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1.
Linear Systems convolution analysis of muscle sodium currents was used to predict the opening rate of sodium channels as a function of time during voltage clamp pulses. If open sodium channel lifetimes are exponentially distributed, the channel opening rate corresponding to a sodium current obtained at any particular voltage, can be analytically obtained using a simple equation, given single channel information about the mean open-channel lifetime and current.Predictions of channel opening rate during voltage clamp pulses show that sodium channel inactivation arises coincident with a decline in channel opening rate.Sodium currents pharmacologically modified with Chloramine-T treatment so that they do not inactivate, show a predicted sustained channel opening rate.Large depolarizing voltage clamp pulses produce channel opening rate functions that resemble gating currents.The predicted channel opening rate functions are best described by kinetic models for Na channels which confer most of the charge movement to transitions between closed states.Comparisons of channel opening rate functions with gating currents suggests that there may be subtypes of Na channel with some contributing more charge movement per channel opening than others.Na channels open on average, only once during the transient period of Na activation and inactivation.After transiently opening during the activation period and then closing by entering the inactivated state, Na channels reopen if the voltage pulse is long enough and contribute to steady-state currents.The convolution model overestimates the opening rate of channels contributing to the steady-state currents that remain after the transient early Na current has subsided.  相似文献   

2.
Ionic selectivity of the sodium channel of frog skeletal muscle   总被引:4,自引:4,他引:0       下载免费PDF全文
The ionic selectivity of the Na channel to a variety of metal and organic cations is studied in frog semitendinosus muscle. Na channel currents are measured under voltage clamp conditions in fibers bathed in solutions with all Na+ replaced by a test ion. Permeability ratios are calculated from measured reversal potentials using the Goldman-Hodgkin-Katz equation. The permeability sequence was Na+ approximately Li+ approximately hydroxylammonium greater than hydrazinium greater than ammonium greater than guanidinium greater than K+ greater than aminoguanidinium in the ratios 1:0.96:0.94:0.31:0.11:0.093:0.048:0.031. No inward currents were observed for Ca++, methylammonium, methylguanidinium, tetraethylammonium, and tetramethylammonium. The results are consistent with the Hille model of the Na channel selectivity filter of the node of Ranvier and suggest that the selectivity filter of the two channels is the same.  相似文献   

3.
Open channel properties of canine cardiac Purkinje cell Na+ channels were studied with single channel cell-attached recording and with whole cell macroscopic current recording in internally perfused cells. Single channel currents and membrane currents increased with an increase in Na+ concentration, but showed evidence of saturation. Assuming first-order binding, the Km for Na+ was 370 mM. PCs/PNa was 0.020 and PK/PNa was 0.094. The current-voltage relationship for single channels showed prominent flattening in the hyperpolarizing direction. This flattening was accentuated by 10 mM Ca2+ and was greatly reduced in O mM Ca2+, indicating that the rectification was a consequence of Ca2+ block of the Na+ channels. A similar instantaneous current-voltage relationship was seen for the whole cell membrane currents. These results demonstrate that the cardiac channel shows substantial Ca2+ block, although it is relatively insensitive to tetrodotoxin. The Na+ and Ca2+ binding properties could be modeled by the four-barrier Eyring rate theory model, with similar values to those reported for the neuroblastoma Na+ channel (Yamamoto, D.,J.Z. Yeh, and T. Narahashi, 1984, Biophys J., 45:337-344).  相似文献   

4.
In this paper we report the direct measurement of rare Na channel events that occur during the cardiac action potential, viz., channels that open at the upstroke and remain open throughout the plateau and early repolarization phase. The technique we use allows us to record channel activity and action potentials at the same time; thus, we are certain of when the Na channels open and when they finally close. The slow Na channels have the same voltage dependence, single-channel conductance, and TTX sensitivity as the fast Na channels, and they conduct Li. It therefore seems likely that the fast and the slow currents flow through the same channel. If this interpretation is correct, then the Na channel not only initiates the action potential but also helps to maintain its plateau. It is possible that the slow Na currents represent a separate collection of channels rather than a low-probability state of the fast Na channels. Regardless of which interpretation is correct, the present experiments allow us to assess the effect of the slow currents on action potential shape and on sustained Na entry.  相似文献   

5.
Slow currents through single sodium channels of the adult rat heart   总被引:18,自引:6,他引:12       下载免费PDF全文
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   

6.
We investigated the cellular and molecular mechanisms underlying arrhythmias in heart failure. A genetically engineered mouse lacking the expression of the muscle LIM protein (MLP-/-) was used in this study as a model of heart failure. We used electrocardiography and patch clamp techniques to examine the electrophysiological properties of MLP-/- hearts. We found that MLP-/- myocytes had smaller Na+ currents with altered voltage dependencies of activation and inactivation and slower rates of inactivation than control myocytes. These changes in Na+ currents contributed to longer action potentials and to a higher probability of early afterdepolarizations in MLP-/- than in control myocytes. Western blot analysis suggested that the smaller Na+ current in MLP-/- myocytes resulted from a reduction in Na+ channel protein. Interestingly, the blots also revealed that the alpha-subunit of the Na+ channel from the MLP-/- heart had a lower average molecular weight than in the control heart. Treating control myocytes with the sialidase neuraminidase mimicked the changes in voltage dependence and rate of inactivation of Na+ currents observed in MLP-/- myocytes. Neuraminidase had no effect on MLP-/- cells thus suggesting that Na+ channels in these cells were sialic acid-deficient. We conclude that deficient glycosylation of Na+ channel contributes to Na+ current-dependent arrhythmogenesis in heart failure.  相似文献   

7.
Currents through delayed rectifier-type K+ channels in Schwann cells cultured from rabbit sciatic nerve were studied with patch-clamp techniques. When the internal and external solutions contained physiological concentrations of sodium, the amplitude of these outward currents declined as the cell was depolarized to potentials above about +40 mV, despite the increased driving force. This reduction in the amplitude of outward K+ currents was observed in many cells before the subtraction of leakage currents; it was also observed for ensemble currents recorded in outside-out patches. It was therefore not the result of a leak-subtraction artefact nor of inadequate voltage-clamp control. Several lines of evidence also suggested that it was not the result of the extracellular accumulation of K+. By contrast, when the Na+ ion concentration of the internal solution was nominally zero, the reduction in the amplitude of outward K+ currents at positive membrane potentials was not observed. The apparent amplitude of single-channel currents through two types of K+ channel was reduced by 30 mM internal Na+, apparently as the result of a rapid 'flickery' block. The results suggest that channel block by internal Na+ is largely responsible for the negative slope conductance seen in current-voltage plots of whole-cell K+ currents at positive membrane potentials. In addition, our analysis of single-channel currents suggests that the current-voltage curve for a delayed rectifier channel in rabbit Schwann cells (in the absence of internal Na+) is roughly linear with internal and external K+ concentrations of 140 mM and 5.6 mM, respectively.  相似文献   

8.
Elementary Na+ currents through single cardiac Na+ channels were recorded at 19 degrees C in patch clamp experiments with cultured neonatal rat cardiocytes. The metabolites of the glycolytic pathway, 2,3-diphosphoglycerate and glyceraldehyde phosphate, were identified as a novel class of modulators of Na+ channel activity. In micromolar concentrations (1-10 mumol/liter), their presence at the cytoplasmic membrane face increased the number of sequential openings during depolarization and prolonged the conductive channel state. As found after ensemble averaging, the decay kinetics of reconstructed macroscopic Na+ currents became retarded and slow Na+ inactivation may have been evoked. Both metabolites attenuated the rundown of channel activity that regularly develops after patch excision in the inside-out patch configuration. It is tempting to assume that interference with Na+ inactivation is the mode of action underlying the increase in single-channel activity.  相似文献   

9.
The effects of a neurotoxin, purified from the venom of the scorpion Leiurus quinquestriatus, on the ionic currents of toad single myelinated fibers were studied under voltage-clamp conditions. Unlike previous investigations using crude scorpion venom, purified Leiurus toxin II alpha at high concentrations (200-400 nM) did not affect the K currents, nor did it reduce the peak Na current in the early stages of treatment. The activation of the Na channel was unaffected by the toxin, the activation time course remained unchanged, and the peak Na current vs. voltage relationship was not altered. In contrast, Na channel inactivation was considerably slowed and became incomplete. As a result, a steady state Na current was maintained during prolonged depolarizations of several seconds. These steady state Na currents had a different voltage dependence from peak Na currents and appeared to result from the opening of previously inactivated Na channels. The opening kinetics of the steady state current were exponential and had rates approximately 100-fold slower than the normal activation processes described for transitions from the resting state to the open state. In addition, the dependence of the peak Na current on the potential of preceding conditioning pulses was also dramatically altered by toxin treatment; this parameter reached a minimal value near a membrane potential of -50 mV and then increased continuously to a "plateau" value at potentials greater than +50 mV. The amplitude of this plateau was dependent on toxin concentration, reaching a maximum value equal to approximately 50% of the peak current; voltage-dependent reversal of the toxin's action limits the amplitude of the plateauing effect. The measured plateau effect was half-maximum at a toxin concentration of 12 nM, a value quite similar to the concentration producing half of the maximum slowing of Na channel inactivation. The results of Hill plots for these actions suggest that one toxin molecule binds to one Na channel. Thus, the binding of a single toxin molecule probably both produces the steady state currents and slows the Na channel inactivation. We propose that Leiurus toxin inhibits the conversion of the open state to inactivated states in a voltage-dependent manner, and thereby permits a fraction of the total Na permeability to remain at membrane potentials where inactivation is normally complete.  相似文献   

10.
Properties of "creep currents" in single frog atrial cells   总被引:1,自引:5,他引:1  
Changes in membrane current in response to an elevation of [Na]i were studied in enzymatically dispersed frog atrial cells. Na loading by either intracellular dialysis or exposure to the Na ionophore monensin produces changes in membrane current that resemble the "creep currents" originally observed in cardiac Purkinje fibers during exposure to low-K solutions. Na loading induces a transient outward current during depolarizing voltage-clamp pulses, followed by an inward current in response to repolarization back to the holding potential. In contrast to cardiac Purkinje fibers, Na loading of frog atrial cells induces creep currents without accompanying transient inward currents. Creep currents induced by Na loading are insensitive to K channel antagonists like Cs and 4-aminopyridine; they are not influenced by doses of Ca channel antagonists that abolish iCa, but are sensitive to changes in [Ca]o or [Na]o. A comparison of the time course of development of inward creep currents are not tail currents associated with iCa. Inward creep currents can also be induced by experimental interventions that increase the iCa amplitude. Exposure to isoproterenol enhances the iCa amplitude and induces inward creep currents; both can be attenuated by Ca channel antagonists. Both inward and outward creep currents are blocked by low doses of La, independently of La's ability to block iCa. It is concluded that (a) creep currents are not mediated by voltage-gated Na, Ca, or K channels or by an electrogenic Na,K pump; (b) inward creep currents induced either by Na loading or in response to an increase in the amplitude of iCa are triggered by an elevation of [Ca]i; and (c) creep currents may be generated by either an electrogenic Na/Ca exchange mechanism or by a nonselective cation channel activated by [Ca]i.  相似文献   

11.
Pharmacological and kinetic analysis of K channel gating currents   总被引:3,自引:2,他引:1       下载免费PDF全文
We have measured gating currents from the squid giant axon using solutions that preserve functional K channels and with experimental conditions that minimize Na channel contributions to these currents. Two pharmacological agents were used to identify a component of gating current that is associated with K channels. Low concentrations of internal Zn2+ that considerably slow K channel ionic currents with no effect on Na channel currents altered the component of gating current associated with K channels. At low concentrations (10-50 microM) the small, organic, dipolar molecule phloretin has several reported specific effects on K channels: it reduces K channel conductance, shifts the relationship between channel conductance and membrane voltage (Vm) to more positive potentials, and reduces the voltage dependence of the conductance-Vm relation. The K channel gating charge movements were altered in an analogous manner by 10 microM phloretin. We also measured the dominant time constants of the K channel ionic and gating currents. These time constants were similar over part of the accessible voltage range, but at potentials between -40 and 0 mV the gating current time constants were two to three times faster than the corresponding ionic current values. These features of K channel function can be reproduced by a simple kinetic model in which the channel is considered to consist of two, two-state, nonidentical subunits.  相似文献   

12.
Permeation characteristics of gramicidin conformers.   总被引:3,自引:3,他引:0       下载免费PDF全文
To investigate the molecular origin of decreased conductance in variant gramicidin channels, we examined the current-voltage (IV) characteristics of single Val1-gramicidin A channels. Unlike standard channels, all variant channels showed pronounced rectification even though bathing solutions were symmetrical. Moreover, channels of lower conductance consistently showed more pronounced rectification. Analysis within the framework of a three-barrier, two-site, single-filing model indicates that the shape of the variant channel IVs could be best explained by an increase in binding affinity near one of the two channel entrances. This conclusion was further tested by characterizing single channel IVs in bi-ionic solutions having different cationic species at each channel entrance. In Cs/Na bi-ionic solutions, reversal potentials of variant channels often differed by a small but significant amount from those of standard channels. When a membrane potential was applied, the ionic currents tended to be reduced more when flowing from the Na+ side than the Cs+ side. These observations support the conclusion that variant channels have increased binding affinity at one end of the channel. Furthermore, H+ currents were increased while Ag+ currents were unaltered for most variant channels exhibiting decreased Na+ or Cs+ currents. The increased H+ conductance argues against long-range coulombic forces as the basis for decreased Na+ or Cs+ conductance while the normal Ag+ conductance suggests that the binding site field strength increases by a change in carbonyl geometry at the channel entrance.  相似文献   

13.
The P segments of the voltage-dependent Na+ channel line the outer mouth and selectivity filter of the pore. The residues that form the cytoplasmic mouth of the pore of the channel have not been identified. To study the structure of the inner pore mouth, the presumed selectivity filter residues (D400, E755, K1237, and A1529), and three amino acids just amino-terminal to each of these residues in the rat skeletal muscle Na+ channel, were mutated to cysteine and expressed in tsA 201 cells. These amino acids are predicted (by analogy to K+ channels) to be on the cytoplasmic side of the putative selectivity filter residues. Inward and outward Na+ currents were measured with the whole-cell configuration of the patch-clamp technique. Cysteinyl side-chain accessibility was gauged by sensitivity to Cd2+ block and by reactivity with methanethiosulfonate (MTS) reagents applied to both the inside and the outside of the cell. Outward currents through the wild-type and all of the mutant channels were unaffected by internal Cd2+ (100 microM). Similarly, 1 mM methanethiosulfonate ethylammonium (MTSEA) applied to the inside of the membrane did not affect wild-type or mutant outward currents. However, two mutants amino-terminal to the selectivity position in domain III (F1236C and T1235C) and one in domain IV (S1528C) were blocked with high affinity by external Cd2+. The Na+ current through F1236C and S1528C channels was inhibited by MTSEA applied to the outside of the cell. The accessibility of these mutants to externally applied cysteinyl ligands indicates that the side chains of the mutated residues face outward rather than inward. The K+ channel model of the P segments as protein loops that span the selectivity region is not applicable to the Na+ channel.  相似文献   

14.
The electrophysiological properties of the cation channel of the purified nicotinic acetylcholine receptor (AChR) reconstituted in planar lipid bilayers were characterized. Single-channel currents were activated by acetylcholine, carbamylcholine and suberyldicholine. The single channel conductance (28 pS in 0.3 M NaCl) was ohmic and independent of the agonist. Single channel currents increased with Na+ concentration to a maximum conductance of 95 pS and showed a half-saturation point of 395 mM. The apparent ion selectivity sequence, derived from single-channel current recordings, is: NH+4 greater than Cs+ greater than Rb+ greater than or equal to Na+ Cl-, F-, SO2-(4). The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of at least two distinct open states. The time constants depend on the choice of agonist, being consistently longer for suberyldicholine than for carbamylcholine. Similar channel properties were recorded in bilayers formed from monolayers at the tip of patch pipets . Single-channel currents occur in paroxysms of channel activity followed by quiescent periods. This pattern is more pronounced as the agonist concentration increases, and is reflected in histograms of channel-opening frequencies. Computer simulations with a three-state model, consisting of two closed (unliganded and liganded) and one open state, do not resemble the recorded pattern of channel activity, especially at high agonist concentration. Inclusion of a desensitized liganded state reproduces the qualitative features of channel recordings. The occurrence of paroxysms of channel activity thus seems to result from the transit of AChR through its active conformation, from which it can open several times before desensitizing.  相似文献   

15.
16.
We propose a reaction model for the palytoxin-sodium-potassium (PTX-Na(+)/K(+)) pump complex. The model, which is similar to the Albers-Post model for Na(+)/K(+)-ATPase, is used to elucidate the effect of PTX on Na(+)/K(+)-ATPase during the enzyme interactions with Na(+) and/or K(+) ions. Conformational substates and reactions for the pump are incorporated into the Albers-Post model to represent enzymes with or without bound PTX. A mathematical model based on the reaction scheme is used in simulations modeling experimental studies of PTX-induced ionic currents. Our simulations suggest that (i) extracellular Na(+) as well as K(+) promotes PTX-induced channel blockage; (ii) extracellular K(+) accelerates PTX unbinding; and (iii) K(+) occlusion in the PTX-pump complex is essential for describing the PTX-induced current dynamics.  相似文献   

17.
Summary The membrane ofParamecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation,fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that theParamecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca2+ concentration. The function of the Ca-dependent Na conductance is discussed.  相似文献   

18.
Kilic G  Lindau M 《Biophysical journal》2001,80(3):1220-1229
We investigated the voltage dependence of membrane capacitance of pituitary nerve terminals in the whole-terminal patch-clamp configuration using a lock-in amplifier. Under conditions where secretion was abolished and voltage-gated channels were blocked or completely inactivated, changes in membrane potential still produced capacitance changes. In terminals with significant sodium currents, the membrane capacitance showed a bell-shaped dependence on membrane potential with a peak at approximately -40 mV as expected for sodium channel gating currents. The voltage-dependent part of the capacitance showed a strong correlation with the amplitude of voltage-gated Na+ currents and was markedly reduced by dibucaine, which blocks sodium channel current and gating charge movement. The frequency dependence of the voltage-dependent capacitance was consistent with sodium channel kinetics. This is the first demonstration of sodium channel gating currents in single pituitary nerve terminals. The gating currents lead to a voltage- and frequency-dependent capacitance, which can be well resolved by measurements with a lock-in amplifier. The properties of the gating currents are in excellent agreement with the properties of ionic Na+ currents of pituitary nerve terminals.  相似文献   

19.
Tetrodotoxin (TTX)-sensitive Na currents were examined in single dissociated ventricular myocytes from neonatal rats. Single channel and whole cell currents were measured using the patch-clamp method. The channel density was calculated as 2/micron 2, which agreed with our usual finding of four channels per membrane patch. At 20 degrees C, the single channel conductance was 20 pS. The open time distributions were fit by a single-exponential function with a mean open time of approximately 1.0 ms at membrane potentials from -60 to -40 mV. Averaged single channel and whole cell currents were similar when scaled and showed both fast and slow rates of inactivation. The inactivation and activation gating shifted quickly to hyperpolarized potentials for channels in cell-attached as well as excised patches, whereas a much slower shift occurred in whole cells. Slowly inactivating currents were present in both whole cell and single channel current measurements at potentials as positive as -40 mV. In whole cell measurements, the potential range could be extended, and slow inactivation was present at potentials as positive as -10 mV. The curves relating steady state activation and inactivation to membrane potential had very little overlap, and slow inactivation occurred at potentials that were positive to the overlap. Slow inactivation is in this way distinguishable from the overlap or window current, and the slowly inactivating current may contribute to the plateau of the rat cardiac action potential. On rare occasions, a second set of Na channels having a smaller unit conductance and briefer duration was observed. However, a separate set of threshold channels, as described by Gilly and Armstrong (1984. Nature [Lond.]. 309:448), was not found. For the commonly observed Na channels, the number of openings in some samples far exceeded the number of channels per patch and the latencies to first opening or waiting times were not sufficiently dispersed to account for the slowly inactivating currents: the slow inactivation was produced by channel reopening. A general model was developed to predict the number of openings in each sample. Models in which the number of openings per sample was due to a dispersion of waiting times combined with a rapid transition from an open to an absorbing inactivated state were unsatisfactory and a model that was more consistent with the results was identified.  相似文献   

20.
We explore the effects of stochastic sodium (Na) channel activation on the variability and dynamics of spiking and bursting in a model neuron. The complete model segregates Hodgin-Huxley-type currents into two compartments, and undergoes applied current-dependent bifurcations between regimes of periodic bursting, chaotic bursting, and tonic spiking. Noise is added to simulate variable, finite sizes of the population of Na channels in the fast spiking compartment.During tonic firing, Na channel noise causes variability in interspike intervals (ISIs). The variance, as well as the sensitivity to noise, depend on the model's biophysical complexity. They are smallest in an isolated spiking compartment; increase significantly upon coupling to a passive compartment; and increase again when the second compartment also includes slow-acting currents. In this full model, sufficient noise can convert tonic firing into bursting.During bursting, the actions of Na channel noise are state-dependent. The higher the noise level, the greater the jitter in spike timing within bursts. The noise makes the burst durations of periodic regimes variable, while decreasing burst length duration and variance in a chaotic regime. Na channel noise blurs the sharp transitions of spike time and burst length seen at the bifurcations of the noise-free model. Close to such a bifurcation, the burst behaviors of previously periodic and chaotic regimes become essentially indistinguishable.We discuss biophysical mechanisms, dynamical interpretations and physiological implications. We suggest that noise associated with finite populations of Na channels could evoke very different effects on the intrinsic variability of spiking and bursting discharges, depending on a biological neuron's complexity and applied current-dependent state. We find that simulated channel noise in the model neuron qualitatively replicates the observed variability in burst length and interburst interval in an isolated biological bursting neuron.  相似文献   

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