scholarly journals Zn2(+)-induced subconductance events in cardiac Na+ channels prolonged by batrachotoxin. Current-voltage behavior and single-channel kinetics.

1991 ◽  
Vol 97 (1) ◽  
pp. 117-142 ◽  
Author(s):  
L Schild ◽  
A Ravindran ◽  
E Moczydlowski
Keyword(s):  
Dwell Time ◽  
Single Channel ◽  
Kinetic Scheme ◽  
Dissociation Rate ◽  
Canine Heart ◽  
Na Channels ◽  
Channel Kinetics ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Site Blocking

The mechanism of voltage-dependent substate production by external Zn2+ in batrachotoxin-modified Na+ channels from canine heart was investigated by analysis of the current-voltage behavior and single-channel kinetics of substate events. At the single-channel level the addition of external Zn2+ results in an increasing frequency of substate events with a mean duration of approximately 15-25 ms for the substate dwell time observed in the range of -70 to +70 mV. Under conditions of symmetrical 0.2 M NaCl, the open state of cardiac Na+ channels displays ohmic current-voltage behavior in the range of -90 to +100 mV, with a slope conductance of 21 pS. In contrast, the Zn2(+)-induced substate exhibits significant outward rectification with a slope conductance of 3.1 pS in the range of -100 to -50 mV and 5.1 pS in the range of +50 to +100 mV. Analysis of dwell-time histograms of substate events as a function of Zn2+ concentration and voltage led to the consideration of two types of models that may explain this behavior. Using a simple one-site blocking model, the apparent association rate for Zn2+ binding is more strongly voltage dependent (decreasing e-fold per +60 mV) than the Zn2+ dissociation rate (increasing e-fold per +420 mV). However, this simple blocking model cannot account for the dependence of the apparent dissociation rate on Zn2+ concentration. To explain this result, a four-state kinetic scheme involving a Zn2(+)-induced conformational change from a high conductance conformation to a substate conformation is proposed. This model, similar to one introduced by Pietrobon et al. (1989. J. Gen. Physiol. 94:1-24) for H(+)-induced substate behavior in L-type Ca2+ channels, is able to simulate the kinetic and equilibrium behavior of the primary Zn2(+)-induced substate process in heart Na+ channels. This model implies that binding of Zn2+ greatly enhances conversion of the open, ohmic channel to a low conductance conformation with an asymmetric energy profile for Na+ permeation.

scholarly journals Block of the cyclic GMP-gated channel of vertebrate rod and cone photoreceptors by l-cis-diltiazem.

1992 ◽  
Vol 100 (5) ◽  
pp. 783-801 ◽  
Author(s):  
L W Haynes
Keyword(s):  
Binding Site ◽  
Single Molecule ◽  
Single Channel ◽  
Inward Rectification ◽  
Pipette Solution ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Extracellular Side ◽  
Current Voltage Relation ◽  
Cytoplasmic Side

Inside-out patches were excised from catfish rod or cone outer segments. Single channel and macroscopic currents were recorded from GMP-gated channels activated by 1 mM cGMP in low divalent buffered saline. Currents were blocked by the application of micromolar concentrations of l-cis-diltiazem to the cytoplasmic side of the patch. The concentration dependence of block indicated that a single molecule was sufficient to block a channel and that all channels were susceptible to block. The dissociation constant for the rod channel was an order of magnitude smaller than for the cone channel, but the voltage dependence of block was nearly identical. The macroscopic current-voltage relation in the presence of blocker was inwardly rectifying and superficially resembled voltage-dependent block by an impermeant blocker occluding the ion-conducting pore of the channel. Block by diltiazem acting from the extracellular side of the channel was investigated by including 5 microM diltiazem in the recording pipette solution. The macroscopic current-voltage relation again showed inward rectification, inconsistent with the idea that diltiazem acts by occluding the pore at the external side. The kinetics of block by diltiazem applied to the intra- and extracellular side were measured in cone patches containing only a single channel. The unbinding rates were similar in both cases, suggesting a single binding site. Differences in the binding rate were consistent with greater accessibility to the binding site from the cytoplasmic side. Block from the cytoplasmic side was independent of pH, suggesting that the state of ionization of diltiazem was not related to its ability to block the channel in a voltage-dependent fashion. These observations are inconsistent with a pore-occluding blocker, but could be explained if the hydrophobic portion of diltiazem partitioned into the hydrophobic core of the channel protein, perhaps altering the gating of the channel.

scholarly journals Intracellular calcium strongly potentiates agonist-activated TRPC5 channels

2009 ◽  
Vol 133 (5) ◽  
pp. 525-546 ◽  
Author(s):  
Nathaniel T. Blair ◽  
J. Stefan Kaczmarek ◽  
David E. Clapham
Keyword(s):  
Single Channel ◽  
Reversal Potential ◽  
Fold Increase ◽  
Receptor Activation ◽  
Brain Regions ◽  
Repetitive Firing ◽  
Dependent Manner ◽  
Open Probability ◽  
Current Voltage ◽  
Voltage Dependent

TRPC5 is a calcium (Ca2+)-permeable nonselective cation channel expressed in several brain regions, including the hippocampus, cerebellum, and amygdala. Although TRPC5 is activated by receptors coupled to phospholipase C, the precise signaling pathway and modulatory signals remain poorly defined. We find that during continuous agonist activation, heterologously expressed TRPC5 currents are potentiated in a voltage-dependent manner (∼5-fold at positive potentials and ∼25-fold at negative potentials). The reversal potential, doubly rectifying current–voltage relation, and permeability to large cations such as N-methyl-d-glucamine remain unchanged during this potentiation. The TRPC5 current potentiation depends on extracellular Ca2+: replacement by Ba2+ or Mg2+ abolishes it, whereas the addition of 10 mM Ca2+ accelerates it. The site of action for Ca2+ is intracellular, as simultaneous fura-2 imaging and patch clamp recordings indicate that potentiation is triggered at ∼1 µM [Ca2+]. This potentiation is prevented when intracellular Ca2+ is tightly buffered, but it is promoted when recording with internal solutions containing elevated [Ca2+]. In cell-attached and excised inside-out single-channel recordings, increases in internal [Ca2+] led to an ∼10–20-fold increase in channel open probability, whereas single-channel conductance was unchanged. Ca2+-dependent potentiation should result in TRPC5 channel activation preferentially during periods of repetitive firing or coincident neurotransmitter receptor activation.

scholarly journals Voltage-dependent blockade of muscle Na+ channels by guanidinium toxins.

1984 ◽  
Vol 84 (5) ◽  
pp. 687-704 ◽  
Author(s):  
E Moczydlowski ◽  
S Hall ◽  
S S Garber ◽  
G S Strichartz ◽  
C Miller
Keyword(s):  
Voltage Dependence ◽  
Single Channel ◽  
Membrane Vesicles ◽  
Hydroxyl Groups ◽  
Binding Affinities ◽  
Na Channels ◽  
Plasma Membrane Vesicles ◽  
Toxin Binding ◽  
Voltage Dependent ◽  
Toxin Receptor

Na+ channels from rat muscle plasma membrane vesicles were inserted into neutral planar phospholipid bilayers and were activated by batrachotoxin. Single channel blocking events induced by the addition of various guanidinium toxins were analyzed to derive the rates of channel-toxin association and dissociation. Blocking by tetrodotoxin, saxitoxin, and six natural saxitoxin derivatives containing sulfate or hydroxyl groups were studied. Although the binding affinities vary over 2,000-fold, all of the toxins exhibit identical voltage dependence of the blocking reactions, regardless of the toxin's net charge. The results suggest that the voltage dependence of toxin binding is due to a voltage-dependent conformational equilibrium of the toxin receptor, rather than to direct entry of the charged toxin molecule into the applied transmembrane electric field.

scholarly journals Voltage-dependent gating of veratridine-modified Na channels.

1986 ◽  
Vol 87 (1) ◽  
pp. 25-46 ◽  
Author(s):  
M D Leibowitz ◽  
J B Sutro ◽  
B Hille
Keyword(s):  
Time Course ◽  
Drug Binding ◽  
Na Channels ◽  
Current Voltage ◽  
Instantaneous Current ◽  
Activation Gating ◽  
Voltage Dependent ◽  
Ca Ions ◽  
Rate Of Recovery ◽  
Gating Process

Na channels of frog muscle fibers treated with 100 microM veratridine became transiently modified after a train of repetitive depolarizations. They open and close reversibly with a gating process whose midpoint lies 93 mV more negative than the midpoint of normal activation gating and whose time course shows no appreciable delay in the opening or closing kinetics but still requires more than two kinetic states. Like normal activation, the voltage dependence of the modified gating can be shifted by changing the bathing Ca2+ concentration. The instantaneous current-voltage relation of veratridine-modified channels is curved at potentials negative to -90 mV, as if external Ca ions produced a voltage-dependent block but also permeated. Modified channels probably carry less current than normal ones. When the concentration of veratridine is varied between 5 and 100 microM, the initial rate of modification during a pulse train is directly proportional to the concentration, while the rate of recovery from modification after the train is unaffected. These are the properties expected if drug binding and modification of channels can be equated. Hyperpolarizations that close modified channels slow unbinding. Allethrin and DDT also modify channels. They bind and unbind far faster than veratridine does, and their binding requires open channels.

Cadmium-induced blockade of the cardiac fast Na channels in calf Purkinje fibres

1985 ◽  
Vol 223 (1233) ◽  
pp. 475-484 ◽  
Keyword(s):  
Single Channel ◽  
Na Channels ◽  
Na Current ◽  
Purkinje Fibres ◽  
Peak Current ◽  
Inward Current ◽  
Current Voltage ◽  
Channel Blocking ◽  
Current Reduction ◽  
Current Kinetic

The fast transient inward current elicited by depolarizations above about –60 mV in calf Purkinje fibres was found to be depressed by Cd in concentrations less than 1 mm. The Cd-sensitive current, which strongly depended on external Na, was recorded in the presence of 2 mm MnCl 2 and was blocked by TTX , indicating that a contamination from slow Ca-dependent currents could be discounted. The current reduction caused by Cd was also observed in nominally Ca-free solutions. The Cd-induced depression of the fast Na current was not accompanied by changes in the current kinetic parameters, as revealed by comparing inactivation curves and peak current voltage relations at different Cd concentrations, and could be attributed to a voltage-independent channel blocking action. Half-blockade occurred at 0.182±0.06 mm ( n = 4). Plots of peak current amplitude as a function of the Cd concentration showed that the cooperation of two Cd ions was required to block a single channel.

scholarly journals Volume-Dependent Atp-Conductive Large-Conductance Anion Channel as a Pathway for Swelling-Induced Atp Release

2001 ◽  
Vol 118 (3) ◽  
pp. 251-266 ◽  
Author(s):  
Ravshan Z. Sabirov ◽  
Amal K. Dutta ◽  
Yasunobu Okada
Keyword(s):  
Single Channel ◽  
Atp Release ◽  
Anion Channel ◽  
Time Dependent ◽  
Whole Cell ◽  
Dependent Inactivation ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Cell Current ◽  
Whole Cell Current

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl− channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around ±25 mV. The whole-cell current was selective for anions and sensitive to Gd3+. In on-cell patches, single-channel events appeared with a lag period of ∼15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at −20 to 0 mV. The channel in inside-out patches had the unitary conductance of ∼400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC50 of 12.3 mM and an electric distance (δ) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC50 of 12.9 mM and δ of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with PATP/PCl of 0.09. The single-channel activity was sensitive to Gd3+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

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 Na+ Occupancy and Mg2+ Block of the N-Methyl-d-Aspartate Receptor Channel

2001 ◽  
Vol 117 (3) ◽  
pp. 275-286 ◽  
Author(s):  
Yongling Zhu ◽  
Anthony Auerbach
Keyword(s):  
Membrane Potential ◽  
Dissociation Constant ◽  
Rate Constant ◽  
Single Channel ◽  
Dissociation Rate ◽  
Equilibrium Dissociation Constant ◽  
Voltage Dependent ◽  
Lock In Effect ◽  
Equilibrium Dissociation ◽  
Competing Ions

The effect of extracellular and intracellular Na+ on the single-channel kinetics of Mg2+ block was studied in recombinant NR1-NR2B NMDA receptor channels. Na+ prevents Mg2+ access to its blocking site by occupying two sites in the external portion of the permeation pathway. The occupancy of these sites by intracellular, but not extracellular, Na+ is voltage-dependent. In the absence of competing ions, Mg2+ binds rapidly (>108 M−1s−1, with no membrane potential) to a site that is located 0.60 through the electric field from the extracellular surface. Occupancy of one of the external sites by Na+ may be sufficient to prevent Mg2+ dissociation from the channel back to the extracellular compartment. With no membrane potential; and in the absence of competing ions, the Mg2+ dissociation rate constant is >10 times greater than the Mg2+ permeation rate constant, and the Mg2+ equilibrium dissociation constant is ∼12 μM. Physiological concentrations of extracellular Na+ reduce the Mg2+ association rate constant ∼40-fold but, because of the “lock-in” effect, reduce the Mg2+ equilibrium dissociation constant only ∼18-fold.

Gap junction channel gating at bass retinal electrical synapses

1996 ◽  
Vol 13 (6) ◽  
pp. 1049-1057 ◽  
Author(s):  
Chengbiao Lu ◽  
Douglas G. McMahon
Keyword(s):  
Time Constant ◽  
Time Course ◽  
Single Channel ◽  
Horizontal Cells ◽  
Electrical Synapses ◽  
Dependent Inactivation ◽  
Current Voltage ◽  
Voltage Dependent ◽  
Current Voltage Relationship ◽  
Voltage Relationship

AbstractTo further characterize the properties of retinal horizontal cell electrical synapses, we have studied the gating characteristics of gap junctions between cone-driven horizontal cells from the hybrid striped bass retina using double whole-cell voltage-clamp techniques. In a total of 105 cell pairs, the macroscopic conductance ranged from 0.4–100 nS with most cell pairs exhibiting junctional conductances between 10 and 30 nS. The junctional current-voltage relationship showed that peak or instantaneous currents (Iinst) were linear within the Vj range of ±100 mV and that steady-state junctional currents (Iss) exhibited rectification with increasing voltage beginning around ±30–40 mV Vj. The normalized junctional current-voltage relationship was well fit by a two-state Boltzmann distribution, with an effective gating charge of 1.9 charges/channel, a half-maximal voltage of approximately ±55 mV, and a normalized residual conductance of 0.28. The decay of junctional current followed a single exponential time course with the time constant decreasing with increasing Vj. Recovery of junctional current from voltage-dependent inactivation takes about 1 s following a pulse of 80 mV, and is about five times slower than the inactivation time course at the same Vj. Single-channel analysis showed that the unitary conductance of junctional channels is 50–70 pS. The overall open probability decreased in a voltage-dependent manner. Both the mean channel open time and the frequency of channel opening decreased, while the channel closure time increased. The ratio of closed time/total recording time significantly increased as Vj increased. Increased Vj reduced the number of events at all levels and shifted the unitary conductance to a lower level. Kinetic analysis of channel open duration showed that the distribution of channel open times was best fit by two exponentials and increased Vj significantly reduced the slower time constant. These results indicate that bass retina horizontal cells exhibit voltage-dependent inactivation of macroscopic junctional current. The inactivation occurs at the single-channel level mainly by increasing the rate of closure of voltage-sensitive channels.

scholarly journals Coupling and cooperativity in voltage activation of a limited-state BK channel gating in saturating Ca2+

2010 ◽  
Vol 135 (5) ◽  
pp. 461-480 ◽  
Author(s):  
Christopher Shelley ◽  
Xiaowei Niu ◽  
Yanyan Geng ◽  
Karl L. Magleby
Keyword(s):  
Single Channel ◽  
Coupling Factor ◽  
Bk Channels ◽  
Bk Channel ◽  
Voltage Sensor ◽  
Channel Kinetics ◽  
Voltage Sensors ◽  
Gating Mechanisms ◽  
Voltage Dependent ◽  
Single Channel Currents

Voltage-dependent gating mechanisms of large conductance Ca2+ and voltage-activated (BK) channels were investigated using two-dimensional maximum likelihood analysis of single-channel open and closed intervals. To obtain sufficient data at negative as well as positive voltages, single-channel currents were recorded at saturating Ca2+ from BK channels mutated to remove the RCK1 Ca2+ and Mg2+ sensors. The saturating Ca2+ acting on the Ca2+ bowl sensors of the resulting BKB channels increased channel activity while driving the gating into a reduced number of states, simplifying the model. Five highly constrained idealized gating mechanisms based on extensions of the Monod-Wyman-Changeux model for allosteric proteins were examined. A 10-state model without coupling between the voltage sensors and the opening/closing transitions partially described the voltage dependence of Po but not the single-channel kinetics. With allowed coupling, the model gave improved descriptions of Po and approximated the single-channel kinetics; each activated voltage sensor increased the opening rate approximately an additional 23-fold while having little effect on the closing rate. Allowing cooperativity among voltage sensors further improved the description of the data: each activated voltage sensor increased the activation rate of the remaining voltage sensors approximately fourfold, with little effect on the deactivation rate. The coupling factor was decreased in models with cooperativity from ∼23 to ∼18. Whether the apparent cooperativity among voltage sensors arises from imposing highly idealized models or from actual cooperativity will require additional studies to resolve. For both cooperative and noncooperative models, allowing transitions to five additional brief (flicker) closed states further improved the description of the data. These observations show that the voltage-dependent single-channel kinetics of BKB channels can be approximated by highly idealized allosteric models in which voltage sensor movement increases Po mainly through an increase in channel opening rates, with limited effects on closing rates.

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