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 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.

scholarly journals Mechanism of Tacrine Block at Adult Human Muscle Nicotinic Acetylcholine Receptors

2002 ◽  
Vol 120 (3) ◽  
pp. 369-393 ◽  
Author(s):  
Richard J. Prince ◽  
Richard A. Pennington ◽  
Steven M. Sine
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Binding Sites ◽  
Acetylcholine Receptors ◽  
Open Channel ◽  
Single Channel ◽  
Dependent Manner ◽  
Open Probability ◽  
Sequential Model ◽  
Hek 293 ◽  
Voltage Dependent

We used single-channel kinetic analysis to study the inhibitory effects of tacrine on human adult nicotinic receptors (nAChRs) transiently expressed in HEK 293 cells. Single channel recording from cell-attached patches revealed concentration- and voltage-dependent decreases in mean channel open probability produced by tacrine (IC50 4.6 μM at −70 mV, 1.6 μM at −150 mV). Two main effects of tacrine were apparent in the open- and closed-time distributions. First, the mean channel open time decreased with increasing tacrine concentration in a voltage-dependent manner, strongly suggesting that tacrine acts as an open-channel blocker. Second, tacrine produced a new class of closings whose duration increased with increasing tacrine concentration. Concentration dependence of closed-times is not predicted by sequential models of channel block, suggesting that tacrine blocks the nAChR by an unusual mechanism. To probe tacrine's mechanism of action we fitted a series of kinetic models to our data using maximum likelihood techniques. Models incorporating two tacrine binding sites in the open receptor channel gave dramatically improved fits to our data compared with the classic sequential model, which contains one site. Improved fits relative to the sequential model were also obtained with schemes incorporating a binding site in the closed channel, but only if it is assumed that the channel cannot gate with tacrine bound. Overall, the best description of our data was obtained with a model that combined two binding sites in the open channel with a single site in the closed state of the receptor.

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.

Possible nicotinic receptor-mediated modulation of synaptic transmission in nucleus of the solitary tract

1997 ◽  
Vol 272 (3) ◽  
pp. R869-R873
Author(s):  
T. Shiraki ◽  
A. Toyoda ◽  
H. Sugino ◽  
A. Hori ◽  
S. Kobayashi
Keyword(s):  
Synaptic Transmission ◽  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Signal Transmission ◽  
Neuronal Cell ◽  
Cell Voltage ◽  
Extracellular Potassium ◽  
Solitary Tract ◽  
Single Channel Currents

Signal transmission from afferent nerves to neurons in the nucleus of the solitary tract (NTS) may be mediated partially by nicotinic acetylcholine receptors (nAChRs). Here, we investigated nAChR-mediated signal transmission using rat NTS slices. First, we characterized nAChRs by obtaining patch-clamp recordings from NTS neuronal cell bodies. Under whole cell voltage-clamp conditions at -60 mV, application of nicotine induced an inward current, and this effect was blocked by hexamethonium. In outside-out patch recordings, nicotine was seen to induce a hexamethonium-sensitive single-channel current. Second, we investigated nAChR-mediated signal transmission. Fast synaptic transmission mediated by nAChRs was not detected. The action of diffusible acetylcholine (ACh) on nAChRs was then tested using the outside-out patches excised from NTS neurons as probes for ACh. When the patch was placed at a distance of 20-30 microm from the cell body, single-channel currents were recorded, and these were inhibited by hexamethonium. The frequency of channel opening was increased by high-extracellular potassium concentration solution suggesting the voltage-dependent release ofACh that acts on nAChRs. These results suggested that nAChR-mediated signal transmission from sensory afferents to NTS neurons is in part mediated by diffusible ACh.

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.

Zinc Potentiates Neuronal Nicotinic Receptors by Increasing Burst Duration

2008 ◽  
Vol 99 (2) ◽  
pp. 999-1007 ◽  
Author(s):  
Bernard Hsiao ◽  
Karla B. Mihalak ◽  
Karl L. Magleby ◽  
Charles W. Luetje
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Large Fraction ◽  
Burst Duration ◽  
Dependent Manner ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Open Probability ◽  
Neuronal Nicotinic Receptors ◽  
Channel Current

Micromolar zinc potentiates neuronal nicotinic acetylcholine receptors (nAChRs) in a subtype-dependent manner. Zinc potentiates receptor function even at saturating agonist concentrations, without altering the receptor desensitization rate. Potentiation could occur through an increase in the number of available receptors, an increase in single-channel current amplitude, or an increase in single-channel open probability. To distinguish among these possibilities, we examined rat neuronal nAChRs expressed in Xenopus oocytes. Blockade of a large fraction of ACh activated α4β4 or α4β2 receptors by the open channel blocker hexamethonium failed to change the extent of potentiation by zinc, suggesting that zinc does not change the number of available receptors. The single-channel amplitudes of ACh (1 μM) activated α4β4 receptors in outside-out patches were similar in the absence and the presence of 100 μM zinc (3.0 ± 0.1 and 2.9 ± 0.1 pA, respectively). To determine the effect of zinc on single-channel open probability, we examined α4β4 receptors in cell-attached patches. The open probability at 100 nM ACh (0.011 ± 0.002) was increased 4.5-fold by 100 μM zinc (0.050 ± 0.008), accounting for most of the potentiation observed at the whole cell level. The increase in open probability was due to an increase in burst duration, which increased from 207 ± 38 ms in the absence of zinc to 830 ± 189 ms in the presence of zinc. Our results suggest that potentiation of neuronal nAChRs by zinc is due to a stabilization of the bursting states of the receptor.

Isoflurane Modulation of Neuronal Nicotinic Acetylcholine Receptors Expressed in Human Embryonic Kidney Cells

2005 ◽  
Vol 102 (1) ◽  
pp. 76-84 ◽  
Author(s):  
Megumi Yamashita ◽  
Takashi Mori ◽  
Keiichi Nagata ◽  
Jay Z. Yeh ◽  
Toshio Narahashi
Keyword(s):  
Nicotinic Acetylcholine Receptors ◽  
Acetylcholine Receptors ◽  
Single Channel ◽  
Single Channels ◽  
Kidney Cells ◽  
Neuronal Nicotinic Acetylcholine Receptors ◽  
Open Probability ◽  
Human Embryonic Kidney Cells ◽  
Open Time ◽  
The Mean

Background It is well established that neuronal nicotinic acetylcholine receptors (nAChRs) are sensitive to inhalational anesthetics. The authors previously reported that halothane potently blocked alpha4beta2-type nAChRs of rat cortical neurons. However, the effect of isoflurane, which is widely used clinically, on nAChRs largely remains to be seen. The authors studied the effects of isoflurane as compared with sevoflurane and halothane on the human alpha4beta2 nAChRs expressed in human embryonic kidney cells. Methods The whole-cell and single-channel patch clamp techniques were used to record currents induced by acetylcholine. Results Isoflurane, sevoflurane, and halothane suppressed the acetylcholine-induced currents in a concentration-dependent manner with 50% inhibitory concentrations of 67.1, 183.3, and 39.8 microM, respectively, which correspond to 0.5 minimum alveolar concentration or less. When anesthetics were coapplied with acetylcholine, isoflurane and sevoflurane decreased the apparent affinity of receptor for acetylcholine, but halothane, in addition, decreased the maximum acetylcholine current. When isoflurane was preapplied and coapplied, its inhibitory action was independent of acetylcholine concentration. Isoflurane blocked the nAChR in both resting and activated states. Single-channel analyses revealed that isoflurane at 84 microM decreased the mean open time and burst duration without inducing "flickering" during channel openings. Isoflurane increased the mean closed time. As a result, the open probability of single channels was greatly reduced by isoflurane. Conclusions Isoflurane, sevoflurane, and halothane potently blocked the alpha4beta2 nAChR. Isoflurane suppression of whole-cell acetylcholine currents was a result of decreases in the open time, burst duration, and open probability and an increase in the closed time of single channels. The high sensitivity of neuronal nAChRs to inhalational anesthetics is expected to play an important role in several stages of anesthesia.

K+ channel currents in basolateral membrane of distal convoluted tubule of rabbit kidney

1989 ◽  
Vol 256 (2) ◽  
pp. F246-F254 ◽  
Author(s):  
J. Taniguchi ◽  
K. Yoshitomi ◽  
M. Imai
Keyword(s):  
Single Channel ◽  
Basolateral Membrane ◽  
Distal Convoluted Tubule ◽  
Rabbit Kidney ◽  
K Channel ◽  
Kinetic Properties ◽  
Open Probability ◽  
K Channels ◽  
Single Channel Currents ◽  
Channel Currents

To examine the nature of ion-conductive pathways in the basolateral membrane of rabbit distal convoluted tubule (DCT), we recorded single-channel currents from the tubule segment isolated from collagenase-treated kidney. Using cell-attached patch pipettes filled with 130 mM KCl, 5.4 mM CaCl2, and 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.4), we observed K+ channels in the basolateral membrane of DCT, having two different single-channel conductances of 48.7 +/- 1.4 (n = 9) and 60.6 +/- 1.4 pS (n = 7). Both types of channels were completely blocked by 0.1 mM BaCl2. Both channels have same open probability of approximately 0.5 at the intrinsic basolateral membrane voltage and were recorded with similar incidence. Mean open and closed times were 31.5 +/- 5.2 and 41.3 +/- 16.0 ms for the smaller channel, and 31.5 +/- 5.1 and 36.7 +/- 8.7 ms for the larger channel, respectively. These kinetic properties did not show any clear voltage dependence in both channels. Because of apparent similarity of channel kinetics, it is possible that both activities might represent different states of the same channel. For definite conclusion, however, further investigations are necessary. In three recordings from 54 successful patches, we observed a flickering channel with rapid kinetics, which was insensitive to 1 meq/l Ba2+. The conductance of this channel was 76.6 pS (n = 2). The extrapolated zero current voltage was 76.0 mV (n = 2), indicating that this channel is permeable to K+. From these results, we suggest that K+ channels constitute conductive pathways for K+ in the basolateral membrane of rabbit DCT.

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