scholarly journals Agonist-dependent Single Channel Current and Gating in α4β2δ and α1β2γ2S GABAA Receptors

2008 ◽  
Vol 131 (2) ◽  
pp. 163-181 ◽  
Author(s):  
Angelo Keramidas ◽  
Neil L. Harrison
Keyword(s):  
Single Channel ◽  
Reaction Scheme ◽  
Mammalian Brain ◽  
Receptor Subtype ◽  
Kinetic Properties ◽  
Open Probability ◽  
Electrophysiological Recordings ◽  
Acid Type ◽  
Single Channel Activity ◽  
Open States

The family of γ-aminobutyric acid type A receptors (GABAARs) mediates two types of inhibition in the mammalian brain. Phasic inhibition is mediated by synaptic GABAARs that are mainly comprised of α1, β2, and γ2 subunits, whereas tonic inhibition is mediated by extrasynaptic GABAARs comprised of α4/6, β2, and δ subunits. We investigated the activation properties of recombinant α4β2δ and α1β2γ2S GABAARs in response to GABA and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3(2H)-one (THIP) using electrophysiological recordings from outside-out membrane patches. Rapid agonist application experiments indicated that THIP produced faster opening rates at α4β2δ GABAARs (β ∼1600 s−1) than at α1β2γ2S GABAARs (β ∼ 460 s−1), whereas GABA activated α1β2γ2S GABAARs more rapidly (β ∼1800 s−1) than α4β2δ GABAARs (β < 440 s−1). Single channel recordings of α1β2γ2S and α4β2δ GABAARs showed that both channels open to a main conductance state of ∼25 pS at −70 mV when activated by GABA and low concentrations of THIP, whereas saturating concentrations of THIP elicited ∼36 pS openings at both channels. Saturating concentrations of GABA elicited brief (<10 ms) openings with low intraburst open probability (PO ∼ 0.3) at α4β2δ GABAARs and at least two “modes” of single channel bursting activity, lasting ∼100 ms at α1β2γ2S GABAARs. The most prevalent bursting mode had a PO of ∼0.7 and was described by a reaction scheme with three open and three shut states, whereas the “high” PO mode (∼0.9) was characterized by two shut and three open states. Single channel activity elicited by THIP in α4β2δ and α1β2γ2S GABAARs occurred as a single population of bursts (PO ∼0.4–0.5) of moderate duration (∼33 ms) that could be described by schemes containing two shut and two open states for both GABAARs. Our data identify kinetic properties that are receptor-subtype specific and others that are agonist specific, including unitary conductance.

Characterization of RyR1-slow, a ryanodine receptor specific to slow-twitch skeletal muscle

2000 ◽  
Vol 279 (5) ◽  
pp. R1889-R1898 ◽  
Author(s):  
Jeffery Morrissette ◽  
Le Xu ◽  
Alexandra Nelson ◽  
Gerhard Meissner ◽  
Barbara A. Block
Keyword(s):  
Skeletal Muscle ◽  
Single Channel ◽  
Fiber Type ◽  
Ryanodine Receptors ◽  
Open Probability ◽  
Dependent Inhibition ◽  
Single Channel Activity ◽  
Intracellular Ca ◽  
Slow Twitch ◽  
Fast Twitch

Two distinct skeletal muscle ryanodine receptors (RyR1s) are expressed in a fiber type–specific manner in fish skeletal muscle (11). In this study, we compare [3H]ryanodine binding and single channel activity of RyR1-slow from fish slow-twitch skeletal muscle with RyR1-fast and RyR3 isolated from fast-twitch skeletal muscle. Scatchard plots indicate that RyR1-slow has a lower affinity for [3H]ryanodine when compared with RyR1-fast. In single channel recordings, RyR1-slow and RyR1-fast had similar slope conductances. However, the maximum open probability (Po) of RyR1-slow was threefold less than the maximum Po of RyR1-fast. Single channel studies also revealed the presence of two populations of RyRs in tuna fast-twitch muscle (RyR1-fast and RyR3). RyR3 had the highest Po of all the RyR channels and displayed less inhibition at millimolar Ca2+. The addition of 5 mM Mg-ATP or 2.5 mM β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) to the channels increased the Po and [3H]ryanodine binding of both RyR1s but also caused a shift in the Ca2+ dependency curve of RyR1-slow such that Ca2+-dependent inactivation was attenuated. [3H]ryanodine binding data also showed that Mg2+-dependent inhibition of RyR1-slow was reduced in the presence of AMP-PCP. These results indicate differences in the physiological properties of RyRs in fish slow- and fast-twitch skeletal muscle, which may contribute to differences in the way intracellular Ca2+ is regulated in these muscle types.

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 Mechanical gating of the auditory transduction channel TMC1 involves the fourth and sixth transmembrane helices

2022 ◽  
Author(s):  
Nurunisa Akyuz ◽  
K. Domenica Karavitaki ◽  
Bifeng Pan ◽  
Panos I. Tamvakologos ◽  
Kelly P. Brock ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Hair Cells ◽  
Single Channel ◽  
Transmembrane Helices ◽  
Open Probability ◽  
Transmembrane Channel ◽  
Force Sensitivity ◽  
Electrophysiological Recordings ◽  
Auditory Transduction ◽  
Pore Lining

The transmembrane channel-like (TMC) 1 and 2 proteins play a central role in auditory transduction, forming ion channels that convert sound into electrical signals. However, the molecular mechanism of their gating remains unknown. Here, using predicted structural models as a guide, we probed the effects of twelve mutations on the mechanical gating of the transduction currents in native hair cells of Tmc1/2-null mice expressing virally introduced TMC1 variants. Whole-cell electrophysiological recordings revealed that mutations within the pore-lining transmembrane (TM) helices 4 and 6 modified gating, reducing the force sensitivity or shifting the open probability of the channels, or both. For some of the mutants, these changes were accompanied by a change in single-channel conductance. Our observations are in line with a model wherein conformational changes in the TM4 and TM6 helices are involved in the mechanical gating of the transduction channel.

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.

Differential regulation of voltage-gated K+ channels by oxidized and reduced pyridine nucleotide coenzymes

2005 ◽  
Vol 288 (2) ◽  
pp. C366-C376 ◽  
Author(s):  
Srinivas M. Tipparaju ◽  
Nina Saxena ◽  
Si-Qi Liu ◽  
Rajiv Kumar ◽  
Aruni Bhatnagar
Keyword(s):  
Redox State ◽  
Redox Regulation ◽  
Single Channel ◽  
Pyridine Nucleotide ◽  
Pyridine Nucleotides ◽  
Open Probability ◽  
Kv Channels ◽  
Open Time ◽  
Dependent Inactivation ◽  
Single Channel Activity

The activity of the voltage-sensitive K+ (Kv) channels varies as a function of the intracellular redox state and metabolism, and several Kv channels act as oxygen sensors. However, the mechanisms underlying the metabolic and redox regulation of these channels remain unclear. In this study we investigated the regulation of Kv channels by pyridine nucleotides. Heterologous expression of Kvα1.5 in COS-7 cells led to the appearance of noninactivating currents. Inclusion of 0.1–1 mM NAD+ or 0.03–0.5 mM NADP+ in the internal solution of the patch pipette did not affect Kv currents. However, 0.5 and 1 mM NAD+ and 0.1 and 0.5 mM NADP+ prevented inactivation of Kv currents in cells transfected with Kvα1.5 and Kvβ1.3 and shifted the voltage dependence of activation to depolarized potentials. The Kvβ-dependent inactivation of Kvα currents was also decreased by internal pipette perfusion of the cell with 1 mM NAD+. The Kvα1.5-Kvβ1.3 currents were unaffected by the internal application of 0.1 mM NADPH or 0.1 or 1 mM NADH. Excised inside-out patches from cells expressing Kvα1.5-Kvβ1.3 showed transient single-channel activity. The mean open time and the open probability of these currents were increased by the inclusion of 1 mM NAD+ in the perfusate. These results suggest that NAD(P)+ prevents Kvβ-mediated inactivation of Kv currents and provide a novel mechanism by which pyridine nucleotides could regulate specific K+ currents as a function of the cellular redox state [NAD(P)H-to-NAD(P)+ ratio].

scholarly journals Adenosine and anoxia reduce N-methyl-D-aspartate receptor open probability in turtle cerebrocortex.

1998 ◽  
Vol 201 (2) ◽  
pp. 289-297 ◽  
Author(s):  
L T Buck ◽  
P E Bickler
Keyword(s):  
Membrane Potential ◽  
Nmda Receptor ◽  
Ion Channel ◽  
Single Channel ◽  
Tissue Injury ◽  
Brain Slices ◽  
Current Amplitude ◽  
Mammalian Brain ◽  
Open Probability ◽  
Experimental Conditions

During normoxia, glutamate and the glutamate family of ion channels play a key role in mediating rapid excitatory synaptic transmission in the central nervous system. However, during hypoxia, intracellular [Ca2+] increases to neurotoxic levels, mediated largely by the N-methyl-D-aspartate (NMDA) subfamily of glutamate receptors. Adenosine has been shown to decrease the magnitude of the hypoxia-induced increase in [Ca2+]i in mammalian brain slices, delaying tissue injury. Turtle brain is remarkably tolerant of anoxia, maintaining a pre-anoxic [Ca2+]i while cerebral adenosine levels increase 12-fold. Employing cell-attached single-channel patch-clamp techniques, we studied the effect of adenosine (200 micromol l-1) and anoxia on NMDA receptor open probability (Popen) and current amplitude. After 60 min of anoxic perfusion, channel Popen decreased by 65 % (from 6.8+/-1.6 to 2.4+/-0.8 %) an effect that could also be achieved with a normoxic perfusion of 200 micromol l-1 adenosine (Popen decreased from 5.8+/-1.1 to 2.3+/-1.2 %). The inclusion of 10 micromol l-1 8-phenyltheophylline, an A1 receptor blocker, prevented the adenosine- and anoxia-induced decrease in Popen. Mean single-channel current amplitude remained at approximately 2.7+/-0.23 pA under all experimental conditions. To determine whether a change in the membrane potential could be part of the mechanism by which Popen decreases, membrane and threshold potential were measured following each experiment. Membrane potential did not change significantly under any condition, ranging from -76.8 to -80.6 mV. Therefore, during anoxia, NMDA receptors cannot be regulated by Mg2+ in a manner dependent on membrane potential. Threshold potentials did decrease significantly following 60 min of anoxic or adenosine perfusion (control -33.3+/-1.9 mV, anoxia -28.4+/-1.5 mV, adenosine -23.4+/-2.8 mV). We conclude that anoxia modulates NMDA receptor activity and that adenosine plays a key role in mediating this change. This is the first direct measurement of ion channel activity in anoxic turtle brain and demonstrates that ion channel regulation is part of the naturally evolved anoxic defence mechanism of this species.

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.

Acetylcholine-sensitive potassium channels in human atrial myocytes

1990 ◽  
Vol 259 (6) ◽  
pp. H1730-H1735 ◽  
Author(s):  
R. Sato ◽  
I. Hisatome ◽  
J. A. Wasserstrom ◽  
C. E. Arentzen ◽  
D. H. Singer
Keyword(s):  
Single Channel ◽  
K Channel ◽  
Channel Activity ◽  
Atrial Myocytes ◽  
Open Probability ◽  
Human Atrial Myocytes ◽  
Open Time ◽  
Patch Pipette ◽  
Single Channel Activity ◽  
Gamma S

Single channel recording techniques were used to study acetylcholine (ACh)-sensitive K+ channel activity in human atrial myocytes isolated from specimens obtained during corrective cardiac surgery. Under conditions of cell-attached patch, the presence of ACh in the patch pipette activated K+ channels. Single channel activity occurred in periodic bursts. The channels exhibited a slope conductance of 46 +/- 2 pS inwardly (means +/- SD, n = 4). During a burst, both open and closed time histograms were fitted by a single exponential curve, suggesting the existence of one open and one closed state during a burst. Open probability increased directly with ACh concentration without affecting open time. The channel could be activated by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) (in the presence and absence of ACh in the pipette, respectively). Slope conductance, the response to GTP and GTP gamma S, and the independence of activation from Ca2+ were similar to those for other species. In contrast, sensitivity to ACh appeared diminished compared with frog atrial myocytes.

scholarly journals Gain-of-Function Mutations in the MEC-4 DEG/ENaC Sensory Mechanotransduction Channel Alter Gating and Drug Blockade

2007 ◽  
Vol 129 (2) ◽  
pp. 161-173 ◽  
Author(s):  
Austin L. Brown ◽  
Silvia M. Fernandez-Illescas ◽  
Zhiwen Liao ◽  
Miriam B. Goodman
Keyword(s):  
Single Channel ◽  
Large Family ◽  
Amino Acid Side Chain ◽  
Open Probability ◽  
Gain Of Function ◽  
Diffusion Limited ◽  
Touch Sensation ◽  
Additional Support ◽  
Open States ◽  
Heteromeric Channels

MEC-4 and MEC-10 are the pore-forming subunits of the sensory mechanotransduction complex that mediates touch sensation in Caenorhabditis elegans (O'Hagan, R., M. Chalfie, and M.B. Goodman. 2005. Nat. Neurosci. 8:43–50). They are members of a large family of ion channel proteins, collectively termed DEG/ENaCs, which are expressed in epithelial cells and neurons. In Xenopus oocytes, MEC-4 can assemble into homomeric channels and coassemble with MEC-10 into heteromeric channels (Goodman, M.B., G.G. Ernstrom, D.S. Chelur, R. O'Hagan, C.A. Yao, and M. Chalfie. 2002. Nature. 415:1039–1042). To gain insight into the structure–function principles that govern gating and drug block, we analyzed the effect of gain-of-function mutations using a combination of two-electrode voltage clamp, single-channel recording, and outside-out macropatches. We found that mutation of A713, the d or degeneration position, to residues larger than cysteine increased macroscopic current, open probability, and open times in homomeric channels, suggesting that bulky residues at this position stabilize open states. Wild-type MEC-10 partially suppressed the effect of such mutations on macroscopic current, suggesting that subunit–subunit interactions regulate open probability. Additional support for this idea is derived from an analysis of macroscopic currents carried by single-mutant and double-mutant heteromeric channels. We also examined blockade by the diuretic amiloride and two related compounds. We found that mutation of A713 to threonine, glycine, or aspartate decreased the affinity of homomeric channels for amiloride. Unlike the increase in open probability, this effect was not related to size of the amino acid side chain, indicating that mutation at this site alters antagonist binding by an independent mechanism. Finally, we present evidence that amiloride block is diffusion limited in DEG/ENaC channels, suggesting that variations in amiloride affinity result from variations in binding energy as opposed to accessibility. We conclude that the d position is part of a key region in the channel functionally and structurally, possibly representing the beginning of a pore-forming domain.

scholarly journals Single-channel analysis of functional epithelial sodium channel (ENaC) stability at the apical membrane of A6 distal kidney cells

2008 ◽  
Vol 295 (5) ◽  
pp. F1519-F1527 ◽  
Author(s):  
Ling Yu ◽  
My N. Helms ◽  
Qiang Yue ◽  
Douglas C. Eaton
Keyword(s):  
Protein Trafficking ◽  
Half Life ◽  
Single Channel ◽  
Apical Membrane ◽  
Brefeldin A ◽  
Open Probability ◽  
Individual Subunit ◽  
Single Channel Activity ◽  
Total Body ◽  
The Stability

Epithelial sodium channels (ENaC) play an essential role in maintaining total body fluid and electrolyte homeostasis. As such, abnormal expression of ENaC at the cell surface is linked to several important human diseases. Although the stability of ENaC subunits has been extensively studied by protein biochemical analysis, the half-life of the functional channel in the apical membrane remains controversial. Because the functional stability of the multisubunit channel may be more physiologically relevant than the stability of individual subunit proteins, we performed studies of functional ENaC channels using A6 epithelial cells, a Xenopus laevis distal nephron cell line. We recorded single-channel activity in over 400 cells with the translation blockers cycloheximide (CHX) or puromycin, as well as the intracellular protein trafficking inhibitors brefeldin A (BFA) or nocodazole. Our cell-attached, single-channel recordings allow us to quantify the channel density in the apical membrane, as well as to determine channel open probability ( Po) from control (untreated) cells and from cells at different times of drug treatment. The data suggest that the half-life of ENaC channels is ∼3.5 h following puromycin, BFA, and nocodazole treatment. Furthermore, these three drugs had no significant effect on the Po of ENaC for at least 6 h after exposure. A decrease in apical channel number and Po was observed following 2 h of CHX inhibition of protein synthesis, and the apparent channel half-life was closer to 1.5 h following CHX treatment. Treatment of cells with the translation inhibitors does not alter the expression of the protease furin, and therefore changes in protease activity cannot explain changes in ENaC Po. Confocal images show that BFA and nocodazole both disrupt most of the Golgi apparatus after 1-h exposure. In cells with the Golgi totally disrupted by overnight exposure to BFA, 20% of apical ENaC channels remained functional. This result suggests that ENaC is delivered to the apical membrane via a pathway that might bypass the Golgi vesicular trafficking pathway, or that there might be two pools of channels with markedly different half-lives in the apical membrane.

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