scholarly journals Mechanism of gating of T-type calcium channels.

1990 ◽  
Vol 96 (3) ◽  
pp. 603-630 ◽  
Author(s):  
C F Chen ◽  
P Hess
Keyword(s):  
Single Channel ◽  
Ca Channels ◽  
3T3 Fibroblasts ◽  
Channel Inactivation ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Single Burst ◽  
Opening And Closing ◽  
Rate Limiting ◽  
Kinetics Of

We have analyzed the gating kinetics of T-type Ca channels in 3T3 fibroblasts. Our results show that channel closing, inactivation, and recovery from inactivation each include a voltage-independent step which becomes rate limiting at extreme potentials. The data require a cyclic model with a minimum of two closed, one open, and two inactivated states. Such a model can produce good fits to our data even if the transitions between closed states are the only voltage-dependent steps in the activating pathway leading from closed to inactivated states. Our analysis suggests that the channel inactivation step, as well as the direct opening and closing transitions, are not intrinsically voltage sensitive. Single-channel recordings are consistent with this scheme. As expected, each channel produces a single burst per opening and then inactivates. Comparison of the kinetics of T-type Ca current in fibroblasts and neuronal cells reveals significant differences which suggest that different subtypes of T-type Ca channels are expressed differentially in a tissue specific manner.

scholarly journals Multiple effects of KPQ deletion mutation on gating of human cardiac Na+ channels expressed in mammalian cells

1998 ◽  
Vol 274 (5) ◽  
pp. H1643-H1654 ◽  
Author(s):  
Rashmi Chandra ◽  
C. Frank Starmer ◽  
Augustus O. Grant
Keyword(s):  
Mammalian Cells ◽  
Single Channel ◽  
Deletion Mutation ◽  
Recovery From Inactivation ◽  
Voltage Dependent ◽  
Rate Of Recovery ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Kinetics Of ◽  
Rate Of Activation

Several aspects of the effect of the KPQ deletion mutation on Na+ channel gating remain unresolved. We have analyzed the kinetics of the early and late currents by recording whole cell and single-channel currents in a human embryonic kidney (HEK) cell line (HEK293) expressing wild-type and KPQ deletion mutation in cardiac Na+ channels. The rate of inactivation increased three- to fivefold between −40 and −80 mV in the mutant channel. The rate of recovery from inactivation was increased twofold. Two modes of gating accounted for the late current: 1) isolated brief openings with open times that were weakly voltage dependent and the same as the initial transient and 2) bursts of opening with highly voltage-dependent prolonged open times. Latency to first opening was accelerated, suggesting an acceleration of the rate of activation. The ΔKPQ mutation has multiple effects on activation and inactivation. The aggregate effects may account for the increased susceptibility to arrhythmias.

scholarly journals Structure–Function Relations of the First and Fourth Predicted Extracellular Linkers of the Type IIa Na+/Pi Cotransporter

2004 ◽  
Vol 124 (5) ◽  
pp. 475-488 ◽  
Author(s):  
Colin Ehnes ◽  
Ian C. Forster ◽  
Katja Kohler ◽  
Andrea Bacconi ◽  
Gerti Stange ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Reaction Rates ◽  
Transport Pathway ◽  
Extracellular Medium ◽  
Voltage Range ◽  
Substituted Cysteine Accessibility ◽  
Voltage Dependent ◽  
Opposite Behavior ◽  
Rate Limiting ◽  
Kinetics Of

The putative first intracellular and third extracellular linkers are known to play important roles in defining the transport properties of the type IIa Na+-coupled phosphate cotransporter (Kohler, K., I.C. Forster, G. Stange, J. Biber, and H. Murer. 2002b. J. Gen. Physiol. 120:693–705). To investigate whether other stretches that link predicted transmembrane domains are also involved, the substituted cysteine accessibility method (SCAM) was applied to sites in the predicted first and fourth extracellular linkers (ECL-1 and ECL-4). Mutants based on the wild-type (WT) backbone, with substituted novel cysteines, were expressed in Xenopus oocytes, and their function was assayed by isotope uptake and electrophysiology. Functionally important sites were identified in both linkers by exposing cells to membrane permeant and impermeant methanethiosulfonate (MTS) reagents. The cysteine modification reaction rates for sites in ECL-1 were faster than those in ECL-4, which suggested that the latter were less accessible from the extracellular medium. Generally, a finite cotransport activity remained at the end of the modification reaction. The change in activity was due to altered voltage-dependent kinetics of the Pi-dependent current. For example, cys substitution at Gly-134 in ECL-1 resulted in rate-limiting, voltage-independent cotransport activity for V ≤ −80 mV, whereas the WT exhibited a linear voltage dependency. After cys modification, this mutant displayed a supralinear voltage dependency in the same voltage range. The opposite behavior was documented for cys substitution at Met-533 in ECL-4. Modification of cysteines at two other sites in ECL-1 (Ile-136 and Phe-137) also resulted in supralinear voltage dependencies for hyperpolarizing potentials. Taken together, these findings suggest that ECL-1 and ECL-4 may not directly form part of the transport pathway, but specific sites in these linkers can interact directly or indirectly with parts of NaPi-IIa that undergo voltage-dependent conformational changes and thereby influence the voltage dependency of cotransport.

scholarly journals Shaker potassium channel gating. I: Transitions near the open state.

1994 ◽  
Vol 103 (2) ◽  
pp. 249-278 ◽  
Author(s):  
T Hoshi ◽  
W N Zagotta ◽  
R W Aldrich
Keyword(s):  
Time Course ◽  
Single Channel ◽  
Activation Process ◽  
Time Data ◽  
Shaker Potassium Channel ◽  
Voltage Dependent ◽  
Average Current ◽  
Shaker Potassium Channels ◽  
Kinetics Of ◽  
Deactivation Process

Kinetics of single voltage-dependent Shaker potassium channels expressed in Xenopus oocytes were studied in the absence of fast N-type inactivation. Comparison of the single-channel first latency distribution and the time course of the ensemble average current showed that the activation time course and its voltage dependence are largely determined by the transitions before first opening. The open dwell time data are consistent with a single kinetically distinguishable open state. Once the channel opens, it can enter at least two closed states which are not traversed frequently during the activation process. The rate constants for the transitions among these closed states and the open state are nearly voltage-independent at depolarized voltages (> -30 mV). During the deactivation process at more negative voltages, the channel can close directly to a closed state in the activation pathway in a voltage-dependent fashion.

scholarly journals Large Peptide Permeation Through a Membrane Channel: Understanding Protamine Translocation Through CymA from Klebsiella Oxytoca

2020 ◽  
Author(s):  
Sushil Pangeni ◽  
Jigneshkumar Dahyabhai Prajapati ◽  
Jayesh Arun Bafna ◽  
Nilam Mohamed ◽  
Werner M. Nau ◽  
...  
Keyword(s):  
Lipid Bilayers ◽  
Klebsiella Oxytoca ◽  
Limiting Factor ◽  
Membrane Channel ◽  
Voltage Dependent ◽  
Dynamics Simulations ◽  
Rate Limiting ◽  
Kinetics Of ◽  
Zero Voltage ◽  
Large Peptide

Quantifying the passage of the large peptide protamine (Ptm) across CymA, a passive channel for cyclodextrin uptake, is in the focus of this study. Using a reporter-pair based fluorescence membrane assay we detected the entry of Ptm into liposomes containing CymA. The kinetics of the Ptm entry was independent of its concentration suggesting that the permeation across CymA is the rate-limiting factor. Furthermore, we reconstituted single CymA channels into planar lipid bilayers and recorded the ion current fluctuations in the presence of Ptm. To this end, we were able to resolve the voltage-dependent entry of single Ptm peptide molecules into the channel. Extrapolation to zero voltage revealed about 1-2 events per second and long dwell times, in agreement with the liposome study. Applied-field and steered molecular dynamics simulations provided an atomistic view on the permeation. It can be concluded that a concentration gradient of 1 M Ptm leads to a translocation rate of about 1 molecule per second and per channel.

scholarly journals Kinetics of the Opening and Closing of Individual Excitability-Inducing Material Channels in a Lipid Bilayer

1974 ◽  
Vol 63 (6) ◽  
pp. 707-721 ◽  
Author(s):  
Gerald Ehrenstein ◽  
Robert Blumenthal ◽  
Ramon Latorre ◽  
Harold Lecar
Keyword(s):  
Lipid Bilayers ◽  
Ionic Current ◽  
Energy Difference ◽  
Excitable Membranes ◽  
Voltage Dependent ◽  
Ion Conducting ◽  
The Many ◽  
Approach To Steady State ◽  
Opening And Closing ◽  
Kinetics Of

The kinetics of the opening and closing of individual ion-conducting channels in lipid bilayers doped with small amounts of excitability-inducing material (EIM) are determined from discrete fluctuations in ionic current. The kinetics for the approach to steady-state conductance during voltage clamp are determined for lipid bilayers containing many EIM channels. The two sets of measurements are found to be consistent, verifying that the voltage-dependent conductance of the many-channel EIM system arises from the opening and closing of individual EIM channels. The opening and closing of the channels are Poisson processes. Transition rates for these processes vary exponentially with applied potential, implying that the energy difference between the open and closed states of an EIM channel is linearly proportional to the transmembrane electric field. A model incorporating the above properties of the EIM channels predicts the observed voltage dependence of ionic conductance and conductance relaxation time, which are also characteristic of natural electrically excitable membranes.

scholarly journals Inactivation of Gating Currents of L-Type Calcium Channels

1998 ◽  
Vol 111 (6) ◽  
pp. 807-823 ◽  
Author(s):  
Roman Shirokov ◽  
Gonzalo Ferreira ◽  
Jianxun Yi ◽  
Eduardo Ríos
Keyword(s):  
Charge Movement ◽  
Ca Channels ◽  
Mobile Charge ◽  
Gating Currents ◽  
Recovery From Inactivation ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Separate Channel ◽  
Α2δ Subunit ◽  
Modal Change

In studies of gating currents of rabbit cardiac Ca channels expressed as α1C/β2a or α1C/β2a/α2δ subunit combinations in tsA201 cells, we found that long-lasting depolarization shifted the distribution of mobile charge to very negative potentials. The phenomenon has been termed charge interconversion in native skeletal muscle (Brum, G., and E. Ríos. 1987. J. Physiol. (Camb.). 387:489–517) and cardiac Ca channels (Shirokov, R., R. Levis, N. Shirokova, and E. Ríos. 1992. J. Gen. Physiol. 99:863–895). Charge 1 (voltage of half-maximal transfer, V1/2 ≃ 0 mV) gates noninactivated channels, while charge 2 (V1/2 ≃ −90 mV) is generated in inactivated channels. In α1C/β2a cells, the available charge 1 decreased upon inactivating depolarization with a time constant τ ≃ 8, while the available charge 2 decreased upon recovery from inactivation (at −200 mV) with τ ≃ 0.3 s. These processes therefore are much slower than charge movement, which takes <50 ms. This separation between the time scale of measurable charge movement and that of changes in their availability, which was even wider in the presence of α2δ, implies that charges 1 and 2 originate from separate channel modes. Because clear modal separation characterizes slow (C-type) inactivation of Na and K channels, this observation establishes the nature of voltage-dependent inactivation of L-type Ca channels as slow or C-type. The presence of the α2δ subunit did not change the V1/2 of charge 2, but sped up the reduction of charge 1 upon inactivation at 40 mV (to τ ≃ 2 s), while slowing the reduction of charge 2 upon recovery (τ ≃ 2 s). The observations were well simulated with a model that describes activation as continuous electrodiffusion (Levitt, D. 1989. Biophys. J. 55:489–498) and inactivation as discrete modal change. The effects of α2δ are reproduced assuming that the subunit lowers the free energy of the inactivated mode.

scholarly journals Kinetics of 9-aminoacridine block of single Na channels.

1984 ◽  
Vol 84 (3) ◽  
pp. 361-377 ◽  
Author(s):  
D Yamamoto ◽  
J Z Yeh
Keyword(s):  
Rate Constant ◽  
Voltage Dependence ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

scholarly journals State-Dependent Inactivation of the α1g T-Type Calcium Channel

1999 ◽  
Vol 114 (2) ◽  
pp. 185-202 ◽  
Author(s):  
Jose R. Serrano ◽  
Edward Perez-Reyes ◽  
Stephen W. Jones
Keyword(s):  
Kinetic Model ◽  
Hek 293 ◽  
Voltage Sensors ◽  
State Dependent ◽  
State Recovery ◽  
Recovery From Inactivation ◽  
293 Cells ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Kinetics Of

We have examined the kinetics of whole-cell T-current in HEK 293 cells stably expressing the α1G channel, with symmetrical Na+i and Na+o and 2 mM Ca2+o. After brief strong depolarization to activate the channels (2 ms at +60 mV; holding potential −100 mV), currents relaxed exponentially at all voltages. The time constant of the relaxation was exponentially voltage dependent from −120 to −70 mV \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{e-fold\;for}}\;31\;{\mathrm{mV}};\;{\mathrm{{\tau}}}\;=\;2.5\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}{\mathrm{{\tau}}}\;=\;12{\raisebox{1mm}{\line(1,0){6}}}17\;{\mathrm{ms\;from}}-40\;{\mathrm{to}}\;+60\;{\mathrm{mV}}\end{equation*}\end{document}. This suggests a mixture of voltage-dependent deactivation (dominating at very negative voltages) and nearly voltage-independent inactivation. Inactivation measured by test pulses following that protocol was consistent with open-state inactivation. During depolarizations lasting 100–300 ms, inactivation was strong but incomplete (∼98%). Inactivation was also produced by long, weak depolarizations \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;220\;{\mathrm{ms\;at}}\;-80\;{\mathrm{mV}};\;{\mathrm{V}}_{1/2}\;=\;-82\;{\mathrm{mV}})\end{equation*}\end{document}, which could not be explained by voltage-independent inactivation exclusively from the open state. Recovery from inactivation was exponential and fast \documentclass[10pt]{article}\usepackage{amsmath}\usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy}\usepackage{mathrsfs}\usepackage[Euler]{upgreek}\pagestyle{empty}\oddsidemargin -1.0in\begin{document}\begin{equation*}({\mathrm{{\tau}}}\;=\;85\;{\mathrm{ms\;at}}\;-100\;{\mathrm{mV}})\end{equation*}\end{document}, but weakly voltage dependent. Recovery was similar after 60-ms steps to −20 mV or 600-ms steps to −70 mV, suggesting rapid equilibration of open- and closed-state inactivation. There was little current at −100 mV during recovery from inactivation, consistent with ≤8% of the channels recovering through the open state. The results are well described by a kinetic model where inactivation is allosterically coupled to the movement of the first three voltage sensors to activate. One consequence of state-dependent inactivation is that α1G channels continue to inactivate after repolarization, primarily from the open state, which leads to cumulative inactivation during repetitive pulses.

scholarly journals Single Channel Properties of Rat Acid–sensitive Ion Channel-1α, -2a, and -3 Expressed in Xenopus Oocytes

2002 ◽  
Vol 120 (4) ◽  
pp. 553-566 ◽  
Author(s):  
Ping Zhang ◽  
Cecilia M. Canessa
Keyword(s):  
Ion Channels ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Acid Sensing Ion Channels ◽  
Voltage Dependent ◽  
Subconductance States ◽  
Kinetics Of ◽  
Channel Properties

The mammalian nervous system expresses proton-gated ion channels known as acid-sensing ion channels (ASICs). Depending on their location and specialization some neurons express more than one type of ASIC where they may form homo- or heteromeric channels. Macroscopic characteristics of the ASIC currents have been described, but little is known at the single channel level. Here, we have examined the properties of unitary currents of homomeric rat ASIC1α, ASIC2a, and ASIC3 expressed in Xenopus oocytes with the patch clamp technique. We describe and characterize properties unique to each of these channels that can be used to distinguish the various types of ASIC channels expressed in mammalian neurons. The amplitudes of the unitary currents in symmetrical Na+ are similar for the three types of channels (23–18 pS) and are not voltage dependent. However, ASIC1α exhibits three subconductance states, ASIC2a exhibits only one, and ASIC3 none. The kinetics of the three types of channels are different: ASIC1α and ASIC2a shift between modes of activity, each mode has different open probability and kinetics. In contrast, the kinetics of ASIC3 are uniform throughout the burst of activity. ASIC1α, ASIC2a, and ASIC3 are activated by external protons with apparent pH50 of 5.9, 5.0, and 5.4, respectively. Desensitization in the continual presence of protons is fast and complete in ASIC1α and ASIC3 (2.0 and 4.5 s−1, respectively) but slow and only partial in ASIC2a (0.045 s−1). The response to external Ca2+ also differs: μM concentrations of extracellular Ca2+ are necessary for proton gating of ASIC3 (EC50 = 0.28 μM), whereas ASIC1α and ASIC2a do not require Ca2+. In addition, Ca2+ inhibits ASIC1α (KD = 9.2 ± 2 mM) by several mechanisms: decrease in the amplitude of unitary currents, shortening of the burst of activity, and decrease in the number of activated channels. Contrary to previous reports, our results indicate that the Ca2+ permeability of ASIC1α is very small.

scholarly journals Voltage-dependent inactivation of T-tubular skeletal calcium channels in planar lipid bilayers.

1991 ◽  
Vol 97 (2) ◽  
pp. 393-412 ◽  
Author(s):  
R Mejía-Alvarez ◽  
M Fill ◽  
E Stefani
Keyword(s):  
Calcium Channels ◽  
Lipid Bilayers ◽  
Single Channel ◽  
Channel Activity ◽  
T Tube ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Kinetics Of ◽  
Channel Properties

Single-channel properties of dihydropyridine (DHP)-sensitive calcium channels isolated from transverse tubular (T-tube) membrane of skeletal muscle were explored. Single-channel activity was recorded in planar lipid bilayers after fusion of highly purified rabbit T-tube microsomes. Two populations of DHP-sensitive calcium channels were identified. One type of channel (noninactivating) was active (2 microM +/- Bay K 8644) at steady-state membrane potentials and has been studied in other laboratories. The second type of channel (inactivating) was transiently activated during voltage pulses and had a very low open probability (Po) at steady-state membrane potentials. Inactivating channel activity was observed in 47.3% of the experiments (n = 84 bilayers). The nonstationary kinetics of this channel was determined using a standard voltage pulse (HP = -50 mV, pulse to 0 mV). The time constant (tau) of channel activation was 23 ms. During the mV). The time constant (tau) of channel activation was 23 ms. During the pulse, channel activity decayed (inactivated) with a tau of 3.7 s. Noninactivating single-channel activity was well described by a model with two open and two closed states. Inactivating channel activity was described by the same model with the addition of an inactivated state as proposed for cardiac muscle. The single-channel properties were compared with the kinetics of DHP-sensitive inward calcium currents (ICa) measured at the cellular level. Our results support the hypothesis that voltage-dependent inactivation of single DHP-sensitive channels contributes to the decay of ICa.

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