scholarly journals Removal of sodium channel inactivation in squid giant axons by n-bromoacetamide.

1978 ◽  
Vol 71 (3) ◽  
pp. 227-247 ◽  
Author(s):  
G S Oxford ◽  
C H Wu ◽  
T Narahashi
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Single Channel ◽  
Sodium Current ◽  
Complete Removal ◽  
Specific Protein ◽  
Peptide Bonds ◽  
Voltage Dependent ◽  
Inactivation Gating ◽  
Sodium Inactivation

The group-specific protein reagents, N-bromacetamide (NBA) and N-bromosuccinimide (NBS), modify sodium channel gating when perfused inside squid axons. The normal fast inactivation of sodium channels is irreversibly destroyed by 1 mM NBA or NBS near neutral pH. NBA apparently exhibits an all-or-none destruction of the inactivation process at the single channel level in a manner similar to internal perfusion of Pronase. Despite the complete removal of inactivation by NBA, the voltage-dependent activation of sodium channels remains unaltered as determined by (a) sodium current turn-on kinetics, (b) sodium tail current kinetics, (c) voltage dependence of steady-state activation, and (d) sensitivity of sodium channels to external calcium concentration. NBA and NBS, which can cleave peptide bonds only at tryptophan, tyrosine, or histidine residues and can oxidize sulfur-containing amino acids, were directly compared with regard to effects on sodium inactivation to several other reagents exhibiting overlapping protein reactivity spectra. N-acetylimidazole, a tyrosine-specific reagent, was the only other compound examined capable of partially mimicking NBA. Our results are consistent with recent models of sodium inactivation and support the involvement of a tyrosine residue in the inactivation gating structure of the sodium channel.

scholarly journals Purified and unpurified sodium channels from eel electroplax in planar lipid bilayers.

1987 ◽  
Vol 90 (3) ◽  
pp. 375-395 ◽  
Author(s):  
E Recio-Pinto ◽  
D S Duch ◽  
S R Levinson ◽  
B W Urban
Keyword(s):  
Sodium Channel ◽  
Lipid Bilayers ◽  
Sodium Channels ◽  
Single Channel ◽  
Planar Bilayers ◽  
Electric Eel ◽  
Voltage Dependent ◽  
Single Channel Activity ◽  
Single Channel Currents ◽  
Channel Currents

Highly purified sodium channel protein from the electric eel, Electrophorus electricus, was reconstituted into liposomes and incorporated into planar bilayers made from neutral phospholipids dissolved in decane. The purest sodium channel preparations consisted of only the large, 260-kD tetrodotoxin (TTX)-binding polypeptide. For all preparations, batrachotoxin (BTX) induced long-lived single-channel currents (25 pS at 500 mM NaCl) that showed voltage-dependent activation and were blocked by TTX. This block was also voltage dependent, with negative potentials increasing block. The permeability ratios were 4.7 for Na+:K+ and 1.6 for Na+:Li+. The midpoint for steady state activation occurred around -70 mV and did not shift significantly when the NaCl concentration was increased from 50 to 1,000 mM. Veratridine-induced single-channel currents were about half the size of those activated by BTX. Unpurified, nonsolubilized sodium channels from E. electricus membrane fragments were also incorporated into planar bilayers. There were no detectable differences in the characteristics of unpurified and purified sodium channels, although membrane stability was considerably higher when purified material was used. Thus, in the eel, the large, 260-kD polypeptide alone is sufficient to demonstrate single-channel activity like that observed for mammalian sodium channel preparations in which smaller subunits have been found.

scholarly journals Interaction of n-alkylguanidines with the sodium channels of squid axon membrane.

1980 ◽  
Vol 76 (3) ◽  
pp. 315-335 ◽  
Author(s):  
G E Kirsch ◽  
J Z Yeh ◽  
J M Farley ◽  
T Narahashi
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Sodium Current ◽  
Time Dependent ◽  
Squid Axon ◽  
Axon Membrane ◽  
Inactivation Mechanism ◽  
Hydrophobic Binding ◽  
Sodium Inactivation ◽  
Guanidino Group

The effects of n-alkylguanidine derivatives on sodium channel conductance were measured in voltage clamped, internally perfused squid giant axons. After destruction of the sodium inactivation mechanism by internal pronase treatment, internal application of n-amylguanidine (0.5 mM) or n-octylguanidine (0.03 mM) caused a time-dependent block of sodium channels. No time-dependent block was observed with shorter chain derivatives. No change in the rising phase of sodium current was seen and the block of steady-state sodium current was independent of the membrane potential. In axons with intact sodium inactivation, an apparent facilitation of inactivation was observed after application of either n-amylguanidine or n-octylguanidine. These results can be explained by a model in which alkylguanidines enter and occlude open sodium channels from inside the membrane with voltage-independent rate constants. Alkylguanidine block bears a close resemblance to natural sodium inactivation. This might be explained by the fact that alkylguanidines are related to arginine, which has a guanidino group and is thought to be an essential amino acid in the molecular mechanism of sodium inactivation. A strong correlation between alkyl chain length and blocking potency was found, suggesting that a hydrophobic binding site exists near the inner mouth of the sodium channel.

Effects of New World scorpion toxins on single-channel and whole cell cardiac sodium currents

1988 ◽  
Vol 254 (3) ◽  
pp. H443-H451 ◽  
Author(s):  
A. Yatani ◽  
G. E. Kirsch ◽  
L. D. Possani ◽  
A. M. Brown
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
New World ◽  
Single Channel ◽  
Sodium Current ◽  
Sodium Currents ◽  
Scorpion Toxins ◽  
Whole Cell ◽  
Cardiac Sodium Channels ◽  
Single Channel Currents

Purified toxins from a North American scorpion, Centruroides noxius (Cn II-10), and a South American scorpion, Tityus serrulatus (Ts-gamma), were tested on cardiac sodium channels using patch-clamp methods to record whole cell and single-channel currents. The two toxins produced similar effects on sodium currents; potassium and calcium currents were not affected. Macroscopic sodium current amplitudes, measured at test potentials greater than -20 mV where the opening probability was high, decreased in a concentration-dependent manner with a half maximum inhibitory concentration of 6 X 10(-8) M. Block was unchanged by repetitive depolarizing pulses. In the presence of scorpion toxin, the currents were rapidly blocked by tetrodotoxin (3 X 10(-5) M). Both toxins shifted the voltage dependence of sodium channel inactivation to more negative potentials. At test potentials between -50 and -70 mV, where the sodium channel opening probability is normally low, both toxins produced an increase in sodium current and slowed the rates of activation and inactivation. At intermediate potentials between -50 and -20 mV the currents in the presence of toxins crossed over the control currents. At a test potential of -20 mV, the toxins decreased single-channel activity and increased the latency to first opening. At a test potential of -60 mV, the toxins significantly prolonged channel open time. The unitary current amplitudes were unchanged at either potential. We conclude that New World scorpion toxins produce apparently complex effects on whole cell currents primarily by retarding activation gating of cardiac sodium channels.

Late Sodium Channel Openings Underlying Epileptiform Activity Are Preferentially Diminished by the Anticonvulsant Phenytoin

1997 ◽  
Vol 77 (6) ◽  
pp. 3021-3034 ◽  
Author(s):  
Michael M. Segal ◽  
Andrea F. Douglas
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Hippocampal Neurons ◽  
Sodium Current ◽  
Epileptiform Activity ◽  
Normal Function ◽  
Anticonvulsant Effect ◽  
Persistent Sodium Current ◽  
Pharmacological Mechanism ◽  
Voltage Dependent

Segal, Michael M. and Andrea F. Douglas. Late sodium channel openings underlying epileptiform activity are preferentially diminished by the anticonvulsant phenytoin. J. Neurophysiol. 77: 3021–3034, 1997. Late openings of sodium channels were observed in outside-out patch recordings from hippocampal neurons in culture. In previous studies of such neurons, a persistent sodium current appeared to underlie the ictal epileptiform activity. All the channel currents were blocked by tetrodotoxin. In addition to the transient openings of sodium channels making up the peak sodium current, there were two types of late channel openings: brief late and burst openings. These late channel openings occurred throughout voltage pulses that lasted 750 ms, producing a persistent sodium current. At −30 mV, this current was 0.4% of the peak current. The late channel openings occurred throughout the physiological range of trans-membrane voltages. The anticonvulsant phenytoin reduced the late channel openings more than the peak currents. The effect on the persistent current was greatest at more depolarized voltages, whereas the effect on peak currents was not substantially voltage dependent. In the presence of 60 μM phenytoin, peak sodium currents at −30 mV were 40–41% of control, as calculated using different methods of analysis. Late currents were 22–24% of control. Phenytoin primarily decreased the number of channel openings, with less effect on the duration of channel openings and no effect on open channel current. This set of findings is consistent with models in which phenytoin binds to the inactivated state of the channel. The preferential effect of phenytoin on the persistent sodium current suggests that an important pharmacological mechanism for a sodium channel anticonvulsant is to reduce late openings of sodium channels, rather than reducing all sodium channel openings. We hypothesize that pharmacological interventions that are most selective in reducing late openings of sodium channels, while leaving early channel openings relatively intact, will be those that produce an anticonvulsant effect while interfering minimally with normal function.

scholarly journals Kinetic analysis of pancuronium interaction with sodium channels in squid axon membranes.

1977 ◽  
Vol 69 (3) ◽  
pp. 293-323 ◽  
Author(s):  
J Z Yeh ◽  
T Narahashi
Keyword(s):  
Membrane Potential ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Rate Constant ◽  
Time Course ◽  
Voltage Dependence ◽  
Sodium Current ◽  
Sodium Concentration ◽  
Internal Concentration ◽  
Sodium Inactivation

The interaction of pancuronium with sodium channels was investigated in squid axons. Sodium current turns on normally but turns off more quickly than the control with pancuronium 0.1-1mM present internally; The sodium tail current associated with repolarization exhibits an initial hook and then decays more slowly than the control. Pancuronium induces inactivation after the sodium inactivation has been removed by internal perfusion of pronase. Such pancuronium-induced sodium inactivation follows a single exponential time course, suggesting first order kinetics which represents the interaction of the pancuronium molecule with the open sodium channel. The rate constant of association k with the binding site is independent of the membrane potential ranging from 0 to 80 mV, but increases with increasing internal concentration of pancuronium. However, the rate constant of dissociation l is independent of internal concentration of pancuronium but decreases with increasing the membrane potential. The voltage dependence of l is not affected by changine external sodium concentration, suggesting a current-independent conductance block, The steady-state block depends on the membrane potential, being more pronounced with increasing depolarization, and is accounted for in terms of the voltage dependence of l. A kinetic model, based on the experimental observations and the assumption on binding kinetics of pancuronium with the open sodium channel, successfully simulates many features of sodium current in the presence of pancuronium.

scholarly journals A molecular link between activation and inactivation of sodium channels.

1995 ◽  
Vol 106 (4) ◽  
pp. 641-658 ◽  
Author(s):  
M E O'Leary ◽  
L Q Chen ◽  
R G Kallen ◽  
R Horn
Keyword(s):  
Sodium Channels ◽  
Voltage Dependence ◽  
Single Channel ◽  
Critical Role ◽  
Channel Measurements ◽  
Wild Type ◽  
Open Probability ◽  
Voltage Dependent ◽  
Steady State Inactivation ◽  
Normal Coupling

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.

Identification of sodium channel subtypes induced in cultured retinal pigment epithelium cells

1995 ◽  
Vol 12 (5) ◽  
pp. 1001-1005 ◽  
Author(s):  
Heather Dawes ◽  
Gail Mandel ◽  
Gary Matthews
Keyword(s):  
Retinal Pigment Epithelium ◽  
Sodium Channel ◽  
Sodium Current ◽  
Pigment Epithelium ◽  
Retinal Pigment ◽  
Rpe Cells ◽  
Voltage Dependent ◽  
Dissociated Cell Culture ◽  
Epithelium Cells ◽  
Neuronal Type

AbstractRecent electrophysiological experiments have shown that retinal pigment epithelium (RPE) cells begin to produce neuronal-type voltage-dependent sodium currents when placed in dissociated cell culture. In this study, the sodium channel types induced in cultured rat RPE cells were identified. Sodium channel mRNAs encoding two distinct alpha subunits were detected in the cultured RPE cells, brain type II/IIA, and a novel rat mRNA which we have termed RET1. These two sodium channel types may correspond to the TTX-sensitive and TTX-insensitive components of sodium current reported previously in cultured rat RPE cells.

scholarly journals Analysis of sodium current fluctuations in the cut-open squid giant axon.

1984 ◽  
Vol 83 (2) ◽  
pp. 133-142 ◽  
Author(s):  
I Llano ◽  
F Bezanilla
Keyword(s):  
Sodium Channel ◽  
Sodium Channels ◽  
Sodium Current ◽  
Giant Axon ◽  
Squid Giant Axon ◽  
Statistical Techniques ◽  
Current Fluctuations ◽  
Small Populations ◽  
Squid Axon ◽  
Single Sodium Channel

Patch pipettes were used to record the current arising from small populations of sodium channels in voltage-clamped cut-open squid axons. The current fluctuations associated with the time-variant sodium conductance were analyzed with nonstationary statistical techniques in order to obtain an estimate for the conductance of a single sodium channel. The results presented support the notion that the open sodium channel in the squid axon has only one value of conductance, 3.5 pS.

scholarly journals Conotoxins as Sensors of Local pH and Electrostatic Potential in the Outer Vestibule of the Sodium Channel

2003 ◽  
Vol 122 (1) ◽  
pp. 63-79 ◽  
Author(s):  
Kwokyin Hui ◽  
Deane McIntyre ◽  
Robert J. French
Keyword(s):  
Lipid Bilayers ◽  
Sodium Channels ◽  
Electrostatic Potential ◽  
Single Channel ◽  
Local Environment ◽  
Bound State ◽  
Channel Changes ◽  
Voltage Dependent ◽  
Charged Ring ◽  
Derivatives Of

We examined the block of voltage-dependent rat skeletal muscle sodium channels by derivatives of μ-conotoxin GIIIA (μCTX) having either histidine, glutamate, or alanine residues substituted for arginine-13. Toxin binding and dissociation were observed as current fluctuations from single, batrachotoxin-treated sodium channels in planar lipid bilayers. R13X derivatives of μCTX only partially block the single-channel current, enabling us to directly monitor properties of both μCTX-bound and -unbound states under different conditions. The fractional residual current through the bound channel changes with pH according to a single-site titration curve for toxin derivatives R13E and R13H, reflecting the effect of changing the charge on residue 13, in the bound state. Experiments with R13A provided a control reflecting the effects of titration of all residues on toxin and channel other than toxin residue 13. The apparent pKs for the titration of residual conductance are shifted 2–3 pH units positive from the nominal pK values for histidine and glutamate, respectively, and from the values for these specific residues, determined in the toxin molecule in free solution by NMR measurements. Toxin affinity also changes dramatically as a function of pH, almost entirely due to changes in the association rate constant, kon. Interpreted electrostatically, our results suggest that, even in the presence of the bound cationic toxin, the channel vestibule strongly favors cation entry with an equivalent local electrostatic potential more negative than −100 mV at the level of the “outer charged ring” formed by channel residues E403, E758, D1241, and D1532. Association rates are apparently limited at a transition state where the pK of toxin residue 13 is closer to the solution value than in the bound state. The action of these unique peptides can thus be used to sense the local environment in the ligand-–receptor complex during individual molecular transitions and defined conformational states.

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