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1.
Statistical analysis of single sodium channels. Effects of N-bromoacetamide 总被引:23,自引:3,他引:20
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Currents were obtained from single sodium channels in outside-out excised patches of membrane from the cell line GH3. The currents were examined in control patches and in patches treated with N- bromoacetamide ( NBA ) to remove inactivation. The single-channel current-voltage relationship was linear over the range -60 to + 10 mV, and was unaffected by NBA . The slope conductance at 9.3 degrees C was 12 pS, and the Q10 for single channel currents was about 1.35. The currents in both control and NBA -treated patches showed evidence of a slow process similar to desensitization in acetylcholine-receptor channels. This process was especially apparent at rapid rates of stimulation (5 Hz), where openings occurred in clusters of records. The clustering of records with and without openings was analyzed by runs analysis, which showed a statistically significant trend toward nonrandom ordering in the responses of channels to voltage pulses. NBA made this nonrandom pattern more apparent. The probability that an individual channel was "hibernating" during an activating depolarization was estimated by a maximum likelihood method. The lifetime of the open state was also estimated by a maximum likelihood method, and was examined as a function of voltage. In control patches the open time was mildly voltage-dependent, showing a maximum at about -50 mV. In NBA -treated patches the open time was greater than in the control case and increased monotonically with depolarization; it asymptotically approached that of the control patches at hyperpolarized potentials. By comparing channel open times in control and NBA -treated patches, we determined beta A and beta I, the rate constants for closing activation gates and fast inactivation gates. Beta I was an exponential function of voltage, increasing e-fold for 34 mV. beta A had the opposite voltage dependence. The probability of an open channel closing its fast inactivation gate, rather than its activation gate, increased linearly with depolarization from -60 to -10 mV. These results indicate that inactivation is inherently voltage dependent. 相似文献
2.
Single Na channel currents were compared in ventricular myocytes and cortical neurons of neonatal rats using the gigaseal patch-clamp method to determine whether tissue-specific differences in gating can be detected at the single-channel level. Single-channel currents were recorded in cell-attached and excised membrane patches at test potentials of -70 to -20 mV and at 9-11 degrees C. In both cell-attached and excised patches brain Na channel mean open time progressively increased from less than 1 ms at -70 mV to approximately 2 ms at -20 mV. Near threshold, single openings with dispersed latencies were observed. By contrast, in cell-attached patches, heart Na channel mean open time peaked near -50 mV, was three times brain Na channel mean open time, and declined continuously to approximately 2 ms at -20 mV. Near threshold, openings occurred frequently usually as brief bursts lasting several milliseconds and rarely as prolonged bursts lasting tens of milliseconds. Unlike what occurs in brain tissue where excision did not change gating, in excised heart patches both the frequency of prolonged bursting and the mean open time of single units increased markedly. Brain and cardiac Na channels can therefore be distinguished on the basis of their mean open times and bursting characteristics. 相似文献
3.
Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current. 相似文献
4.
5.
William A. Catterall 《Journal of bioenergetics and biomembranes》1996,28(3):219-230
Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium during electrical signals in cell membranes. Their principal subunits are members of a gene family and can function as voltage-gated ion channels by themselves. They are expressed in association with one or more auxiliary subunits which increase functional expression and modify the functional properties of the principal subunits. Structural elements which are required for voltage-dependent activation, selective ion conductance, and inactivation have been identified, and their mechanisms of action are being explored through mutagenesis, expression in heterologous cells, and functional analysis. These experiments reveal that these two channels are built on a common structural theme with variations appropriate for functional specialization of each channel type. 相似文献
6.
Currents through single cardiac sodium channels have been measured in inside-out patches from guinea pig ventricular cells. To abolish the fast inactivation, Na channels were modified by DPI 201–106. In symmetrical Na solutions, a diminution of outward sodium currents can be observed that depends on the intracellular magnesium concentration and the membrane potential. Inward currents were not altered by the concentrations of magnesium used (between 0 and 22.5 mmol/1). In Mg free solutions a linear current-voltage relation can also be measured in the range of outward Na currents. At +60 mV (symmetrical Na solutions, single channel conductance 24 pS) a half maximal block of cardiac Na channels by intracellular magnesium was found at 2.1 mmol/l. From the analysis of single channel current-voltage relationships the concentration and voltage-dependent block by intracellular magnesium of cardiac sodium channels could be described as binding of Mg at one site with a K
d
value of 5.1 mmol/1 at 0 mV. The site is located at an electrical distance of 0.18 from the inside.
Offprint requests to: B. Nilius 相似文献
7.
Voltage-dependent calcium block of normal and tetramethrin-modified single sodium channels 总被引:26,自引:9,他引:17
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The mechanisms by which external Ca ions block sodium channels were studied by a gigaohm seal patch clamp method using membranes excised from N1E-115 neuroblastoma cells. Tetramethrin was used to prolong the open time of single channels so that the current-voltage relationship could be readily determined over a wide range of membrane potentials. Comparable experiments were performed in the absence of tetramethrin. Increasing external Ca ions from 0.18 to 9.0 mM reduced the single channel conductance without causing flickering. From the dose-response relation the dissociation constant for Ca block at 0 mV was estimated to be 32.4 +/- 1.05 mM. The block was intensified by hyperpolarization. The voltage dependence indicates that Ca ions bind to sodium channels at a site located 37 +/- 2% of the electrical distance from the outside. The current increased with increasing external Na concentrations but showed a saturation; the concentration for half-maximal saturation was estimated to be 185 mM at -50 mV and 204 mM at 0 mV. A model consisting of a one-ion pore with four barriers and three wells can account for the observations that deviate from the independence principle, namely, the saturation of current, block by Ca ions, and rectification in current-voltage relationship. The results suggest that the Ca-induced decrease of the macroscopic sodium current results from a reduced single sodium channel conductance. 相似文献
8.
The effects of pH of the external medium on amplitude of currents through single sodium channels at the membrane of cultured neuroblastoma cells were investigated in mice belonging to strain C 1300, clone N18A-1. Currents through single sodium channels in isolated membrane segments (outside-out configuration) were registered with normal (7.2) and reduced (5.4) pH levels in the external medium. Reducing the pH to 5.4 was found to decrease current amplitude reversibly by about twofold (–10 to –30 mV for test potentials). Findings would confirm that the depression of macroscopic sodium currents produced by reducing the pH of the extracellular solution is due to a decline in ionic flow through single open sodium channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 21, No. 1, pp. 101–105, January–February, 1989. 相似文献
9.
Aconitine-induced modification of single sodium channels in neuroblastoma cell membrane 总被引:1,自引:0,他引:1
Aconitine-modified sodium channels in the neuroblastoma cell membrane were investigated with patch-clamp technique in outside-out configuration. When aconitine (0.1 mmol/l) was present in the pipette solution two types of modified single sodium channels were observed. The first type showed openings with normal amplitude (slope conductance 15.5 pS) and bursting behaviour. The second type of modified channel openings was characterized with low amplitude (slope conductance 2.8 pS) and longer open time as comparing to unmodified channels. The low-amplitude channels were shown to have altered ion selectivity: they were permeable to NH4+. Both populations of aconitine-modified channels could be blocked by tetrodotoxin. In contrast to macroscopic current experiments (Mozhayeva et al. 1977) the development of aconitine modification was not affected by repetitive stimulation and external application of the agent had no effect on single sodium channels in outside-out membrane patch. 相似文献
10.
On the stochastic properties of single ion channels 总被引:38,自引:0,他引:38
D Colquhoun A G Hawkes 《Proceedings of the Royal Society of London. Series B, Containing papers of a Biological character. Royal Society (Great Britain)》1981,211(1183):205-235
It is desirable to be able to predict, from a specified mechanism, the appearance of currents that flow through single ion channels (a) to enable interpretation of experiments in which single channel currents are observed, and (b) to allow physical meaning to be attached to the results observed in kinetic (noise and relaxation) experiments in which the aggregate of many single channel currents is observed. With this object, distributions (and the means) are derived for the length of the sojourn in any specified subset of states (e.g. all shut states). In general these are found to depend not only on the state in which the sojourn starts, but also on the state that immediately follows the sojourn. The methods described allow derivation of the distribution of, for example, (a) the number of openings, and total length of the burst of openings, that may occur during a single occupancy, and (b) the apparent gap between such bursts. The methods are illustrated by their application to two simple theories of agonist action. The Castillo-Katz (non-cooperative) mechanism predicts, for example, that the number of openings per occupancy, and the apparent burst length, are independent of agonist concentration whereas a simple cooperative mechanism predicts that both will increase with agonist concentration. 相似文献
11.
The kidney is responsible for the maintenance of an organism's body solute and water balance (i.e., Na+ homeostasis). The distal nephron and the cortical collecting duct (CCD) (an example of a tight epithelium) are important sites of regulatory control over the rate of Na+ reabsorption. The Na+ channel, a specialized protein located in the apical membrane of CCD cells, is the specific site of transepithelial Na+ movement. Na+ entry into the cell across the apical membrane occurs by passive diffusion of Na+ down an electrochemical gradient. We have used the patch-voltage clamp method to examine single-channel conductance events of the amiloride-sensitive apical Na+ channel in A6 cells, a model of CCD. Two types of Na+ channel were identified. One type was characterized by low selectivity (Na+ to K+) and high conductance, the other by high selectivity and low conductance. The type and frequency of channel observed depended on the transporting state of the epithelium. In a tissue with poor transport rates, the low-selectivity type of channel was prevalent (the other type of channel was present, but in a very low density). Therefore, the poorly transporting tissue had an overall low apical Na+ conductance. In a tissue with high transport rates, the highly selective channel appeared to be predominant. In this case the net result was a highly Na+ conductive apical membrane. 相似文献
12.
A study of properties of batrachotoxin modified sodium channels 总被引:4,自引:0,他引:4
A further analysis of the effects of the steroidal alkaloid batrachotoxin (BTX) on sodium channels in frog node of Ranvier has been carried out under voltage-clamp conditions. The main properties of modified channels as compared with those of normal ones are as follows: The rate of channel closing is drastically decreased, whereas that of opening is changed slightly if at all; The steady-state voltage dependence of channel activation is shifted towards more negative potentials by 60-70 mV; Currents through modified channels do not show a decay during maintained depolarization as it is typical for normal channels. However modified channels retain the ability to partial inactivation as shown by experiments with depolarizing prepulses; Sodium against potassium selectivity beyond--20 mV suggesting either nonhomogeneity of the modified channels as for their kinetic and selectivity properties or potential-dependence of ionic selectivity for each channel; The selectivity sequence determined from peak current reversal potential measurements is as follows: H: Na :NH4:K = 528:1:0.47: :0.19; The effective pK value of proton block is decreased by about 0.4; 7) The sensitivity of the channels to tetrodotoxin (TTX) block is practically unchanged. 相似文献
13.
The currents through single Na+ channels from the sarcolemma of ventricular cells dissociated from adult rat hearts were studied using the patch-clamp technique. All patches had several Na+ channels; most had 5-10, while some had up to 50 channels. At 10 degrees C, the conductance of the channel was 9.8 pS. The mean current for sets of many identical pulses inactivated exponentially with a time constant of 1.7 +/- 0.6 ms at -40 mV. Careful examination of the mean currents revealed a small, slow component of inactivation at pulse potentials ranging from -60 to -30 mV. The time constant of the slow component was between 8 and 14 ms. The channels that caused the slow component had the same conductance and reversal potential as the fast Na+ currents and were blocked by tetrodotoxin. The slow currents appear to have been caused by repeated openings of one or more channels. The holding potential influenced the frequency with which such channel reopening occurred. The slow component was prominent during pulses from a holding potential of -100 mV, while it was very small during pulses from -140 mV. Ultraslow currents through the Na+ channel were observed occasionally in patches that had large numbers of channels. They consisted of bursts of 10 or more sequential openings of a single channel and lasted for up to 150 ms. We conclude that the single channel data cannot be explained by standard models, even those that have two inactivated states or two open states of the channel. Our results suggest that Na+ channels can function in several different "modes," each with a different inactivation rate. 相似文献
14.
Single sodium channel currents were analysed in cell attached patches from single ventricular cells of guinea pig hearts in the presence of a novel cardiotonic compound DPI 201-106. The mean single channel conductance of DPI-treated Na channels was not changed by DPI (20.8 +/- 4 pS, control, 3 patches; 21.3 +/- 1 pS with DPI, 5 mumol/1,3 patches). DPI voltage-dependently prolongs the cardiac sodium channel openings by removal of inactivation at potentials positive to -40 mV. At potentials negative to -40 mV a clustering of short openings at the very beginning of the depolarizing voltage steps can be observed causing a transient time course of the averaged currents. Long openings induced an extremely slow inactivation. Short openings, long openings and nulls appeared in groups referring to a modal gating behaviour of DPI-treated sodium channels. DPI-modified Na channels showed a monotonously prolonged mean open time with increased depolarizing voltage steps, e.g. the open state probability within a sweep was increased. However, the number of non-empty sweeps was decreased with the magnitude of the depolarizing steps, e.g. the probability of the channel being open as calculated from the averaged currents was voltage-dependently decreased by DPI (50% decrease at -50.7 +/- 9 9 mV, 3 patches). Short and long openings of DPI-modified channels could be separated by variation of the holding potential. The occurrence of long Na channel openings was much more suppressed by reducing the holding potential (half maximum inactivation at -112 +/- 8 mV, 4 patches) than that of short openings (half maximum inactivation at -88 +/- 8 mV, 4 patches). Otherwise, short living openings completely disappeared at potentials positive to -40 mV where the occurrence of long openings was favoured. The differential voltage dependence of blocking and activating effects of DPI on cardiac Na channels as well as the differential voltage dependence of the appearance of short and long openings refers to a modal gating behaviour of cardiac Na channels. 相似文献
15.
Kinetic analysis of single sodium channels from canine cardiac Purkinje cells 总被引:7,自引:3,他引:4
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Single sodium channel events were recorded from cell-attached patches on single canine cardiac Purkinje cells at 10-13 degrees C. Data from four patches containing two to four channels and one patch with one channel were selected for quantitative analysis. The channels showed prominent reopening behavior at voltages near threshold, and the number of reopenings declined steeply with depolarization. Mean channel open time was a biphasic function of voltage with the maximum value (1-1.5 ms) occurring between -50 and -40 mV and lower values at more and at less hyperpolarized levels. Inactivation without opening was also prominent near threshold, and this occurrence also declined with depolarization. The waiting time distributions and the probability of being open showed voltage and time dependence as expected from whole-cell current studies. The results were analyzed in terms of a five-state Markovian kinetic model using both histogram analysis and a maximum likelihood method to estimate kinetic parameters. The kinetic parameters of the model fits were similar to those of GH3 pituitary cells (Horn, R., and C. A. Vandenberg. 1984. Journal of General Physiology. 84:505-534) and N1E115 neuroblastoma cells (Aldrich, R. W., and C. F. Stevens. Journal of Neuroscience. 7:418-431). Both histogram and maximum likelihood analysis implied that much of the voltage dependence of cardiac Na current is in its activation behavior, with inactivation showing modest voltage dependence. 相似文献
16.
Properties of aconitine-modified sodium channels in single cells of mouse ventricular myocardium 总被引:4,自引:0,他引:4
Effects of the plant alkaloid Aconitine on the kinetics of sodium channels were studied in enzymatically isolated single cells of the mouse ventricular myocardium. Aconitine (1 mumol/l) induced a prolongation of the 90% repolarization of action potentials from 52.4 +/- 3.7 ms to 217.0 +/- 12.5 ms. Delayed terminal repolarization and oscillatory afterpotentials preceded spontaneous activity with high frequencies. Peak sodium currents were diminished from 28.0 +/- 9.0 to 14.0 +/- 6.0 nA. The reversal potential of the sodium current was shifted from 16.0 +/- 11.0 to -8.0 +/- 6.0 mV (52.5 mmol/l extracellular sodium concentration) suggesting a decreased selectivity of the Aconitine-modified Na channels. The m-affinity-curves were shifted 31 mV towards more negative potentials at a constant slope. The h affinity-curves were shifted in the same direction by 13 mV. The slope parameter of the h affinity-voltage relationship was enlarged from 9.1 +/- 2.2 mV to 15.6 +/- 4.4 mV. Shifts in m affinity and h affinity resulted in an increased "window". The alkaloid modified channels inactivated extremely slowly at potentials negative to -40 mV, but showed a fast and complete inactivation at potentials positive to -40 mV. 相似文献
17.
A. V. Sudarikova V. I. Chubinsky-Nadezhdin Yu. A. Negulyaev E. A. Morachevskaya 《Cell and Tissue Biology》2009,3(5):461-469
In the present paper, functional properties of nonvoltage-gated sodium channels in K562 cells were studied after cholesterol
depletion, i.e., under conditions of the destruction of microdomains (rafts). For cholesterol depletion, cells were incubated
with methyl-beta-cyclodextrin (MbCD), an oligosaccharide that selectively binds sterols. Single currents through sodium channels
were recorded in cell-attached and inside-out experiments using the patch-clamp technique. After incubation with MbCD (2.5
or 5 mM), the activation of sodium channels in response to cytochalasin B or D was observed in both native cells and membrane
fragments. Biophysical characteristics of sodium channels in cholesterol-depleted K562 cells were close to those in control;
unitary conductance was 12 pS. Inside-out experiments with the use of globular actin have indicated that filament assembly
on cytoplasmic membrane side causes an inactivation of sodium channels in the modified cells. These data imply that sodium
channels in K562 cells are not associated with cholesterol-rich membrane microdomains. Possible mechanisms of the interaction
of the plasma membrane and the cortical cytoskeleton are discussed. 相似文献
18.
Fast and slow gating of sodium channels encoded by a single mRNA 总被引:13,自引:0,他引:13
We investigated the kinetics of rat brain type III Na+ currents expressed in Xenopus oocytes. We found distinct patterns of fast and slow gating. Fast gating was characterized by bursts of longer openings. Traces with slow gating occurred in runs with lifetimes of 5 and 30 s and were separated by periods with lifetimes of 5 and 80 s. Cycling of fast and slow gating was present in excised outside-out patches at 10 degrees C, suggesting that metabolic factors are not essential for both forms of gating. It is unlikely that more than one population of channels was expressed, as patches with purely fast or purely slow gating were not observed. We suggest that structural mechanisms for fast and slow gating are encoded in the primary amino acid sequence of the channel protein. 相似文献
19.
Umami taste is elicited by monosodium glutamate (MSG), a compound consisting of two potent taste stimuli, Na(+) and glutamate. In rat fungiform taste cells, amiloride-sensitive epithelial sodium channels (ENaCs) mediate Na(+) transduction, while glutamate is transduced by a combination of ionotropic and metabotropic glutamate receptors. We used giga-seal whole-cell recording to determine if responses to glutamate and Na(+) occur in the same taste cells. Approximately 68% of the cells tested responded to amiloride, indicating that they express functional ENaCs. Responses to glutamate occurred in about 58% of the cells tested. Interestingly, responses to glutamate occurred in the subset of cells that also responded to amiloride, indicating that glutamate receptors are located preferentially in the same taste cells that also express ENaCs. Further experiments showed that amiloride did not suppress responses to glutamate under voltage-clamp conditions. Taken together, the data suggest that although ENaCs are not involved directly in glutamate transduction, their co-localization with glutamate receptors provides a substrate for the cellular integration of these independent pathways. Copyright Copyright 1999 S. Karger AG, Basel 相似文献