Kinetic evidence for two Na channels in frog skeletal muscle

The kinetics of voltage-clamped sodium currents were studied in frog skeletal muscle. Sodium currents in frog skeletal muscle activate and inactivate following an initial delay in response to a depolarizing voltage pulse. Inactivation occurs via a double exponential decay exhibiting fast and slow components for virtually all depolarizing pulses used.The deactivation of Na currents exhibits two exponential components, one decaying rapidly, while the other decays slowly in time; the relative amplitude of the two components changes with the duration of the activating pulse. The two deactivation phases remain after pharmacological elimination of inactivation.In individual fibers, the percent amplitude of the slow inactivation component correlates with the percent amplitude of the slow deactivation component.Tetrodotoxin differentially blocks the slow deactivation component.These observations are interpreted as the activation, inactivation and deactivation of two subtypes (fast and slow) of Na channels.Studies of the slow deactivation phase magnitude vs the duration of the eliciting pulse provide a way to determine the kinetics of the slow Na channel in muscle.Ammonium substitution for Na in the Ringer produces a voltage dependent activation and inactivation of current which exhibits only one decay phase, and eliminates the slow decay phase of current, suggesting that adjustments of the ionic environment of the channels can mask the presence of one of the channel subtypes.     

Slow currents through single sodium channels of the adult rat heart

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.     

Cardiac Na currents and the inactivating, reopening, and waiting properties of single cardiac Na channels

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.     

Effects of benzocaine on the kinetics of normal and batrachotoxin-modified Na channels in frog node of Ranvier.

The effects of benzocaine (0.5-1 mM) on normal Na currents, and on Na current and gating charge movement (Q) of batrachotoxin (BTX)-modified Na channels were analyzed in voltage-clamped frog node of Ranvier. Without BTX treatment the decay of Na current during pulses to between -40 and 0 mV could be decomposed into two exponential components both in the absence and in the presence of benzocaine. Benzocaine did not significantly alter the inactivation time constant of either component, but reduced both their amplitudes. The amplitude of the slow inactivating component was more decreased by benzocaine than the amplitude of the fast one, leading to an apparently faster decline of the overall Na current. After removal of Na inactivation and charge movement immobilization by BTX, benzocaine decreased the amplitude of INa with no change in time course. INa, QON, and QOFF were all reduced by the same factor. The results suggest that the rate of reaction of benzocaine with its receptor is slow compared to the rates of channel activation and inactivation. The differential effects of benzocaine on the two components of Na current inactivation in normal channels can be explained assuming two types of channel with different rates of inactivation and different affinities for the drug.     

Two modes of gating during late Na+ channel currents in frog sartorius muscle

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.     

Patch and whole cell calcium currents recorded simultaneously in snail neurons

The flow of Ca ions through single Ca channels has been examined. The gigaseal method was used on identifiable snail neurons that were voltage clamped using a two-microelectrode voltage clamp method. Average Ca patch currents and whole cell currents have similar time courses. They are affected similarly by changes in temperature. The differences in amplitude and inactivation between Ba and Ca whole cell currents were present in the patch records. The stationary noise spectra recorded from ensembles of multichannel patches have two components with fast and slow time constants equivalent to two components in the whole cell tail current relaxations. Elementary current amplitudes measured from the variance-mean relationship and from noise spectra gave values comparable to measurements from single channels. The single channel I-V relationship was curvilinear and the maximum slope conductance in 40 mM Cao was 7 pS. The amplitude of unitary currents was unchanged at long times when inactivation had occurred; hence depletion is not involved in this process. Channel density was approximately 3 microns-2 and was the same for Ba and Ca currents. The whole cell asymmetry currents gave very large values for the gating charge per channel. Changes in temperature from 29 to 9 degrees C had only a slight effect on the two Ca tail current tau's at potentials where turn-on of patch and whole cell currents was markedly slowed and the peak amplitudes were reduced by one-third. Single channel recordings were obtained at these two temperatures, and the mean open time and the fast component of the closed times were scarcely affected. Unit amplitudes were reduced by 30% and the slow closed time component was doubled. Therefore, peak currents and the slow closed time component was doubled. Therefore, peak currents were reduced partly as a result of the reduction in unit amplitude, but mainly as a result of a reduction in opening probability, the latter arising from an increase of the long closed times. It is concluded that the behavior of single Ca channels in membrane patches is the same as it is in whole cells. Cooling from 29 to 9 degrees C acts primarily on transitions among closed states and has little effect on the open to closed transition.     

Na+ permeation and block of hERG potassium channels

The inactivation gating of hERG channels is important for the channel function and drug-channel interaction. Whereas hERG channels are highly selective for K+, we have found that inactivated hERG channels allow Na+ to permeate in the absence of K+. This provides a new way to directly monitor and investigate hERG inactivation. By using whole cell patch clamp method with an internal solution containing 135 mM Na+ and an external solution containing 135 mM NMG+, we recorded a robust Na+ current through hERG channels expressed in HEK 293 cells. Kinetic analyses of the hERG Na+ and K+ currents indicate that the channel experiences at least two states during the inactivation process, an initial fast, less stable state followed by a slow, more stable state. The Na+ current reflects Na+ ions permeating through the fast inactivated state but not through the slow inactivated state or open state. Thus the hERG Na+ current displayed a slow inactivation as the channels travel from the less stable, fast inactivated state into the more stable, slow inactivated state. Removal of fast inactivation by the S631A mutation abolished the Na+ current. Moreover, acceleration of fast inactivation by mutations T623A, F627Y, and S641A did not affect the hERG Na+ current, but greatly diminished the hERG K+ current. We also found that external Na+ potently blocked the hERG outward Na+ current with an IC50 of 3.5 mM. Mutations in the channel pore and S6 regions, such as S624A, F627Y, and S641A, abolished the inhibitory effects of external Na+ on the hERG Na+ current. Na+ permeation and blockade of hERG channels provide novel ways to extend our understanding of the hERG gating mechanisms.     

Development of ionic currents of zebrafish slow and fast skeletal muscle fibers

Voltage-gated Na+ and K+ channels play key roles in the excitability of skeletal muscle fibers. In this study we investigated the steady-state and kinetic properties of voltage-gated Na+ and K+ currents of slow and fast skeletal muscle fibers in zebrafish ranging in age from 1 day postfertilization (dpf) to 4-6 dpf. The inner white (fast) fibers possess an A-type inactivating K+ current that increases in peak current density and accelerates its rise and decay times during development. As the muscle matured, the V50s of activation and inactivation of the A-type current became more depolarized, and then hyperpolarized again in older animals. The activation kinetics of the delayed outward K+ current in red (slow) fibers accelerated within the first week of development. The tail currents of the outward K+ currents were too small to allow an accurate determination of the V50s of activation. Red fibers did not show any evidence of inward Na+ currents; however, white fibers expressed Na+ currents that increased their peak current density, accelerated their inactivation kinetics, and hyperpolarized their V50 of inactivation during development. The action potentials of white fibers exhibited significant changes in the threshold voltage and the half width. These findings indicate that there are significant differences in the ionic current profiles between the red and white fibers and that a number of changes occur in the steady-state and kinetic properties of Na+ and K+ currents of developing zebrafish skeletal muscle fibers, with the most dramatic changes occurring around the end of the first day following egg fertilization.     

Gating of cardiac Na+ channels in excised membrane patches after modification by alpha-chymotrypsin.

Single cardiac Na+ channels were investigated after intracellular proteolysis to remove the fast inactivation process in an attempt to elucidate the mechanisms of channel gating and the role of slow inactivation. Na+ channels were studied in inside-out patches excised from guinea-pig ventricular myocytes both before and after very brief exposure (2-4 min) to the endopeptidase, alpha-chymotrypsin. Enzyme exposure times were chosen to maximize removal of fast inactivation and to minimize potential nonspecific damage to the channel. After proteolysis, the single channel current-voltage relationship was approximately linear with a slope conductance of 18 +/- 2.5 pS. Na+ channel reversal potentials measured before and after proteolysis by alpha-chymotrypsin were not changed. The unitary current amplitude was not altered after channel modification suggesting little or no effect on channel conductance. Channel open times were increased after removal of fast inactivation and were voltage-dependent, ranging between 0.7 (-70 mV) and 3.2 (-10 mV) ms. Open times increased with membrane potential reaching a maximum at -10 mV; at more positive membrane potentials, open times decreased again. Fast inactivation appeared to be completely removed by alpha-chymotrypsin and slow inactivation became more apparent suggesting that fast and slow inactivation normally compete, and that fast inactivation dominates in unmodified channels. This finding is not consistent with a slow inactivated state that can only be entered through the fast inactivated state, since removal of fast inactivation does not eliminate slow inactivation. The data indicate that cardiac Na+ channels can enter the slow inactivated state by a pathway that bypasses the fast inactivated state and that the likelihood of entering the slow inactivated state increases after removal of fast inactivation.     

Subunit dependence of Na channel slow inactivation and open channel block in cerebellar neurons

Purkinje and cerebellar nuclear neurons both have Na currents with resurgent kinetics. Previous observations, however, suggest that their Na channels differ in their susceptibility to entering long-lived inactivated states. To compare fast inactivation, slow inactivation, and open-channel block, we recorded voltage-clamped, tetrodotoxin-sensitive Na currents in Purkinje and nuclear neurons acutely isolated from mouse cerebellum. In nuclear neurons, recovery from all inactivated states was slower, and open-channel unblock was less voltage-dependent than in Purkinje cells. To test whether specific subunits contributed to this differential stability of inactivation, experiments were repeated in Na(V)1.6-null (med) mice. In med Purkinje cells, recovery times were prolonged and the voltage dependence of open-channel block was reduced relative to control cells, suggesting that availability of Na(V)1.6 is quickly restored at negative potentials. In med nuclear cells, however, currents were unchanged, suggesting that Na(V)1.6 contributes little to wild-type nuclear cells. Extracellular Na(+) prevented slow inactivation more effectively in Purkinje than in nuclear neurons, consistent with a resilience of Na(V)1.6 to slow inactivation. The tendency of nuclear Na channels to inactivate produced a low availability during trains of spike-like depolarization. Hyperpolarizations that approximated synaptic inhibition effectively recovered channels, suggesting that increases in Na channel availability promote rebound firing after inhibition.     

Metabolites of the glycolytic pathway modulate the activity of single cardiac Na+ channels

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.     

Sodium channels in cultured chick dorsal root ganglion neurons

Isolated Na currents were studied in cultured chick sensory neurons using the patch clamp technique. On membrane depolarization, whole cell currents showed the typical transient and voltage-dependent time course as in nerve fibres. Na currents appeared at about-40 mV and reached maximum amplitude at around-10 mV. At low voltages (-30 to 0 mV), their turning-on was sigmoidal and inactivation developed exponentially. The ratio of inactivation time constants was found to be smaller than in squid axons and comparable to that of mammalian nodes of Ranvier. Peak conductance and steady-state inactivation were strongly voltage-dependent, with maximum slopes at-17 and-40 mV, respectively. The reversal potential was close to the Nernst equilibrium potential, indicating a high degree of ion-selectivity for the channel. Addition of 3M TTX, or replacement of Na by Choline in the external bath, abolished these currents. Internal pronase (1 mg/ml) and N-bromoacetamide (0.4 mM) made inactivation incomplete, with little effect on its rate of decay.Single Na channel currents were studied in outside-out membrane patches, at potentials between-50 and-20 mV. Their activation required large negative holding potentials (-90 mV). They were fully blocked by addition of TTX (3 M) to the external bath. At-40 mV their mean open time was about 2ms and the amplitude distribution could be fitted by a single Gaussian curve, indicating the presence of a homogeneous population of channels with a conductance of 11±2 pS. Probability of opening increased and latency to first opening decreased with increasing depolarization. Inactivation of the channel became faster with stronger depolarizations, as measured from the inactivation time course of sample averages. Internal pronase (0.1 mg/ml) produced effects on inactivation comparable to those on whole cell currents. Openings of the channel had a tendency to occur in bursts and showed little inactivation during pulses of 250 ms duration. The open lifetime of the channel at low potentials (-50,-40 mV) was only three times larger than in control patches, suggesting that Na channels in chick sensory neurons can close several times before entering an inactivating absorbing state.     

Two Fast Transient Current Components during Voltage Clamp on Snail Neurons

Voltage clamp currents from medium sized ganglion cells of Helix pomatia have a fast transient outward current component in addition to the usually observed inward and outward currents. This component is inactivated at normal resting potential. The current, which is carried by K+ ions, may surpass leakage currents by a factor of 100 after inactivation has been removed by hyperpolarizing conditioning pulses. Its kinetics are similar to those of the inward current, except that it has a longer time constant of inactivation. It has a threshold close to resting potential. This additional component is also present in giant cells, where however, it is less prominent. Pacemaker activity is controlled by this current. It was found that inward currents have a slow inactivating process in addition to a fast, Hodgkin-Huxley type inactivation. The time constants of the slow process are similar to those of slow outward current inactivation.     

Voltage-dependent sodium channel function is regulated through membrane mechanics.

Cut-open recordings from Xenopus oocytes expressing either nerve (PN1) or skeletal muscle (SkM1) Na(+) channel alpha subunits revealed slow inactivation onset and recovery kinetics of inward current. In contrast, recordings using the macropatch configuration resulted in an immediate negative shift in the voltage-dependence of inactivation and activation, as well as time-dependent shifts in kinetics when compared to cut-open recordings. Specifically, a slow transition from predominantly slow onset and recovery to exclusively fast onset and fast recovery from inactivation occurred. The shift to fast inactivation was accelerated by patch excision and by agents that disrupted microtubule formation. Application of positive pressure to cell-attached macropatch electrodes prevented the shift in kinetics, while negative pressure led to an abrupt shift to fast inactivation. Simultaneous electrophysiological recording and video imaging of the cell-attached patch membrane revealed that the pressure-induced shift to fast inactivation coincided with rupture of sites of membrane attachment to cytoskeleton. These findings raise the possibility that the negative shift in voltage-dependence and the fast kinetics observed normally for endogenous Na(+) channels involve mechanical destabilization. Our observation that the beta1 subunit causes similar changes in function of the Na(+) channel alpha subunit suggests that beta1 may act through interaction with cytoskeleton.     

Tail currents in the myelinated axon of Xenopus laevis suggest a two-open-state Na channel.

Na tail currents in the myelinated axon of Xenopus laevis were measured at -70 mV after steps to -10 mV. The tail currents were biexponential, comprising a fast and a slow component. The time constant of the slow tail component, analyzed in the time window 0.35-0.50 ms, was independent of step duration, and had a value of 0.23 ms. The amplitude, extrapolated back to time 0, varied, however, with step duration. It reached a peak after 0.7 ms and inactivated relatively slowly (at 2.1 ms the absolute value was reduced by approximately 30%). The amplitude of the fast component, estimated by subtracting the amplitude of the slow component from the calculated total tail current amplitude, reached a peak (three times larger than that of the slow component) after 0.5 ms and inactivated relatively fast (at 2.1 ms it was reduced by approximately 65%). The results were explained by a novel Na channel model, comprising two open states bifurcating from a common closed state and with separate inactivating pathways. A voltage-regulated use of the two pathways explains a number of findings reported in the literature.     

Patch recordings from the electrocytes of Electrophorus electricus. Na currents and PNa/PK variability

Sodium currents were recorded in cell-attached and inside-out patches from the innervated membrane of Electrophorus electrocytes. Electrocytes from Sachs and main electric organs were prepared as described by Pasquale et al. (1986. J. Membr. Biol. 93:195.). Maximal currents in the Sachs organ, measured with 1-2 microns diameter patch pipettes and at room temperature, were in the range of 20 to 300 pA (27 patches) and were obtained near +10 mV. This range of current corresponds to approximately 70 to 1,300 channels in a patch. Maximal current in main organ cells also occurred near +10 mV and were in the range of 100 to 400 pA. Delayed K current was observed in a few patches. The inactivation phase of the currents during maintained depolarizations appears to be a single-exponential relaxation. The time constant decreases from 1 ms near -55 mV to a minimum of 0.3 ms near 0 mV, and then gradually increases with stronger depolarization. The mean currents are half inactivated near -90 mV with an apparent voltage dependence of e-fold per 6 mV. No apparent differences were observed in the decay time course or steady-state inactivation of the currents in the same patch before and after excision. From ensemble fluctuation analysis the peak open probability was found to be approximately 0.5 at +25 mV and increased only gradually with larger depolarizations. The single channel conductances were approximately 20 pS with 200 mM Na outside and 200 mM K inside, and 40 pS in 400 mM solutions. Reversal potentials in the 200 Na parallel 200 K solutions ranged from +51 to +94 mV in multichannel patches, corresponding to selectivity ratios PNa/PK from 8 to 43. Large differences in reversal potentials were seen even among patches from the same cell. Several controls rule out obvious sources of error in the reversal potential measurements. It is concluded that there is heterogeneity in the selectivity properties of the Na channels.     

Level of expression controls modes of gating of a K+ channel.

Several distinct subfamilies of K+ channel genes have been discovered by molecular cloning, however, in some cases the structural differences among them do not account for the diversity of K+ current types, ranging from transient A-type to slowly inactivating delayed rectifier-type, as members within each subfamily have been shown to code for K+ channels of different inactivation kinetics and pharmacological properties. We show that a single K+ channel cDNA of the Shaker subfamily (ShH4) can express in Xenopus oocytes not only a transient A-type K+ current but also, upon increased level of expression, slowly inactivating K+ currents with markedly reduced sensitivity to tetraethylammonium. In correlation with the macroscopic currents there are single-channel gating modes ranging from the fast-inactivation mode which underlies the transient A-type current, to slow-inactivation modes characterized by bursts of longer openings, and corresponding to the slowly inactivating macroscopic currents.     

Fast and slow gating of sodium channels encoded by a single mRNA

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.     

Block by internal Mg2+ causes voltage-dependent inactivation of Kv1.5

Internal Mg2+ blocks many potassium channels including Kv1.5. Here, we show that internal Mg2+ block of Kv1.5 induces voltage-dependent current decay at strongly depolarised potentials that contains a component due to acceleration of C-type inactivation after pore block. The voltage-dependent current decay was fitted to a bi-exponential function (tau(fast) and tau(slow)). Without Mg2+, tau(fast) and tau(slow) were voltage-independent, but with 10 mM Mg2+, tau(fast) decreased from 156 ms at +40 mV to 5 ms at +140 mV and tau(slow) decreased from 2.3 s to 206 ms. With Mg2+, tail currents after short pulses that allowed only the fast phase of decay showed a rising phase that reflected voltage-dependent unbinding. This suggested that the fast phase of voltage-dependent current decay was due to Mg2+ pore block. In contrast, tail currents after longer pulses that allowed the slow phase of decay were reduced to almost zero suggesting that the slow phase was due to channel inactivation. Consistent with this, the mutation R487V (equivalent to T449V in Shaker) or increasing external K+, both of which reduce C-type inactivation, prevented the slow phase of decay. These results are consistent with voltage-dependent open-channel block of Kv1.5 by internal Mg2+ that subsequently induces C-type inactivation by restricting K+ filling of the selectivity filter from the internal solution.     

Stationarity of sodium channel gating kinetics in excised patches from neuroblastoma N1E 115.

Na channel gating parameters in a number of preparations are translated along the voltage axis in excised patches compared to cell attached or whole cell recording. The aim of this study is to determine whether these changes in gating behavior continue over an extended period or, rather, develop rapidly on excision with stationary kinetics thereafter. Average currents were constructed from single-channel records from neuroblastoma N1E 115 at various times after excision, excluding the first 5 min, in eight inside-out excised patches. Single exponentials were fitted to the current decay of the average records, and the mean time constant for each patch was determined. Values were plotted as the percentage difference from these means for each patch against time from excision. Collected results show no obvious trend in values from 5 min to 2 h. Kinetics are stationary, and shifts in Na channel gating parameters along the voltage axis seen in excised as compared to whole cell configuration in neuroblastoma must be complete by the first few minutes after excision. Raising the internal Na concentration reduced the single channel current amplitude, confirming that these are Na channels.