scholarly journals Shaker potassium channel gating. III: Evaluation of kinetic models for activation.

1994 ◽  
Vol 103 (2) ◽  
pp. 321-362 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
R W Aldrich
Keyword(s):  
Steady State ◽  
Conformational Changes ◽  
Time Course ◽  
Single Channel ◽  
Ionic Currents ◽  
Single Step ◽  
Gating Currents ◽  
Open Probability ◽  
Shaker Potassium Channel ◽  
Single Channel Currents

Predictions of different classes of gating models involving identical conformational changes in each of four subunits were compared to the gating behavior of Shaker potassium channels without N-type inactivation. Each model was tested to see if it could simulate the voltage dependence of the steady state open probability, and the kinetics of the single-channel currents, macroscopic ionic currents and macroscopic gating currents using a single set of parameters. Activation schemes based upon four identical single-step activation processes were found to be incompatible with the experimental results, as were those involving a concerted, opening transition. A model where the opening of the channel requires two conformational changes in each of the four subunits can adequately account for the steady state and kinetic behavior of the channel. In this model, the gating in each subunit is independent except for a stabilization of the open state when all four subunits are activated, and an unstable closed conformation that the channel enters after opening. A small amount of negative cooperativity between the subunits must be added to account quantitatively for the dependence of the activation time course on holding voltage.

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 Gating of Recombinant Small-Conductance Ca-activated K+ Channels by Calcium

1998 ◽  
Vol 111 (4) ◽  
pp. 565-581 ◽  
Author(s):  
Birgit Hirschberg ◽  
James Maylie ◽  
John P. Adelman ◽  
Neil V. Marrion
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Cell Types ◽  
K Channel ◽  
Open Probability ◽  
K Channels ◽  
Sensitive Clone ◽  
Single Channel Currents ◽  
Open States ◽  
Small Conductance

Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 μM Ca2+ and was maximal above 2 μM Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was ∼0.6 in 1 μM free Ca2+. A lower open probability of ∼0.05 in 1 μM Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.

scholarly journals Shaker potassium channel gating. II: Transitions in the activation pathway.

1994 ◽  
Vol 103 (2) ◽  
pp. 279-319 ◽  
Author(s):  
W N Zagotta ◽  
T Hoshi ◽  
J Dittman ◽  
R W Aldrich
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Ionic Current ◽  
Activation Process ◽  
Charge Movement ◽  
Open Probability ◽  
Current Time ◽  
Gating Charge

Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associated with a large number of the activation transitions. The equivalent charge movement of 1.1 electronic charges was associated with the closing conformational change. The results were generally consistent with models involving a number of independent and identical transitions with a major exception that the first closing transition is slower than expected as indicated by tail current and OFF gating charge measurements.

scholarly journals Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions.

1991 ◽  
Vol 98 (1) ◽  
pp. 1-17 ◽  
Author(s):  
E Perozo ◽  
C A Vandenberg ◽  
D S Jong ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Slow Component ◽  
Giant Axon ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Delayed Rectifier Current ◽  
K Current ◽  
The Difference ◽  
Single Channel Currents

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.

scholarly journals Mechanism of activation of the prokaryotic channel ELIC by propylamine: A single-channel study

2014 ◽  
Vol 145 (1) ◽  
pp. 23-45 ◽  
Author(s):  
Alessandro Marabelli ◽  
Remigijus Lape ◽  
Lucia Sivilotti
Keyword(s):  
Ion Channel ◽  
Nicotinic Acetylcholine Receptors ◽  
Intermediate State ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Single Channel ◽  
Structural Information ◽  
Kinetic Properties ◽  
Open Probability ◽  
Single Channel Currents

Prokaryotic channels, such as Erwinia chrysanthemi ligand-gated ion channel (ELIC) and Gloeobacter violaceus ligand-gated ion channel, give key structural information for the pentameric ligand-gated ion channel family, which includes nicotinic acetylcholine receptors. ELIC, a cationic channel from E. chrysanthemi, is particularly suitable for single-channel recording because of its high conductance. Here, we report on the kinetic properties of ELIC channels expressed in human embryonic kidney 293 cells. Single-channel currents elicited by the full agonist propylamine (0.5–50 mM) in outside-out patches at −60 mV were analyzed by direct maximum likelihood fitting of kinetic schemes to the idealized data. Several mechanisms were tested, and their adequacy was judged by comparing the predictions of the best fit obtained with the observable features of the experimental data. These included open-/shut-time distributions and the time course of macroscopic propylamine-activated currents elicited by fast theta-tube applications (50–600 ms, 1–50 mM, −100 mV). Related eukaryotic channels, such as glycine and nicotinic receptors, when fully liganded open with high efficacy to a single open state, reached via a preopening intermediate. The simplest adequate description of their activation, the “Flip” model, assumes a concerted transition to a single intermediate state at high agonist concentration. In contrast, ELIC open-time distributions at saturating propylamine showed multiple components. Thus, more than one open state must be accessible to the fully liganded channel. The “Primed” model allows opening from multiple fully liganded intermediates. The best fits of this type of model showed that ELIC maximum open probability (99%) is reached when at least two and probably three molecules of agonist have bound to the channel. The overall efficacy with which the fully liganded channel opens was ∼102 (∼20 for α1β glycine channels). The microscopic affinity for the agonist increased as the channel activated, from 7 mM for the resting state to 0.15 mM for the partially activated intermediate state.

scholarly journals Activation of Shaker Potassium Channels

1998 ◽  
Vol 111 (2) ◽  
pp. 313-342 ◽  
Author(s):  
N.E. Schoppa ◽  
F.J. Sigworth
Keyword(s):  
Single Channel ◽  
Conformational Stability ◽  
Ionic Currents ◽  
Total Charge ◽  
Charge Movement ◽  
Gating Currents ◽  
Shaker Potassium Channel ◽  
Channel Opening ◽  
Tetrameric Structure ◽  
Charge Movements

A functional kinetic model is developed to describe the activation gating process of the Shaker potassium channel. The modeling in this paper is constrained by measurements described in the preceding two papers, including macroscopic ionic and gating currents and single channel ionic currents. These data were obtained from the normally activating wild-type channel as well as a mutant channel V2, in which the leucine at position 382 has been mutated to a valine. Different classes of models that incorporate Shaker's symmetrical tetrameric structure are systematically examined. Many simple gating models are clearly inadequate, but a model that can account for all of the qualitative features of the data has the channel open after its four subunits undergo three transitions in sequence, and two final transitions that reflect the concerted action of the four subunits. In this model, which we call Scheme 3+2′, the channel can also close to several states that are not part of the activation path. Channel opening involves a large total charge movement (10.8 e0), which is distributed among a large number of small steps each with rather small charge movements (between 0.6 and 1.05 e0). The final two transitions are different from earlier steps by having slow backward rates. These steps confer a cooperative mechanism of channel opening at Shaker's activation voltages. In the context of Scheme 3+2′, significant effects of the V2 mutation are limited to the backward rates of the final two transitions, implying that L382 plays an important role in the conformational stability of the final two states.

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.

scholarly journals Hidden Markov Model Analysis of Intermediate Gating Steps Associated with the Pore Gate of Shaker Potassium Channels

2001 ◽  
Vol 118 (5) ◽  
pp. 547-564 ◽  
Author(s):  
Jie Zheng ◽  
Lalitha Vankataramanan ◽  
Fred J. Sigworth
Keyword(s):  
Potassium Channels ◽  
Conformational Changes ◽  
Time Course ◽  
Single Channel ◽  
Hidden Markov ◽  
Voltage Sensitivity ◽  
Open Probability ◽  
Shaker Channels ◽  
Voltage Dependent ◽  
Shaker Potassium Channels

Cooperativity among the four subunits helps give rise to the remarkable voltage sensitivity of Shaker potassium channels, whose open probability changes tenfold for a 5-mV change in membrane potential. The cooperativity in these channels is thought to arise from a concerted structural transition as the final step in opening the channel. Recordings of single-channel ionic currents from certain other channel types, as well as our previous recordings from T442S mutant Shaker channels, however, display intermediate conductance levels in addition to the fully open and closed states. These sublevels might represent stepwise, rather than concerted, transitions in the final steps of channel activation. Here, we report a similar fine structure in the closing transitions of Shaker channels lacking the mutation. Describing the deactivation time course with hidden Markov models, we find that two subconductance levels are rapidly traversed during most closing transitions of chimeric, high conductance Shaker channels. The lifetimes of these levels are voltage-dependent, with maximal values of 52 and 22 μs at −100 mV, and the voltage dependences of transitions among these states suggest that they arise from equivalent conformational changes occurring in individual subunits. At least one subconductance level is found to be traversed in normal conductance Shaker channels. We speculate that voltage-dependent conformational changes in the subunits give rise to changes in a “pore gate” associated with the selectivity filter region of the channel, producing the subconductance states. As a control for the hidden Markov analysis, we applied the same procedures to recordings of the recovery from N-type inactivation in Shaker channels. These transitions are found to be instantaneous in comparison.

scholarly journals Modal affinities of endplate acetylcholine receptors caused by loop C mutations

2015 ◽  
Vol 146 (5) ◽  
pp. 375-386 ◽  
Author(s):  
Ridhima Vij ◽  
Prasad Purohit ◽  
Anthony Auerbach
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptors ◽  
Time Course ◽  
Single Channel ◽  
Equilibrium Constants ◽  
Open Probability ◽  
In The Wild ◽  
Single Channel Currents ◽  
Almost All ◽  
Channel Currents

The time course of the endplate current is determined by the rate and equilibrium constants for acetylcholine receptor (AChR) activation. We measured these constants in single-channel currents from AChRs with mutations at the neurotransmitter-binding sites, in loop C. The main findings are: (a) Almost all perturbations of loop C generate heterogeneity in the channel open probability (“modes”). (b) Modes are generated by different affinities for ACh that can be either higher or lower than in the wild-type receptors. (c) The modes are stable, in so far as each receptor maintains its affinity for at least several minutes. (d) Different agonists show different degrees of modal activity. With the loop C mutation αP197A, there are four modes with ACh but only two with partial agonists. (e) The affinity variations arise exclusively from the αδ-binding site. (f) Substituting four γ-subunit residues into the δ subunit (three in loop E and one in the β5–β5′ linker) reduces modal activity. (g) At each neurotransmitter-binding site, affinity is determined by a core of five aromatic residues. Modes are eliminated by an alanine mutation at δW57 but not at the other aromatics. (h) Modes are eliminated by a phenylalanine substitution at all core aromatics except αY93. The results suggest that, at the αδ agonist site, loop C and the complementary subunit surface can each adopt alternative conformations and interact with each other to influence the position of δW57 with respect to the aromatic core and, hence, affinity.

Effect of Prozac on whole cell ionic currents in lens and corneal epithelia

1995 ◽  
Vol 269 (1) ◽  
pp. C250-C256 ◽  
Author(s):  
J. L. Rae ◽  
A. Rich ◽  
A. C. Zamudio ◽  
O. A. Candia
Keyword(s):  
Potassium Channels ◽  
Single Channel ◽  
Ionic Currents ◽  
Current Amplitude ◽  
Human Lens ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Channel Current ◽  
Single Channel Current ◽  
Ionic Conductances

Prozac (fluoxetine), a compound used therapeutically in humans to combat depression, has substantial effects on ionic conductances in rabbit corneal epithelial cells and in cultured human lens epithelium. In corneal epithelium, it reduces the current due to the large-conductance potassium channels that dominate this preparation. Its effects seem largely to decrease the open probability while leaving the single-channel current amplitude unaltered. In cultured human epithelium, currents from calcium-activated potassium channels and inward rectifiers are unaffected by Prozac. Delayed-rectifier potassium currents are reduced by Prozac in a complicated way that involves both gating and single-channel current amplitude. Fast tetrodotoxin-blockable sodium currents are also decreased by Prozac in this preparation. For all of these ion conductance effects, Prozac concentrations of 10(-5) to 10(-4) M are required. Whereas these levels are 10- to 100-fold higher than the plasma levels achieved in therapeutic use in humans, they are comparable to or less than levels needed for many other blockers of the ionic conductances studied here.

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