with forskolin increased Po of CFTR to approximately 0.5, which resulted from a decrease of the longest closed state while the faster open and closed states were unaffected. Neither corner frequency was affected during stimulation. Recordings from multichannel patches revealed in addition, unique, very long channel openings (high Po mode, average 13 s). Channels exhibiting high Po (i.e., Po approximately 1.0) or low Po (i.e., Po approximately 0.5) gating modes were both present in multichannel recordings, and CFTRs switched modes during stimulation. In addition, the switch to the high Po mode appeared to be a cooperative event for channel pairs. High forskolin concentration (i.e., 10 microM) favored transition into the high Po mode, suggesting a cellularly mediated regulation of model switching due to a fundamental change in configuration of the CFTR. Thus, during stimulation the CFTR increased its activity through two distinct effects: the reduction of the long closed state and modal switching to the high Po mode.

Large-conductance Ca2+-activated potassium channel in mitochondria of endothelial EA.hy926 cells

2013 ◽  
Vol 304 (11) ◽  
pp. H1415-H1427 ◽  
Author(s):  
Piotr Bednarczyk ◽  
Agnieszka Koziel ◽  
Wieslawa Jarmuszkiewicz ◽  
Adam Szewczyk
Keyword(s):  
Potassium Channel ◽  
Mitochondrial Membrane ◽  
Single Channel ◽  
Dependent Manner ◽  
Channel Activity ◽  
Inner Mitochondrial Membrane ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Α Subunit ◽  
Ea.Hy926 Cells

In the present study, we describe the existence of a large-conductance Ca2+-activated potassium (BKCa) channel in the mitochondria of the human endothelial cell line EA.hy926. A single-channel current was recorded from endothelial mitoplasts (i.e., inner mitochondrial membrane) using the patch-clamp technique in the mitoplast-attached mode. A potassium-selective current was recorded with a mean conductance equal to 270 ± 10 pS in a symmetrical 150/150 mM KCl isotonic solution. The channel activity, which was determined as the open probability, increased with the addition of calcium ions and the potassium channel opener NS1619. Conversely, the activity of the channel was irreversibly blocked by paxilline and iberiotoxin, BKCa channel inhibitors. The open-state probability was found to be voltage dependent. The substances known to modulate BKCa channel activity influenced the bioenergetics of mitochondria isolated from human endothelial EA.hy926 cells. In isolated mitochondria, 100 μM Ca2+, 10 μM NS1619, and 0.5 μM NS11021 depolarized the mitochondrial membrane potential and stimulated nonphosphorylating respiration. These effects were blocked by iberiotoxin and paxilline in a potassium-dependent manner. Under phosphorylating conditions, NS1619-induced, iberiotoxin-sensitive uncoupling diverted energy from ATP synthesis during the phosphorylating respiration of the endothelial mitochondria. Immunological analysis with antibodies raised against proteins of the plasma membrane BKCa channel identified a pore-forming α-subunit and an auxiliary β2-subunit of the channel in the endothelial mitochondrial inner membrane. In conclusion, we show for the first time that the inner mitochondrial membrane in human endothelial EA.hy926 cells contains a large-conductance calcium-dependent potassium channel with properties similar to those of the surface membrane BKCa channel.

scholarly journals Naturally Occurring Mutations at the Acetylcholine Receptor Binding Site Independently Alter ACh Binding and Channel Gating

2002 ◽  
Vol 120 (4) ◽  
pp. 483-496 ◽  
Author(s):  
Steven M. Sine ◽  
Xing-Ming Shen ◽  
Hai-Long Wang ◽  
Kinji Ohno ◽  
Won-Yong Lee ◽  
...  
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Rate Constants ◽  
Structural Model ◽  
Single Channel ◽  
Channel Gating ◽  
Congenital Myasthenic Syndrome ◽  
Protein Chain ◽  
Α Subunit ◽  
Closed State

By defining functional defects in a congenital myasthenic syndrome (CMS), we show that two mutant residues, located in a binding site region of the acetylcholine receptor (AChR) epsilon subunit, exert opposite effects on ACh binding and suppress channel gating. Single channel kinetic analysis reveals that the first mutation, εN182Y, increases ACh affinity for receptors in the resting closed state, which promotes sequential occupancy of the binding sites and discloses rate constants for ACh occupancy of the nonmutant αδ site. Studies of the analogous mutation in the δ subunit, δN187Y, disclose rate constants for ACh occupancy of the nonmutant αε site. The second CMS mutation, εD175N, reduces ACh affinity for receptors in the resting closed state; occupancy of the mutant site still promotes gating because a large difference in affinity is maintained between closed and open states. εD175N impairs overall gating, however, through an effect independent of ACh occupancy. When mapped on a structural model of the AChR binding site, εN182Y localizes to the interface with the α subunit, and εD175 to the entrance of the ACh binding cavity. Both εN182Y and εD175 show state specificity in affecting closed relative to desensitized state affinities, suggesting that the protein chain harboring εN182 and εD175 rearranges in the course of receptor desensitization. The overall results show that key residues at the ACh binding site differentially stabilize the agonist bound to closed, open and desensitized states, and provide a set point for gating of the channel.

scholarly journals The β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

1999 ◽  
Vol 113 (3) ◽  
pp. 425-440 ◽  
Author(s):  
Crina M. Nimigean ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Bk Channels ◽  
Bk Channel ◽  
Β Subunit ◽  
Open Probability ◽  
Α Subunit ◽  
Closed Intervals ◽  
Single Channel Analysis ◽  
Channel Analysis ◽  
The Mean

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone.

scholarly journals Single-channel Properties of Human NaV1.1 and Mechanism of Channel Dysfunction in SCN1A-associated Epilepsy

2005 ◽  
Vol 127 (1) ◽  
pp. 1-14 ◽  
Author(s):  
Carlos G. Vanoye ◽  
Christoph Lossin ◽  
Thomas H. Rhodes ◽  
Alfred L. George
Keyword(s):  
Sodium Channel ◽  
Single Channel ◽  
Sodium Current ◽  
Febrile Seizures ◽  
Persistent Sodium Current ◽  
Open Probability ◽  
Α Subunit ◽  
Whole Cell ◽  
Open Time ◽  
Channel Properties

Mutations in genes encoding neuronal voltage-gated sodium channel subunits have been linked to inherited forms of epilepsy. The majority of mutations (>100) associated with generalized epilepsy with febrile seizures plus (GEFS+) and severe myoclonic epilepsy of infancy (SMEI) occur in SCN1A encoding the NaV1.1 neuronal sodium channel α-subunit. Previous studies demonstrated functional heterogeneity among mutant SCN1A channels, revealing a complex relationship between clinical and biophysical phenotypes. To further understand the mechanisms responsible for mutant SCN1A behavior, we performed a comprehensive analysis of the single-channel properties of heterologously expressed recombinant WT-SCN1A channels. Based on these data, we then determined the mechanisms for dysfunction of two GEFS+-associated mutations (R1648H, R1657C) both affecting the S4 segment of domain 4. WT-SCN1A has a slope conductance (17 pS) similar to channels found in native mammalian neurons. The mean open time is ∼0.3 ms in the −30 to −10 mV range. The R1648H mutant, previously shown to display persistent sodium current in whole-cell recordings, exhibited similar slope conductance but had an increased probability of late reopening and a subfraction of channels with prolonged open times. We did not observe bursting behavior and found no evidence for a gating mode shift to explain the increased persistent current caused by R1648H. Cells expressing R1657C exhibited conductance, open probability, mean open time, and latency to first opening similar to WT channels but reduced whole-cell current density, suggesting decreased number of functional channels at the plasma membrane. In summary, our findings define single-channel properties for WT-SCN1A, detail the functional phenotypes for two human epilepsy-associated sodium channel mutants, and clarify the mechanism for increased persistent sodium current induced by the R1648H allele.

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