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
Single channel currents were obtained from voltage-activated sodium channels in outside-out patches of tissue-cultured GH3 cells, a clonal line from rat pituitary gland. In membrane patches where the probability of overlapping openings was low, the open time histograms were well fit by a single exponential. Most analysis was done on a patch with exactly one channel. We found no evidence for multiple open states at -25 and -40 mV, since open times, burst durations, and autocorrelation functions were time independent. Amplitude histograms showed no evidence of multiple conductance levels. We fit the gating with 25 different time-homogeneous Markov chain models having up to five states, using a maximum likelihood procedure to estimate the rate constants. For selected models, this procedure yielded excellent predictions for open time, closed time, and first latency density functions, as well as the probability of the channel being open after a step depolarization, the burst duration distribution, autocorrelation, and the distribution of number of openings per record. The models were compared statistically using likelihood ratio tests and Akaike's information criterion. Acceptable models allowed inactivation from closed states, as well as from the open state. Among the models eliminated as unacceptable by this survey were the Hodgkin-Huxley model and any model requiring a channel to open before inactivating. 相似文献
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 Kd 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
Rapid progress in our understanding of the properties and functions of voltage-gated calcium channels had produced the need for an update to our previous review of calcium inactivation. The major elements of change included in this review are: 1. The existence of multiple forms of voltage-sensitive Ca+ channels, with distinctive single channel properties, thus necessitating a reappraisal of properties deduced from macroscopic current recordings, particularly of the processes of activation and inactivation. 2. The differences in biochemical properties between channel types are reflected in their differences in divalent selectivity, their requirement for metabolic maintenance and their mechanism of inactivation. These properties appear to divide the channels into two categories which may relate to their molecular structures. Further subgroupings, based upon the voltage thresholds, have also been observed. 3. Molecular properties of one class of channels have been elucidated, which correlate with the observed biochemistry of channel modulation and inactivation. 4. An enzymatic process underlying the mechanism of Ca2+-dependent inactivation has been elucidated and may serve as a model for other modulatory systems. The interweaving of the properties of these Ca2+ channels, with their spatial distributions and their influence upon other channel types, acts to transduce and integrate information within cells. 相似文献
In small cell-attached patches containing one and only one Na+ channel, inactivation was studied in three different gating modes, namely, the fast-inactivating F mode and the more slowly inactivating S mode and P mode with similar inactivation kinetics. In each of these modes, ensemble-averaged currents could be fitted with a Hodgkin-Huxley-type model with a single exponential for inactivation (tauh). tauh declined from 1.0 ms at -60 mV to 0.1 ms at 0 mV in the F mode, from 4.6 ms at -40 mV to 1.1 ms at 0 mV in the S mode, and from 4.5 ms at -40 mV to 0.8 ms at +20 mV in the P mode, respectively. The probability of non-empty traces (net), the mean number of openings per non-empty trace (op/tr), and the mean open probability per trace (popen) were evaluated at 4-ms test pulses. net inclined from 30% at -60 mV to 63% at 0 mV in the F mode, from 4% at -90 mV to 90% at 0 mV in the S mode, and from 2% at -60 mV to 79% at +20 mV in the P mode. op/tr declined from 1.4 at -60 mV to 1.1 at 0 mV in the F mode, from 4.0 at -60 mV to 1.2 at 0 mV in the S mode, and from 2.9 at -40 mV to 1.6 at +20 mV in the P mode. popen was bell-shaped with a maximum of 5% at -30 mV in the F mode, 48% at -50 mV in the S mode, and 16% at 0 mV in the P mode. It is concluded that 1) a switch between F and S modes may reflect a functional change of inactivation, 2) a switch between S and P modes may reflect a functional change of activation, 3) tauh is mainly determined by the latency until the first channel opening in the F mode and by the number of reopenings in the S and P modes, 4) at least in the S and P modes, inactivation is independent of pore opening, and 5) in the S mode, mainly open channels inactivate, and in the P mode, mainly closed channels inactivate. 相似文献
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
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
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
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
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
Ba(2+), a doubly charged analogue of K(+), specifically blocks K(+) channels by virtue of electrostatic stabilization in the permeation pathway. Ba(2+) block is used here as a tool to determine the equilibrium binding affinity for various monovalent cations at specific sites in the selectivity filter of a noninactivating mutant of KcsA. At high concentrations of external K(+), the block-time distribution is double exponential, marking at least two Ba(2+) sites in the selectivity filter, in accord with a Ba(2+)-containing crystal structure of KcsA. By analyzing block as a function of extracellular K(+), we determined the equilibrium dissociation constant of K(+) and of other monovalent cations at an extracellular site, presumably S1, to arrive at a selectivity sequence for binding at this site: Rb(+) (3 μM) > Cs(+) (23 μM) > K(+) (29 μM) > NH(4)(+) (440 μM) > Na(+) and Li(+) (>1 M). This represents an unusually high selectivity for K(+) over Na(+), with |ΔΔG(0)| of at least 7 kcal mol(-1). These results fit well with other kinetic measurements of selectivity as well as with the many crystal structures of KcsA in various ionic conditions. 相似文献
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
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
Ionic currents through aconitine-modified sodium channels of the Ranvier node membrane were measured by a voltage clamp method in an external medium free from sodium ions. A shift of pH of the solution below 4.6 led to the appearance of inward ionic currents, whose kinetics and activation region were characteristic of aconitine-modified sodium channels at low pH. These currents were blocked by the local anesthetic benzocaine in a concentration of 2 mM. Experiments with variation of the concentration of Ca++, Tris+, TEA+, and choline+ in acid sodium-free solutions showed that these cations make no appreciable contribution to the inward current. It is concluded that the inward currents observed under these conditions are carried by H+ (or H3O+) through aconitine-modified sodium channels. From the shifts of reversal potentials of the ionic currents the relative permeability (PH/PNa) for H+ was determined: 1059 ± 88. The results agree with the view that the aconitine-modified sodium channel is a relatively wide water pore, and that movement of H+ through it is limited by its binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 508–516, September–October, 1982. 相似文献
Ionic currents through the frog Ranvier node membrane were measured by the voltage clamp method on the membrane of a single myelinated frog's nerve fiber under conditions when Na+ in the external solution was replaced by nonpenetrating cations. When pH fell below 4.0, small (under 0.1 nA) inward currents were found and on the basis of various features (kinetics, region of activation, and blocking by the local anesthetic benzocaine — 1.0 mM) were identified as currents through sodium channels. The results of control experiments with variation of the concentrations of cations in the external solution led to the conclusion that the H+ (or H3O+) ion is the main carrier of the measured inward current. According to the results of measurement of the reversal potential of these currents, the relative permeability of sodium channels for hydrogen ions (PH/PNa) averages 205 ± 14. The results are discussed in terms of a model of the water pore with saturation. It is concluded that the energy barriers for H+ in the sodium channel are low. It was also shown that the velocity of passage of protons through the channel is limited by binding with an acid group.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 14, No. 5, pp. 499–507, September–October, 1982. 相似文献
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