Sequential approach to describe the time course of synaptic channel opening under constant transmitter concentration

1992 ◽  
Vol 158 (4) ◽  
pp. 517-534 ◽  
Author(s):  
E. Buchman ◽  
H. Parnas
Keyword(s):  
Time Course ◽  
Sequential Approach ◽  
Channel Opening

scholarly journals Adaptive behavior of bacterial mechanosensitive channels is coupled to membrane mechanics

2010 ◽  
Vol 135 (6) ◽  
pp. 641-652 ◽  
Author(s):  
Vladislav Belyy ◽  
Kishore Kamaraju ◽  
Bradley Akitake ◽  
Andriy Anishkin ◽  
Sergei Sukharev
Keyword(s):  
Adaptive Behavior ◽  
Time Course ◽  
Prolonged Exposure ◽  
Mechanosensitive Channel ◽  
Membrane Mechanics ◽  
Response Curves ◽  
Channel Opening ◽  
Complex Adaptive ◽  
Excised Patches ◽  
Pressure Adaptation

Mechanosensitive channel of small conductance (MscS), a tension-driven osmolyte release valve residing in the inner membrane of Escherichia coli, exhibits a complex adaptive behavior, whereas its functional counterpart, mechanosensitive channel of large conductance (MscL), was generally considered nonadaptive. In this study, we show that both channels exhibit similar adaptation in excised patches, a process that is completely separable from inactivation prominent only in MscS. When a membrane patch is held under constant pressure, adaptation of both channels is manifested as a reversible current decline. Their dose–response curves recorded with 1–10-s ramps of pressure are shifted toward higher tension relative to the curves measured with series of pulses, indicating decreased tension sensitivity. Prolonged exposure of excised patches to subthreshold tensions further shifts activation curves for both MscS and MscL toward higher tension with similar magnitude and time course. Whole spheroplast MscS recordings performed with simultaneous imaging reveal activation curves with a midpoint tension of 7.8 mN/m and the slope corresponding to ∼15-nm2 in-plane expansion. Inactivation was retained in whole spheroplast mode, but no adaptation was observed. Similarly, whole spheroplast recordings of MscL (V23T mutant) indicated no adaptation, which was present in excised patches. MscS activities tried in spheroplast-attached mode showed no adaptation when the spheroplasts were intact, but permeabilized spheroplasts showed delayed adaptation, suggesting that the presence of membrane breaks or edges causes adaptation. We interpret this in the framework of the mechanics of the bilayer couple linking adaptation of channels in excised patches to the relaxation of the inner leaflet that is not in contact with the glass pipette. Relaxation of one leaflet results in asymmetric redistribution of tension in the bilayer that is less favorable for channel opening.

Kinetic and pharmacological properties of low voltage-activated Ca2+ current in rat clonal (GH3) pituitary cells

1992 ◽  
Vol 68 (1) ◽  
pp. 213-232 ◽  
Author(s):  
J. Herrington ◽  
C. J. Lingle
Keyword(s):  
Time Course ◽  
Low Voltage ◽  
Gh3 Cells ◽  
Pituitary Cells ◽  
Anesthetic Agents ◽  
Channel Opening ◽  
Cell Recording ◽  
Voltage Dependent ◽  
Dependent Processes ◽  
Site Binding

1. Low voltage-activated (LVA) Ca2+ current in clonal (GH3) pituitary cells was studied with the use of the whole-cell recording technique. The use of internal fluoride to facilitate the rundown of high voltage-activated (HVA) Ca2+ current allowed the study of LVA current in virtual isolation. 2. In 10 mM [Ca2+]o, detectable LVA current begins to appear at about -50 mV, with half-maximal activation occurring at -33 mV. The time course of activation was best described by a Hodgkin-Huxley expression with n = 3, suggesting that at least three closed states must be traversed before channel opening. 3. Deactivation was found to vary exponentially with membrane potential between -60 and -160 mV, indicating that channel closing is rate-limited by a single, voltage-dependent transition. 4. Onset and removal of inactivation between -40 and -130 mV were best described by the sum of two exponentials. Between -80 and -130 mV, both components of removal of inactivation showed little voltage dependence, with time constants of approximately 200-300 ms and 1-2 s. At membrane potentials above -40 mV, a single component of inactivation onset was detected. This component was voltage independent between -20 and +20 mV (tau = 22 ms). Thus inactivation of LVA current is best described by multiple, voltage-in-dependent processes. 5. Significant inactivation of LVA current occurred at -65 mV without detectable macroscopic current. This suggests that inactivation is not strictly coupled to channel opening. 6. Peak LVA current increased with increasing [Ca2+]o, with saturation approximately 50 mM. The Ca(2+)-dependence of peak LVA current was reasonably well described by a single-site binding isotherm with half-maximal LVA current at approximately 7 mM. 7. LVA current in GH3 cells was largely resistant to blockade by Ni2+. The relative potency of inorganic cations in blocking GH3 LVA current was (concentrations which produced 50% block): La3+ (2.4 microM) greater than Cd2+ (188 microM) greater than Ni2+ (777 microM). 8. Several organic agents, including putative LVA blockers, HVA current blockers and various anesthetic agents, were tested for their ability to block LVA current. The concentrations that produced 50% block are as follows: nifedipine (approximately 50 microM), D600 (51 microM), diltiazem (131 microM), octanol (244 microM), pentobarbital (985 microM), methoxyflurane (1.41 mM), and amiloride (1.55 mM). Phenytoin and ethosuximide produced 36 and 10% block at 100 microM and 2.5 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Desensitization to ANG II in guinea pig ileum depends on membrane repolarization: role of maxi-K+ channel

1999 ◽  
Vol 277 (4) ◽  
pp. C739-C745 ◽  
Author(s):  
Bagnólia A. Silva ◽  
Viviane L. A. Nouailhetas ◽  
Jeannine Aboulafia
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
K Channel ◽  
Ang Ii ◽  
Guinea Pig Ileum ◽  
Tonic Component ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Sustained Activation

Desensitization of ANG II tonic contractile response of the guinea pig ileum is related to membrane repolarization determined by Ca2+-activated K+(maxi-K+) channel opening. ANG II-stimulated depolarized myocytes presented sustained activation of maxi-K+ channels, characterized by reduction from 415 to 12 ms of the closed time constant. ANG II desensitization was prevented by 100 nM iberiotoxin, being reversible within 30 min. Depolarization by KCl, higher than 4 mM, impaired desensitization, suggesting that the membrane potential must attain a threshold to counteract the repolarization induced by maxi-K+ channel opening. Once this value is attained, there is no time dependency because the desensitization process was shut off by addition of KCl along the time course of the tonic response. In contrast, the sustained ACh tonic component was not altered by these maneuvers. We conclude that desensitization of the ANG II tonic component is foremost due to the opening of maxi-K+ channels, leading to membrane repolarization, thus closing the voltage-dependent Ca2+ channels responsible for the Ca2+ influx that sustains the tonic component in this muscle.

scholarly journals Voltage Clamp Fluorimetry Reveals a Novel Outer Pore Instability in a Mammalian Voltage-gated Potassium Channel

2008 ◽  
Vol 132 (2) ◽  
pp. 209-222 ◽  
Author(s):  
Moninder Vaid ◽  
Thomas W. Claydon ◽  
Saman Rezazadeh ◽  
David Fedida
Keyword(s):  
Voltage Clamp ◽  
Time Course ◽  
Ionic Current ◽  
Voltage Sensor ◽  
Selectivity Filter ◽  
Similar Time ◽  
Voltage Gated ◽  
Kv Channels ◽  
Channel Opening ◽  
Outer Pore

Voltage-gated potassium (Kv) channel gating involves complex structural rearrangements that regulate the ability of channels to conduct K+ ions. Fluorescence-based approaches provide a powerful technique to directly report structural dynamics underlying these gating processes in Shaker Kv channels. Here, we apply voltage clamp fluorimetry, for the first time, to study voltage sensor motions in mammalian Kv1.5 channels. Despite the homology between Kv1.5 and the Shaker channel, attaching TMRM or PyMPO fluorescent probes to substituted cysteine residues in the S3–S4 linker of Kv1.5 (M394C-V401C) revealed unique and unusual fluorescence signals. Whereas the fluorescence during voltage sensor movement in Shaker channels was monoexponential and occurred with a similar time course to ionic current activation, the fluorescence report of Kv1.5 voltage sensor motions was transient with a prominent rapidly dequenching component that, with TMRM at A397C (equivalent to Shaker A359C), represented 36 ± 3% of the total signal and occurred with a τ of 3.4 ± 0.6 ms at +60 mV (n = 4). Using a number of approaches, including 4-AP drug block and the ILT triple mutation, which dissociate channel opening from voltage sensor movement, we demonstrate that the unique dequenching component of fluorescence is associated with channel opening. By regulating the outer pore structure using raised (99 mM) external K+ to stabilize the conducting configuration of the selectivity filter, or the mutations W472F (equivalent to Shaker W434F) and H463G to stabilize the nonconducting (P-type inactivated) configuration of the selectivity filter, we show that the dequenching of fluorescence reflects rapid structural events at the selectivity filter gate rather than the intracellular pore gate.

Dual activation of the cardiac Ca2+ channel alpha 1C-subunit and its modulation by the beta-subunit

1995 ◽  
Vol 268 (3) ◽  
pp. C732-C740 ◽  
Author(s):  
A. Neely ◽  
R. Olcese ◽  
P. Baldelli ◽  
X. Wei ◽  
L. Birnbaumer ◽  
...  
Keyword(s):  
Time Course ◽  
Slow Component ◽  
Ionic Currents ◽  
Ionic Channel ◽  
Relative Increase ◽  
Beta Subunit ◽  
Single Channels ◽  
Gating Currents ◽  
Channel Opening ◽  
Dual Activation

Ca2+ channels are heteromultimeric proteins in which the alpha 1-subunit forms the voltage-dependent Ca(2+)-selective ionic channel. We reported recently that coexpression of the beta-subunit with the cardiac alpha 1-subunit (alpha 1C) facilitates channel opening without affecting either the amplitude or the time course of the gating currents (13). Here we present evidence for the existence of two modes of channel opening. Xenopus oocytes expressing the alpha 1C-subunit alone display two modes of activation as indicated by the double-exponential time course of macroscopic ionic currents and the two open-time distributions of single channels. Coexpression of the beta-subunit potentiates Ca2+ currents by a relative increase of the fast-activating component, an acceleration of the slow component, and a larger proportion of long openings. We propose that multiple modes of gating are encoded in the alpha 1-subunit and that the beta-subunit increases Ca2+ channel opening by favoring a willing mode of gating in which the final transitions leading to channel opening are facilitated. In addition, we show that the carboxy terminus of alpha 1C also modulates the channel-gating behavior.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

scholarly journals Slow Inactivation in Shaker K Channels Is Delayed by Intracellular Tetraethylammonium

2008 ◽  
Vol 132 (6) ◽  
pp. 633-650 ◽  
Author(s):  
Vivian González-Pérez ◽  
Alan Neely ◽  
Christian Tapia ◽  
Giovanni González-Gutiérrez ◽  
Gustavo Contreras ◽  
...  
Keyword(s):  
Time Course ◽  
Inactivation Kinetics ◽  
Type I ◽  
Slow Inactivation ◽  
External Application ◽  
K Channels ◽  
Gating Charge ◽  
Channel Opening ◽  
Activation Gate ◽  
Free Membrane

After removal of the fast N-type inactivation gate, voltage-sensitive Shaker (Shaker IR) K channels are still able to inactivate, albeit slowly, upon sustained depolarization. The classical mechanism proposed for the slow inactivation observed in cell-free membrane patches—the so called C inactivation—is a constriction of the external mouth of the channel pore that prevents K+ ion conduction. This constriction is antagonized by the external application of the pore blocker tetraethylammonium (TEA). In contrast to C inactivation, here we show that, when recorded in whole Xenopus oocytes, slow inactivation kinetics in Shaker IR K channels is poorly dependent on external TEA but severely delayed by internal TEA. Based on the antagonism with internally or externally added TEA, we used a two-pulse protocol to show that half of the channels inactivate by way of a gate sensitive to internal TEA. Such gate had a recovery time course in the tens of milliseconds range when the interpulse voltage was −90 mV, whereas C-inactivated channels took several seconds to recover. Internal TEA also reduced gating charge conversion associated to slow inactivation, suggesting that the closing of the internal TEA-sensitive inactivation gate could be associated with a significant amount of charge exchange of this type. We interpreted our data assuming that binding of internal TEA antagonized with U-type inactivation (Klemic, K.G., G.E. Kirsch, and S.W. Jones. 2001. Biophys. J. 81:814–826). Our results are consistent with a direct steric interference of internal TEA with an internally located slow inactivation gate as a “foot in the door” mechanism, implying a significant functional overlap between the gate of the internal TEA-sensitive slow inactivation and the primary activation gate. But, because U-type inactivation is reduced by channel opening, trapping the channel in the open conformation by TEA would also yield to an allosteric delay of slow inactivation. These results provide a framework to explain why constitutively C-inactivated channels exhibit gating charge conversion, and why mutations at the internal exit of the pore, such as those associated to episodic ataxia type I in hKv1.1, cause severe changes in inactivation kinetics.

scholarly journals The time course of miniature endplate currents and its modification by receptor blockade and ethanol.

1984 ◽  
Vol 83 (3) ◽  
pp. 435-468 ◽  
Author(s):  
T M Linder ◽  
P Pennefather ◽  
D M Quastel
Keyword(s):  
Decay Rate ◽  
Time Constant ◽  
Time Course ◽  
System Analysis ◽  
Decay Phase ◽  
Receptor Interaction ◽  
Voltage Sensitivity ◽  
Receptor Blockade ◽  
Channel Opening ◽  
Alpha Bungarotoxin

Miniature endplate currents (MEPCs) recorded from mouse diaphragms with a point voltage clamp, without inhibition of acetylcholinesterase (AChE) and in the absence of any drug, showed in their decay phase consistent deviations from an exponential time course, consisting of (a) "curvature," a progressive increase of decay rate during most of the decay phase, followed by (b) "late" tails. Both phenomena persisted when MEPCs (and channel lifetime) were prolonged by ethanol. Curvature was increased by muscle fiber depolarization and decreased by hyperpolarization. Receptor blockade by (+)-tubocurarine, alpha-bungarotoxin, hexamethonium, or myasthenic IgG accelerated the decay of the main part of MEPCs and eliminated curvature; the time constant of MEPCs became close to the channel time constant. We conclude that curvature arises from repeated action of ACh with cooperativity in ACh-receptor interaction; the voltage sensitivity of curvature follows from the voltage sensitivity of channel closing. Ethanol, in addition to its effect to prolong channel lifetime, enhances the tendency of ACh to act more than once to open channels before being lost to the system. Analysis of the rising phase of the MEPC, in terms of driving functions, also indicated that ethanol promotes channel opening by ACh; this action can account for a substantial increase of MEPC height by ethanol when MEPCs are made small by receptor blockade. Driving functions were also voltage sensitive, in a manner indicating acceleration of channel opening, but reduction of channel conductance, with hyperpolarization. Poisoning or inhibition of AChE prolonged MEPCs without altering the duration of ionic channels. Since ethanol caused further prolongation of MEPCs after poisoning of AChE, with little change in MEPC height, we conclude that the extension of mean channel lifetime by ethanol is accompanied by a similar extension of ACh binding to receptors. After poisoning of AChE, MEPCs became very variable in time course and the decay rate (tau-1) was correlated with MEPC height with a slope of log tau vs. log height of 0.77 for MEPCs of greater than 60% mean size. This slope is larger than expected from cooperativity in ACh-receptor interaction. Correlation of tau and height of MEPCs also exists when AChE is intact; the slope of log tau vs. log height was 0.12 with or without prolongation of MEPCs by ethanol.

Brevity of the Ca2+ Microdomain and Active Zone Geometry Prevent Ca2+-Sensor Saturation for Neurotransmitter Release

2005 ◽  
Vol 94 (3) ◽  
pp. 1912-1919 ◽  
Author(s):  
Vahid Shahrezaei ◽  
Kerry R. Delaney
Keyword(s):  
Binding Sites ◽  
Neurotransmitter Release ◽  
Time Course ◽  
Receptor Occupancy ◽  
Channel Opening ◽  
Sensor Saturation ◽  
Highly Sensitive ◽  
Equilibrium Dissociation ◽  
Sensor Molecules ◽  
Kinetics Of

The brief time course of the calcium (Ca2+) channel opening combined with the molecular-level colocalization of Ca2+ channels and synaptic vesicles in presynaptic terminals predict sub-millisecond calcium concentration ([Ca2+]) transients of ≥100 μM in the immediate vicinity of the vesicle. This [Ca2+] is much higher than some of the recent estimates for the equilibrium dissociation constant of the Ca2+ sensor(s) that control neurotransmitter release, suggesting release should be close to saturation, yet it is well known that release is highly sensitive to changes in Ca2+ influx. We show that due to the brevity of the Ca2+ influx the binding kinetics of the Ca2+ sensor rather than its equilibrium affinity determine receptor occupancy. For physiologically relevant Ca2+ currents and forward Ca2+ binding rates, the effective affinity of the Ca2+ sensor can be several-fold lower than the equilibrium affinity. Using simple models, we show redundant copies of the binding sites increase effective affinity of the Ca2+ sensor for release. Our results predict that different levels of expression of Ca2+ binding sites could account for apparent differences in Ca2+ sensor affinities between synapses. Using Monte Carlo simulations of Ca2+ dynamics with nanometer resolution, we demonstrate that these kinetic constraints combined with vesicles acting as diffusion barriers can prevent saturation of the Ca2+-sensor(s) for neurotransmitter release. We further show the random positioning of the Ca2+-sensor molecules around the vesicle can result in the emergence of two distinct populations of the vesicles with low and high release probability. These considerations allow experimental evidence for the Ca2+ channel-vesicle colocalization to be reconciled with a high equilibrium affinity for the Ca2+ sensor of the release machinery.

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