scholarly journals Improved resolution of single channel dwell times reveals mechanisms of binding, priming, and gating in muscle AChR

2016 ◽  
Vol 148 (1) ◽  
pp. 43-63 ◽  
Author(s):  
Nuriya Mukhtasimova ◽  
Corrie J.B. daCosta ◽  
Steven M. Sine
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Partial Agonist ◽  
Equilibrium Constants ◽  
Channel Gating ◽  
Kinetic Scheme ◽  
Step Response ◽  
Forward Rate ◽  
Gain Of Function ◽  
Single Channel Currents

The acetylcholine receptor (AChR) from vertebrate skeletal muscle initiates voluntary movement, and its kinetics of activation are crucial for maintaining the safety margin for neuromuscular transmission. Furthermore, the kinetic mechanism of the muscle AChR serves as an archetype for understanding activation mechanisms of related receptors from the Cys-loop superfamily. Here we record currents through single muscle AChR channels with improved temporal resolution approaching half an order of magnitude over our previous best. A range of concentrations of full and partial agonists are used to elicit currents from human wild-type and gain-of-function mutant AChRs. For each agonist–receptor combination, rate constants are estimated from maximum likelihood analysis using a kinetic scheme comprised of agonist binding, priming, and channel gating steps. The kinetic scheme and rate constants are tested by stochastic simulation, followed by incorporation of the experimental step response, sampling rate, background noise, and filter bandwidth. Analyses of the simulated data confirm all rate constants except those for channel gating, which are overestimated because of the established effect of noise on the briefest dwell times. Estimates of the gating rate constants were obtained through iterative simulation followed by kinetic fitting. The results reveal that the agonist association rate constants are independent of agonist occupancy but depend on receptor state, whereas those for agonist dissociation depend on occupancy but not on state. The priming rate and equilibrium constants increase with successive agonist occupancy, and for a full agonist, the forward rate constant increases more than the equilibrium constant; for a partial agonist, the forward rate and equilibrium constants increase equally. The gating rate and equilibrium constants also increase with successive agonist occupancy, but unlike priming, the equilibrium constants increase more than the forward rate constants. As observed for a full and a partial agonist, the gain-of-function mutation affects the relationship between rate and equilibrium constants for priming but not for channel gating. Thus, resolving brief single channel currents distinguishes priming from gating steps and reveals how the corresponding rate and equilibrium constants depend on agonist occupancy.

scholarly journals Fundamental Gating Mechanism of Nicotinic Receptor Channel Revealed by Mutation Causing a Congenital Myasthenic Syndrome

2000 ◽  
Vol 116 (3) ◽  
pp. 449-462 ◽  
Author(s):  
Hai-Long Wang ◽  
Kinji Ohno ◽  
Margherita Milone ◽  
Joan M. Brengman ◽  
Amelia Evoli ◽  
...  
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Gaussian Function ◽  
Congenital Myasthenic Syndrome ◽  
Markov Modeling ◽  
Amphipathic Helix ◽  
Wild Type ◽  
Gating Mechanism ◽  
Myasthenic Syndrome ◽  
Single Channel Currents

We describe the genetic and kinetic defects in a congenital myasthenic syndrome due to the mutation εA411P in the amphipathic helix of the acetylcholine receptor (AChR) ε subunit. Myasthenic patients from three unrelated families are either homozygous for εA411P or are heterozygous and harbor a null mutation in the second ε allele, indicating that εA411P is recessive. We expressed human AChRs containing wild-type or A411P ε subunits in 293HEK cells, recorded single channel currents at high bandwidth, and determined microscopic rate constants for individual channels using hidden Markov modeling. For individual wild-type and mutant channels, each rate constant distributes as a Gaussian function, but the spread in the distributions for channel opening and closing rate constants is greatly expanded by εA411P. Prolines engineered into positions flanking residue 411 of the ε subunit greatly increase the range of activation kinetics similar to εA411P, whereas prolines engineered into positions equivalent to εA411 in β and δ subunits are without effect. Thus, the amphipathic helix of the ε subunit stabilizes the channel, minimizing the number and range of kinetic modes accessible to individual AChRs. The findings suggest that analogous stabilizing structures are present in other ion channels, and possibly allosteric proteins in general, and that they evolved to maintain uniformity of activation episodes. The findings further suggest that the fundamental gating mechanism of the AChR channel can be explained by a corrugated energy landscape superimposed on a steeply sloped energy well.

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 Rate theory calculation of gramicidin single-channel currents using NMR-derived rate constants.

1980 ◽  
Vol 77 (4) ◽  
pp. 2028-2032 ◽  
Author(s):  
D. W. Urry ◽  
C. M. Venkatachalam ◽  
A. Spisni ◽  
P. Lauger ◽  
M. A. Khaled
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Rate Theory ◽  
Single Channel Currents ◽  
Theory Calculation ◽  
Channel Currents

The malonyl gramicidin channel: NMR-derived rate constants and comparison of calculated and experimental single-channel currents

1980 ◽  
Vol 55 (1) ◽  
pp. 29-51 ◽  
Author(s):  
D. W. Urry ◽  
C. M. Venkatachalam ◽  
A. Spisni ◽  
R. J. Bradley ◽  
T. L. Trapane ◽  
...  
Keyword(s):  
Rate Constants ◽  
Single Channel ◽  
Gramicidin Channel ◽  
Single Channel Currents ◽  
Channel Currents

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 NMDA receptor channel gating control by the pre-M1 helix

2020 ◽  
Vol 152 (4) ◽  
Author(s):  
Miranda J. McDaniel ◽  
Kevin K. Ogden ◽  
Steven A. Kell ◽  
Pieter B. Burger ◽  
Dennis C. Liotta ◽  
...  
Keyword(s):  
Nmda Receptor ◽  
Conformational Changes ◽  
Time Course ◽  
Transmembrane Domain ◽  
Single Channel ◽  
De Novo ◽  
Channel Gating ◽  
Open Probability ◽  
Single Channel Currents

The NMDA receptor (NMDAR) is an ionotropic glutamate receptor formed from the tetrameric assembly of GluN1 and GluN2 subunits. Within the flexible linker between the agonist binding domain (ABD) and the M1 helix of the pore-forming transmembrane helical bundle lies a two-turn, extracellular pre-M1 helix positioned parallel to the plasma membrane and in van der Waals contact with the M3 helix thought to constitute the channel gate. The pre-M1 helix is tethered to the bilobed ABD, where agonist-induced conformational changes initiate activation. Additionally, it is a locus for de novo mutations associated with neurological disorders, is near other disease-associated de novo sites within the transmembrane domain, and is a structural determinant of subunit-selective modulators. To investigate the role of the pre-M1 helix in channel gating, we performed scanning mutagenesis across the GluN2A pre-M1 helix and recorded whole-cell macroscopic and single channel currents from HEK293 cell-attached patches. We identified two residues at which mutations perturb channel open probability, the mean open time, and the glutamate deactivation time course. We identified a subunit-specific network of aromatic amino acids located in and around the GluN2A pre-M1 helix to be important for gating. Based on these results, we are able to hypothesize about the role of the pre-M1 helix in other NMDAR subunits based on sequence and structure homology. Our results emphasize the role of the pre-M1 helix in channel gating, implicate the surrounding amino acid environment in this mechanism, and suggest unique subunit-specific contributions of pre-M1 helices to GluN1 and GluN2 gating.

scholarly journals Low pH Potentiates Both Capsaicin Binding and Channel Gating of VR1 Receptors

2003 ◽  
Vol 122 (1) ◽  
pp. 45-61 ◽  
Author(s):  
Sujung Ryu ◽  
Beiying Liu ◽  
Feng Qin
Keyword(s):  
Single Channel ◽  
Channel Gating ◽  
General Mechanism ◽  
Low Concentrations ◽  
Excised Patches ◽  
High Concentrations ◽  
Activation Pathways ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Important Basis

Capsaicin ion channels are highly expressed in peripheral nervous terminals and involved in pain and thermal sensations. One characteristic of the cloned VR1 receptor is its multimodal responses to various types of noxious stimuli. The channel is independently activated by capsaicin and related vanilloids at submicromolar range, by heat above 40°C, and by protons at pH below 6.5. Furthermore, simultaneous applications of two or more stimuli lead to cross sensitization of the receptor, with an apparent increase in the sensitivity to any individual stimulus when applied alone. We studied here the mechanism underlying such cross-sensitization; in particular, between capsaicin and pH, two prototypical stimuli for the channel. By analyzing single-channel currents recorded from excised-patches expressing single recombinant VR1 receptors, we examined the effect of pH on burst properties of capsaicin activation at low concentrations and the effect on gating kinetics at high concentrations. Our results indicate that pH has dual effects on both capsaicin binding and channel gating. Lowering pH enhances the apparent binding affinity of capsaicin, promotes the occurrences of long openings and short closures, and stabilizes at least one of the open conformations of the channel. Our data also demonstrate that capsaicin binding and protonation of the receptor interact allosterically, where the effect of one can be offset by the effect of the other. These results provide important basis to further understand the nature of the activation pathways of the channel evoked by different stimuli as well as the general mechanism underling the cross-sensitization of pain.

scholarly journals An integrated catch-and-hold mechanism activates nicotinic acetylcholine receptors

2012 ◽  
Vol 140 (1) ◽  
pp. 17-28 ◽  
Author(s):  
Snehal Jadey ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Nicotinic Acetylcholine Receptors ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Protein Structures ◽  
Channel Gating ◽  
Structural Basis ◽  
Agonist Binding ◽  
Adult Type

In neuromuscular acetylcholine (ACh) receptor channels (AChRs), agonist molecules bind with a low affinity (LA) to two sites that can switch to high affinity (HA) and increase the probability of channel opening. We measured (by using single-channel kinetic analysis) the rate and equilibrium constants for LA binding and channel gating for several different agonists of adult-type mouse AChRs. Almost all of the variation in the equilibrium constants for LA binding was from differences in the association rate constants. These were consistently below the limit set by diffusion and were substantially different even though the agonists had similar sizes and the same charge. This suggests that binding to resting receptors is not by diffusion alone and, hence, that each binding site can undergo two conformational changes (“catch” and “hold”) that connect three different structures (apo-, LA-bound, and HA-bound). Analyses of ACh-binding protein structures suggest that this binding site, too, may adopt three discrete structures having different degrees of loop C displacement (“capping”). For the agonists we tested, the logarithms of the equilibrium constants for LA binding and LA↔HA gating were correlated. Although agonist binding and channel gating have long been considered to be separate processes in the activation of ligand-gated ion channels, this correlation implies that the catch-and-hold conformational changes are energetically linked and together comprise an integrated process having a common structural basis. We propose that loop C capping mainly reflects agonist binding, with its two stages corresponding to the formation of the LA and HA complexes. The catch-and-hold reaction coordinate is discussed in terms of preopening states and thermodynamic cycles of activation.

scholarly journals Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons.

1995 ◽  
Vol 105 (6) ◽  
pp. 837-859 ◽  
Author(s):  
J L Donnelly ◽  
B S Pallotta
Keyword(s):  
Steady State ◽  
Rat Brain ◽  
Cortical Neurons ◽  
Single Channel ◽  
Channel Gating ◽  
Reversal Potential ◽  
Open Probability ◽  
Histidine Residues ◽  
Gating Model ◽  
Single Channel Currents

The role of histidine residues in the function of N-methyl-D-aspartate (NMDA)-activated channels was tested with the histidine-modifying reagent diethylpyrocarbonate (DEP) applied to cells and membrane patches from rat brain cortical neurons in culture. Channels in excised outside-out patches that were treated with 3 mM DEP for 15-30 s (pH 6.5) showed an average 3.4-fold potentiation in steady state open probability when exposed to NMDA and glycine. Analysis of the underlying alterations in channel gating revealed no changes in the numbers of kinetic states: distributions of open intervals were fitted with three exponential components, and four components described the shut intervals, in both control and DEP-modified channels. However, the distribution of shut intervals was obviously different after DEP treatment, consistent with the single-channel current record. After modification, the proportion of long shut states was decreased while the time constants were largely unaffected. Burst kinetics reflected these effects with an increase in the average number of openings/burst from 1.5 (control) to 2.2 (DEP), and a decrease in the average interburst interval from 54.1 to 38.2 ms. These effects were most likely due to histidine modification because other reagents (n-acetylimidazole and 2,4,6-trinitrobenzene 1-sulfonic acid) that are specific for residues other than histidine failed to reproduce the effects of DEP, whereas hydroxylamine could restore channel open probability to control levels. In contrast to these effects on channel gating, DEP had no effect on average single-channel conductance or reversal potential under bi-ionic (Na+:Cs+) conditions. Inhibition by zinc was also unaffected by DEP. We propose a channel gating model in which transitions between single- and multi-opening burst modes give rise to the channel activity observed under steady state conditions. When adjusted to account for the effects of DEP, this model suggests that one or more extracellular histidine residues involved in channel gating are associated with a single kinetic state.

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