scholarly journals Single Ion Occupancy and Steady-state Gating of Na Channels in Squid Giant Axon

2002 ◽  
Vol 119 (3) ◽  
pp. 235-250 ◽  
Author(s):  
Robert F. Rakowski ◽  
David C. Gadsby ◽  
Paul De Weer
Keyword(s):  
Steady State ◽  
Flux Ratio ◽  
Giant Axon ◽  
Boltzmann Distribution ◽  
Na Channels ◽  
Open Probability ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Effective Valence ◽  
Equilibrium Dissociation

The properties of the small fraction of tetrodotoxin (TTX)-sensitive Na channels that remain open in the steady state were studied in internally dialyzed voltage clamped squid giant axons. The observed Ussing flux ratio exponent (n′) of 0.97 ± 0.03 (calculated from simultaneous measurements of TTX-sensitive current and 22Na efflux) and nonindependent behavior of Na current at high internal [Na] are explained by a one-site (“1s”) permeation model characterized by a single effective binding site within the channel pore in equilibrium with internal Na ions (apparent equilibrium dissociation constant KNai(0) = 0.61 ± 0.08 M). Steady-state open probability of the TTX-sensitive channels can be modeled by the product pap∞, where pa represents voltage-dependent activation described by a Boltzmann distribution with midpoint Va = −7 mV and effective valence za = 3.2 (Vandenberg, C.A., and F. Bezanilla. 1991. Biophys. J. 60:1499–1510) coupled to voltage-independent inactivation by an equilibrium constant (Bezanilla, F., and C.M. Armstrong. 1977. J. Gen. Physiol. 70:549–566) Keq = 770. The factor p∞ represents voltage-dependent inactivation with empirical midpoint V∞= −83 ± 5 mV and effective valence z∞ = 0.55 ± 0.03. The composite pap∞1s model describes the steady-state voltage dependence of the persistent TTX-sensitive current well.

Altered frequency-dependent inactivation and steady-state inactivation of polyglutamine-expanded α1A in SCA6

2007 ◽  
Vol 292 (3) ◽  
pp. C1078-C1086 ◽  
Author(s):  
Haiyan Chen ◽  
Erika S. Piedras-Rentería
Keyword(s):  
Steady State ◽  
Late Onset ◽  
Spinocerebellar Ataxia Type ◽  
Gain Of Function ◽  
Calcium Dependent ◽  
Spinocerebellar Ataxia Type 6 ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Activity Dependent

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease of the cerebellum and inferior olives characterized by a late-onset cerebellar ataxia and selective loss of Purkinje neurons ( 15 , 16 ). SCA6 arises from an expansion of the polyglutamine tract located in exon 47 of the α1A (P/Q-type calcium channel) gene from a nonpathogenic size of 4 to 18 glutamines (CAG4–18) to CAG19–33 in SCA6. The molecular basis of SCA6 is poorly understood. To date, the biophysical properties studied in heterologous systems support both a gain and a loss of channel function in SCA6. We studied the behavior of the human α1A isoform, previously found to elicit a gain of function in disease ( 41 ), focusing on properties in which the COOH terminus of the channel is critical for function: we analyzed the current properties in the presence of β4- and β2a-subunits (both known to interact with the α1A COOH terminus), current kinetics of activation and inactivation, calcium-dependent inactivation and facilitation, voltage-dependent inactivation, frequency dependence, and steady-state activation and inactivation properties. We found that SCA6 channels have decreased activity-dependent inactivation and a depolarizing shift (+6 mV) in steady-state inactivation properties consistent with a gain of function.

scholarly journals Biphasic voltage-dependent inactivation of human NaV1.3, 1.6 and 1.7 Na+channels expressed in rodent insulin-secreting cells

2018 ◽  
Vol 596 (9) ◽  
pp. 1601-1626 ◽  
Author(s):  
Mahdieh Godazgar ◽  
Quan Zhang ◽  
Margarita V. Chibalina ◽  
Patrik Rorsman
Keyword(s):  
Na Channels ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Insulin Secreting Cells

scholarly journals Gating of maxi K+ channels studied by Ca2+ concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder).

1992 ◽  
Vol 99 (6) ◽  
pp. 841-862 ◽  
Author(s):  
F Markwardt ◽  
G Isenberg
Keyword(s):  
Steady State ◽  
Time Course ◽  
Boltzmann Distribution ◽  
Hill Coefficient ◽  
K Channel ◽  
Open Probability ◽  
Apparent Dissociation Constant ◽  
K Channels ◽  
The Mean ◽  
Inside Out

Currents through maxi K+ channels were recorded in inside-out macro-patches. Using a liquid filament switch (Franke, C., H. Hatt, and J. Dudel. 1987. Neurosci, Lett. 77:199-204) the Ca2+ concentration at the tip of the patch electrode ([Ca2+]i) was changed in less than 1 ms. Elevation of [Ca2+]i from less than 10 nM to 3, 6, 20, 50, 320, or 1,000 microM activated several maxi K+ channels in the patch, whereas return to less than 10 nM deactivated them. The time course of Ca(2+)-dependent activation and deactivation was evaluated from the mean of 10-50 sweeps. The mean currents started a approximately 10-ms delay that was attributed to diffusion of Ca2+ from the tip to the K+ channel protein. The activation and deactivation time courses were fitted with the third power of exponential terms. The rate of activation increased with higher [Ca2+]i and with more positive potentials. The rate of deactivation was independent of preceding [Ca2+]i and was reduced at more positive potentials. The rate of deactivation was measured at five temperatures between 16 and 37 degrees C; fitting the results with the Arrhenius equation yielded an energy barrier of 16 kcal/mol for the Ca2+ dissociation at 0 mV. After 200 ms, the time-dependent processes were in a steady state, i.e., there was no sign of inactivation. In the steady state (200 ms), the dependence of channel openness, N.P(o), on [Ca2+]i yielded a Hill coefficient of approximately 3. The apparent dissociation constant, KD, decreased from 13 microM at -50 mV to 0.5 microM at +70 mV. The dependence of N.P(o) on voltage followed a Boltzmann distribution with a maximal P(o) of 0.8 and a slope factor of approximately 39 mV. The results were summarized by a model describing Ca2+- and voltage-dependent activation and deactivation, as well as steady-state open probability by the binding of Ca2+ to three equal and independent sites within the electrical field of the membrane at an electrical distance of 0.31 from the cytoplasmic side.

scholarly journals Temperature Dependence of Fast and Slow Gating Relaxations of ClC-0 Chloride Channels

1997 ◽  
Vol 109 (1) ◽  
pp. 105-116 ◽  
Author(s):  
Michael Pusch ◽  
Uwe Ludewig ◽  
Thomas J. Jentsch
Keyword(s):  
Steady State ◽  
Temperature Dependence ◽  
Chloride Channels ◽  
Single Channel ◽  
Electric Organ ◽  
Boltzmann Distribution ◽  
Temperature Sensitive ◽  
Strong Temperature Dependence ◽  
Open Probability ◽  
Large Gene

The chloride channel from the Torpedo electric organ, ClC-0, is the best studied member of a large gene-family (Jentsch, T.J. 1996. Curr. Opin. Neurobiol. 6:303–310.). We investigate the temperature dependence of both the voltage- and chloride-dependent fast gate and of the slow gate of the “double-barreled” ClC-0 expressed in Xenopus oocytes. Kinetics of the fast gate exhibit only a moderate temperature dependence with a Q10 of 2.2. Steady-state popen of the fast gate is relatively independent of temperature. The slow gate, in contrast, is highly temperature sensitive. Deactivation kinetics at positive voltages are associated with a Q10 of ∼40. Steady-state open probability of the slow gate (popenslow(V)) can be described by a Boltzmann distribution with an apparent gating valence of ≈2 and a variable “offset” at positive voltages. We note a positive correlation of this offset (i.e., the fraction of channels that are not closed by the slow gate) with the amount of expression. This offset is also highly temperature sensitive, being drastically decreased at high temperatures. Paradoxically, the maximum degree of activation of the slow gate also decreases at higher temperatures. The strong temperature dependence of the slow gate was also observed at the single channel level in inside-out patches. The results imply that within a Markovian-type description at least two open and two closed states are needed to describe slow gating. The strong temperature dependence of the slow gate explains the phenotype of several ClC-0 point-mutants described recently by Ludewig et al. (Ludewig, U., T.J. Jentsch, and M. Pusch. 1996. J. Physiol. (Lond.). In press). The large Q10 of slow gating kinetics points to a complex rearrangement. This, together with the correlation of the fraction of noninactivating channels with the amount of expression and the fact that the slow gate closes both protochannels simultaneously suggests that the slow gate is coupled to subunit interaction of the multimeric ClC-0 channel.

Cytoskeleton modulates gating of voltage-dependent sodium channel in heart

1995 ◽  
Vol 269 (1) ◽  
pp. H203-H214 ◽  
Author(s):  
A. I. Undrovinas ◽  
G. S. Shander ◽  
J. C. Makielski
Keyword(s):  
Membrane Proteins ◽  
Actin Polymerization ◽  
Integral Membrane Proteins ◽  
Na Channel ◽  
Na Channels ◽  
Open Probability ◽  
Whole Cell ◽  
Voltage Dependent ◽  
D 20 ◽  
Inside Out

To investigate the role of the cytoskeleton in cardiac Na+ channel gating, the action of cytochalasin D (Cyto-D), an agent that interferes with actin polymerization, was studied by whole cell voltage clamp and cell-attached and inside-out patches from rat and rabbit ventricular cardiac myocytes. Cyto-D (20-40 microM) reduced whole cell peak Na+ current by 20% within 12 min and slowed current decay without affecting steady-state voltage-dependent availability or recovery from inactivation. Brief treatments (< 10-15 min) of cell-attached patches by Cyto-D (20 microM) in the bath induced short bursts of Na+ channel openings and prolonged decays of ensemble-averaged currents. Bursting of the Na+ channel was more pronounced when the cell suspension was pretreated with Cyto-D (20 microM) for 1 h before seal formation. Application of Cyto-D on the cytoplasmic side of inside-out patches resulted in more dramatic gating changes. Peak open probability was reduced by > 50% within 20 min, and long bursts of openings occurred. Washout of Cyto-D did not restore ensemble-averaged current amplitude, but burst duration decreased toward control values. Cyto-D also induced an additional slower component to open and closed times. These results suggest that Cyto-D, through effects on cytoskeleton, induced cardiac Na+ channels to enter a mode characterized by a lower peak open probability but a greater persistent activity as if the inactivation rate was slowed. The cytoskeleton, in addition to localizing integral membrane proteins, apparently also plays a role in regulating specific detailed functions of integral membrane proteins such as the gating of Na+ channels.

Voltage-dependent inactivation of the potassium current of embryonic chick hepatocytes

1991 ◽  
Vol 69 (6) ◽  
pp. 739-745 ◽  
Author(s):  
Ceredwyn E. Hill ◽  
Alvin Shrier
Keyword(s):  
Steady State ◽  
Potassium Current ◽  
Time Course ◽  
Resting Potential ◽  
Embryonic Chick ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Open Development ◽  
Steady State Inactivation ◽  
Time Courses

The whole-cell patch electrode voltage clamp technique was used to study the inactivation properties of the delayed rectifying potassium current of single cultured embryonic chick hepatocytes at 20 °C. The potassium current activates maximally within 250–500 ms of membrane depolarization, after which it decays with a monoexponential time course. Both steady-state activation and inactivation are voltage dependent. Steady-state inactivation declines from 100% at −5 mV to 0 near −70 mV, with half inactivation at −41 mV. At the resting potential (EM) of these cells (−21.5 ± 6.0 mV, n = 36) 6–18% of the IK channels are not inactivated and less than 5% are open. Development and removal of inactivation follow single exponential time courses. The inactivation time constant attains a maximum of around 30 s at −35 mV and is sharply voltage dependent at the EM of these cells. Measurement of EM under current clamp shows random oscillations of 5–10 mV amplitude. We suggest that the voltage- and time-dependent properties of IK, in tandem with a time- and voltage-independent, nonselective current also seen here, would provide the mechanism for a fluctuating EM.Key words: hepatocyte, embryonic, potassium current.

scholarly journals Single channel studies of the phosphorylation of K+ channels in the squid giant axon. I. Steady-state conditions.

1991 ◽  
Vol 98 (1) ◽  
pp. 1-17 ◽  
Author(s):  
E Perozo ◽  
C A Vandenberg ◽  
D S Jong ◽  
F Bezanilla
Keyword(s):  
Steady State ◽  
Single Channel ◽  
Slow Component ◽  
Giant Axon ◽  
Delayed Rectifier ◽  
Open Probability ◽  
Delayed Rectifier Current ◽  
K Current ◽  
The Difference ◽  
Single Channel Currents

Phosphorylation of the delayed rectifier channel of squid potentiates the macroscopic K+ current and slows its activation kinetics. We have studied this phenomenon at the single channel level using the cut-open axon technique under steady-state conditions. In 10 mM external K+/310 mM internal K+ there are predominantly two types of channels present, a 20-pS and a 40-pS channel. In steady state at depolarized potentials, the 40-pS channel was most active, whereas the 20-pS channel tended to disappear due to a slow inactivation process. Two methods were developed to shift the population of channels toward a dephosphorylated state. One method consisted of predialyzing a whole axon with solutions containing no ATP, while recording the currents under axial-wire voltage clamp. A piece of axon was then removed and cut open, and single channel currents were recorded from the cut-open axon. A second method was based on the difference in diffusion coefficients for ATP and proteins such as the endogenous phosphatase. The axon was cut open in a solution that did not contain Ca2+ or Cl- in order to maintain the axoplasm structurally intact and permit endogenous phosphatase to act on the membrane while ATP diffused away, before removing the axoplasm and forming a membrane patch. When dephosphorylating conditions were used, the steady-state open probability of the 40-pS channel at 42 mV was very low (less than 0.0002), and the channel openings appeared as a series of infrequent, short-duration events. The channel activity was increased up to 150-fold by photoreleasing caged ATP inside the patch pipette in the presence of the catalytic subunit of protein kinase A. The sharp increase in open probability could be accounted for by a decrease of the slow component of the closed time distribution from 23 s to 170 ms with little change in the distribution of open times (1-2 ms) and no change in the single channel current amplitude. In voltage-jump experiments the contribution of the 40-pS channel to the delayed rectifier current was often small due to the large values of the latency to the first opening.

scholarly journals Calcium currents in the A7r5 smooth muscle-derived cell line. Increase in current and selective removal of voltage-dependent inactivation by intracellular trypsin.

1991 ◽  
Vol 98 (6) ◽  
pp. 1127-1140 ◽  
Author(s):  
C A Obejero-Paz ◽  
S W Jones ◽  
A Scarpa
Keyword(s):  
Smooth Muscle ◽  
Cell Line ◽  
Single Channel ◽  
Calcium Currents ◽  
Open Probability ◽  
Calcium Channel Inactivation ◽  
Voltage Curve ◽  
Dependent Inactivation ◽  
Voltage Dependent ◽  
Current Voltage Curve

We studied the effects of trypsin on L-type calcium current in the A7r5 smooth muscle cell line. Intracellular dialysis with trypsin increased the whole-cell current up to fivefold. The effect was concentration dependent, and was prevented by soybean trypsin inhibitor. Ensemble analysis indicated an increase in the number of functional channels, and possibly a smaller increase in the open probability, with no change in the single channel current. The shape of the current-voltage curve was unaffected. Trypsin also nearly eliminated inactivation of currents carried by Ba2+, but had little or no effect on the rapid inactivation process in Ca2+, This indicates that trypsin removes voltage-dependent but not Ca(2+)-dependent inactivation, suggesting the existence of distinct protein domains for these two mechanisms of calcium channel inactivation.

scholarly journals Transfer of twelve charges is needed to open skeletal muscle Na+ channels.

1995 ◽  
Vol 106 (6) ◽  
pp. 1053-1068 ◽  
Author(s):  
B Hirschberg ◽  
A Rovner ◽  
M Lieberman ◽  
J Patlak
Keyword(s):  
Skeletal Muscle ◽  
Membrane Potential ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Fast Inactivation ◽  
Open Probability ◽  
Single Channel Analysis ◽  
Voltage Dependent ◽  
Function Models

Voltage-dependent Na+ channels are thought to sense membrane potential with fixed charges located within the membrane's electrical field. Measurement of open probability (Po) as a function of membrane potential gives a quantitative indication of the number of such charges that move through the field in opening the channel. We have used single-channel recording to measure skeletal muscle Na+ channel open probability at its most negative extreme, where channels may open as seldom as once per minute. To prevent fast inactivation from masking the voltage dependence of Po, we have generated a clone of the rat skeletal muscle Na+ channel that is lacking in fast inactivation (IFM1303QQQ). Using this mutant channel expressed in Xenopus oocytes, and the extra resolution afforded by single-channel analysis, we have extended the resolution of the hyperpolarized tail of the Po curve by four orders of magnitude. We show that previous measurements, which indicated a minimum of six effective gating charges, may have been made in a range of Po values that had not yet arrived at its limiting slope. In our preparation, a minimum of 12 charges must function in the activation gating of the channel. Our results will require reevaluation of kinetic models based on six charges, and they have major implications for the interpretation of S4 mutagenesis studies and structure/function models of the Na+ channel.

Meperidine and Lidocaine Block of Recombinant Voltage-Dependent Na+Channels 

1999 ◽  
Vol 91 (5) ◽  
pp. 1481-1481 ◽  
Author(s):  
Larry E. Wagner ◽  
Michael Eaton ◽  
Salas S. Sabnis ◽  
Kevin J. Gingrich
Keyword(s):  
Steady State ◽  
Local Anesthesia ◽  
Local Anesthetic ◽  
Voltage Dependence ◽  
Tonic Inhibition ◽  
Na Channel ◽  
Mutant Form ◽  
Na Channels ◽  
Voltage Dependent ◽  
Steady State Inactivation

Background The opioid meperidine induces spinal anesthesia and blocks nerve action potentials, suggesting it is a local anesthetic. However, whether it produces effective clinical local anesthesia in peripheral nerves remains unclear. Classification as a local anesthetic requires clinical local anesthesia but also blockade of voltage-dependent Na+ channels with characteristic features (tonic and phasic blockade and a negative shift in the voltage-dependence of steady-state inactivation) involving an intrapore receptor. The authors tested for these molecular pharmacologic features to explore whether meperidine is a local anesthetic. Methods The authors studied rat skeletal muscle mu1 (RSkM1) voltage-dependent Na+ channels or a mutant form heterologously coexpressed with rat brain Na+ channel accessory beta1, subunit in Xenopus oocytes. Polymerase chain reaction was used for mutagenesis, and mutations were confirmed by sequencing. Na+ currents were measured using a two-microelectrode voltage clamp. Meperidine and the commonly used local anesthetic lidocaine were applied to oocytes in saline solution at room temperature. Results Meperidine and lidocaine produced tonic current inhibition with comparable concentration dependence. Meperidine caused phasic current inhibition in which the concentration-response relationship was shifted to fivefold greater concentration relative to lidocaine. Meperidine and lidocaine negatively shifted the voltage dependence of steady-state inactivation. Mutation of a putative local anesthetic receptor reduced phasic inhibition by meperidine and lidocaine and tonic inhibition by lidocaine, but not meperidine tonic inhibition. Conclusions Meperidine blocks Na+ channels with molecular pharmacologic features of a local anesthetic. The findings support classification of meperidine as a local anesthetic but with less overall potency than lidocaine.

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