Gating in iodate-modified single cardiac Na+ channels

Summary Elementary Na⁺ currents were recorded at 19°C during 220-msec lasting step depolarizations in cell-attached and inside-out patches from cultured neonatal rat cardiocytes in order to study the modifying influence of iodate, bromate and glutaraldehyde on single cardiac Na⁺ channels.Iodate (10 mmol/liter) removed Na⁺ inactivation and caused repetitive, burst-like channel activity after treating the cytoplasmic channel surface. In contrast to normal Na⁺ channels under control conditions, iodate-modified Na⁺ channels attain two conducting states, a short-lasting one with a voltage-independent lifetime close to 1 msec and, likewise tested between –50 and +10 mV, a long-lasting one being apparently exponentially dependent on voltage. Channel modification by bromate (10 mmol/liter) and glutaraldehyde (0.5 mmol/liter) also included the occurrence of two open states. Also, burst duration depended apparently exponentially on voltage and increased when shifting the membrane in the positive direction, but there was no evidence for two bursting states. Chemically modified Na⁺ channels retain an apparently normal unitary conductance (12.8±0.5 pS). Of the two substates observed, one of them is remarkable in that it is mostly attained from full-state openings and is very short living in nature; the voltage-independent lifetime was close to 2 msec. Despite removal of inactivation, open probability progressively declined during membrane depolarization. The underlying deactivation process is strongly voltage sensitive but, in contrast to slow Na⁺ inactivation, responds to a voltage shift in the positive direction with a retardation in kinetics. Chemically modified Na⁺ channels exhibit a characteristic bursting state much shorter than in DPI-modified Na⁺ channels, a difference not consistent with the hypothesis of common kinetic properties in noninactivating Na⁺ channels.     

Gating properties of cardiac Na+ channels in cell-free conditions

Summary In patches from neonatal rat heart myocytes, elementary Na⁺ currents were recorded at near threshold potentials in order to compare cardiac Na⁺ channels kinetics in the cell-attached mode with those in the inside-out mode.The transition from cell-attached to cell-free recording conditions caused a small prolongation of the conductive state of about 20%. This appeared within 8 min after, patch excision regardless of the anionic composition (in mmol/liter) at the cytoplasmic membrane surface: 20 Cl^– plus 120 aspartate, 140 Cl^–, or 140 F^–. Prolonged exposure (up to 50 min) to cell-free conditions evoked no additional changes and, specifically, left the monoexponential open-time distribution unchanged. Increased burst activity only developed in the cytoplasmic presence of F^–, indicating that it is this artificial anion which influences reopening, but not the isolation of the Na⁺ channels from their natural environmentper se. The mean number of openings per sequence (increase by a factor of 1.23±0.04) and _decay, of reconstructed macroscopicI _Na (increase by a factor of 1.32±0.06) responded rather weakly to F^–. Cooling from 19 to 9°C accentuated this F^– effect significantly and led, at –65 mV, to pronounced burst activity. Moreover, the combined influence of F^– and cooling induced a second. long-lasting and sometimes dominating open state. It is concluded that isolated cardiac Na⁺ channels largely, preserve their intrinsic kinetic properties when facing a cytoplasmic environment with a quasi-physiological anionic composition.     

Inactivation of cloned Na channels expressed in Xenopus oocytes

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.     

Gating of gap junction channels.

Gap junctional conductance ( gj ) in various species is gated by voltage and intracellular pH (pHi). In amphibian embryos, gj is reduced to half by a 14 mV transjunctional voltage ( Vj ), a change that in fish embryo requires approximately 28 mV. Crayfish septate axon and pairs of dissociated rat myocytes show no voltage dependence of gj over a range of Vj greater than +/- 50 mV. In fish and amphibian blastomeres , gj is steeply decreased by decrease in pHi (n, Hill coefficient: 4.5) and the apparent pKH (7.3) is in the physiological range. In crayfish septate axon the pKH is lower (6.7) and the curve is less steep (n = 2.7). Rises in cytoplasmic Ca can also decrease gj but much higher concentrations are required (greater than 0.1 mM in fish blastomeres). Voltage and pH gates on gap junctions in amphibian embryos appear independent. In squid blastomeres pH gates exhibit some sensitivity to potential, both transjunctional and between inside and outside. A pharmacology of gap junctions is being developed: certain agents block gj directly (aldehydes, alcohols, NEM in crayfish); others block by decreasing pHi (esters that are hydrolyzed by intrinsic esterases, NEM in vertebrates, and, as in the experiments demonstrating the effect of pHi, weak acids). Certain agents block pH sensitivity without affecting voltage dependence (retinoic acid, glutaraldehyde, EEDQ), further indicating separateness of pH and voltage gates. These studies demonstrate a dynamics of gap junctional conductance and variability in gating in a series of possibly homologous membrane channels.     

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.     

Inactivation of single cardiac Na+ channels in three different gating modes.

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.     

Gating currents in Shaker K+ channels. Implications for activation and inactivation models.

We have studied ionic and gating currents in mutant and wild-type Shaker K+ channels to investigate the mechanisms of channel activation and the relationship between the voltage sensor of the channel and its inactivation particle. The turn on of the gating current shows a rising phase, indicating that the hypothetical identical activation subunits are not independent. Hyperpolarizing prepulses indicate that most of the voltage-dependence occurs in the transitions between closed states. The open-to-closed transition is voltage independent, as suggested by the presence of a rising phase in the off gating currents. In Shaker channels showing fast inactivation, the off gating charge is partially immobilized as a result of depolarizing pulses that elicit inactivation. In mutant channels lacking inactivation, the charge is recovered quickly at the end of the pulse. Internal TEA mimics the inactivation particle in its behavior but the charge immobilization is established faster and is complete. We conclude that the activation mechanism cannot be due to the movement of identical independent gating subunits, each undergoing first order transitions, and that the inactivation particle is responsible for charge immobilization in this channel.     

Gating kinetics of batrachotoxin-modified Na+ channels in the squid giant axon. Voltage and temperature effects.

The gating kinetics of batrachotoxin-modified Na+ channels were studied in outside-out patches of axolemma from the squid giant axon by means of the cut-open axon technique. Single channel kinetics were characterized at different membrane voltages and temperatures. The probability of channel opening (Po) as a function of voltage was well described by a Boltzmann distribution with an equivalent number of gating particles of 3.58. The voltage at which the channel was open 50% of the time was a function of [Na+] and temperature. A decrease in the internal [Na+] induced a shift to the right of the Po vs. V curve, suggesting the presence of an integral negative fixed charge near the activation gate. An increase in temperature decreased Po, indicating a stabilization of the closed configuration of the channel and also a decrease in entropy upon channel opening. Probability density analysis of dwell times in the closed and open states of the channel at 0 degrees C revealed the presence of three closed and three open states. The slowest open kinetic component constituted only a small fraction of the total number of transitions and became negligible at voltages greater than -65 mV. Adjacent interval analysis showed that there is no correlation in the duration of successive open and closed events. Consistent with this analysis, maximum likelihood estimation of the rate constants for nine different single-channel models produced a preferred model (model 1) having a linear sequence of closed states and two open states emerging from the last closed state. The effect of temperature on the rate constants of model 1 was studied. An increase in temperature increased all rate constants; the shift in Po would be the result of an increase in the closing rates predominant over the change in the opening rates. The temperature study also provided the basis for building an energy diagram for the transitions between channel states.     

Inactivation of the sodium channel. II. Gating current experiments

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.     

Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.     

Gating of voltage-dependent potassium channels

Activation of voltage-dependent ion channels is primarily controlled by the applied potential difference across the membrane. For potassium channels the Drosophila Shaker channel has served as an archetype of all other potassium channels in studies of activation mechanisms. In the Shaker potassium channel much of the voltage sensitivity is conferred by the S4 transmembrane helix, which contains seven positively charged residues. During gating, the movement of these charges produces gating currents. Mutagenic and fluorescence studies indicate that four of these residues are particularly important and contribute to the majority of gating charge, R362, R365, R368 and R371. The channel is thought to dwell in several closed states prior to opening. Ionic-charge pairing with negatively charged residues in the S2 and S3 helices is thought to be important in regulating these closed states and detailed kinetic models have attempted to define the kinetics and charge of the transitions between these states. Neutral residues throughout the S4 and S5 helices are thought to control late steps in channel opening and may have important roles in modulating the stability of the open state and late closed states. In response to depolarization, the S4 helix is thought to undergo a rotational translation and this movement is also important in studies of the movement of the pore helices, S5 and S6, during opening. This review will examine residues that are important during activation as well as kinetic models that have attempted to quantitatively define the activation pathway of voltage-dependent potassium channels.     

Inactivation of Kv2.1 potassium channels.

We report here several unusual features of inactivation of the rat Kv2.1 delayed rectifier potassium channel, expressed in Xenopus oocytes. The voltage dependence of inactivation was U-shaped, with maximum inactivation near 0 mV. During a maintained depolarization, development of inactivation was slow and only weakly voltage dependent (tau = 4 s at 0 mV; tau = 7 s at +80 mV). However, recovery from inactivation was strongly voltage dependent (e-fold for 20 mV) and could be rapid (tau = 0.27 s at -140 mV). Kv2.1 showed cumulative inactivation, where inactivation built up during a train of brief depolarizations. A single maintained depolarization produced more steady-state inactivation than a train of pulses, but there could actually be more inactivation with the repeated pulses during the first few seconds. We term this phenomenon "excessive cumulative inactivation." These results can be explained by an allosteric model, in which inactivation is favored by activation of voltage sensors, but the open state of the channel is resistant to inactivation.     

N-type Inactivation Features of Kv4.2 Channel Gating

We examined whether the N-terminus of Kv4.2 A-type channels (4.2NT) possesses an autoinhibitory N-terminal peptide domain, which, similar to the one of Shaker, mediates inactivation of the open state. We found that chimeric Kv2.1(4.2NT) channels, where the cytoplasmic Kv2.1 N-terminus had been replaced by corresponding Kv4.2 domains, inactivated relatively fast, with a mean time constant of 120 ms as compared to 3.4 s in Kv2.1 wild-type. Notably, Kv2.1(4.2NT) showed features typically observed for Shaker N-type inactivation: fast inactivation of Kv2.1(4.2NT) channels was slowed by intracellular tetraethylammonium and removed by N-terminal truncation (Δ40). Kv2.1(4.2NT) channels reopened during recovery from inactivation, and recovery was accelerated in high external K⁺. Moreover, the application of synthetic N-terminal Kv4.2 and ShB peptides to inside-out patches containing slowly inactivating Kv2.1 channels mimicked N-type inactivation. Kv4.2 channels, after fractional inactivation, mediated tail currents with biphasic decay, indicative of passage through the open state during recovery from inactivation. Biphasic tail current kinetics were less prominent in Kv4.2/KChIP2.1 channel complexes and virtually absent in Kv4.2Δ40 channels. N-type inactivation features of Kv4.2 open-state inactivation, which may be suppressed by KChIP association, were also revealed by the finding that application of Kv4.2 N-terminal peptide accelerated the decay kinetics of both Kv4.2Δ40 and Kv4.2/KChIP2.1 patch currents. However, double mutant cycle analysis of N-terminal inactivating and pore domains indicated differences in the energetics and structural determinants between Kv4.2 and Shaker N-type inactivation.     

Gating at the selectivity filter of ion channels that conduct Na+ and K+ ions

The NaK channel is a cation selective channel with similar permeability for K⁺ and Na⁺. The available crystallographic structure of wild-type (WT) NaK is usually associated with a conductive state of the channel. Here, potential of mean force for complete conduction events of Na⁺ and K⁺ ions through NaK show that: i), large energy barriers prevent the passage of ions through the WT NaK structure, ii), the barriers are correlated to the presence of a hydrogen bond between Asp-66 and Asn-68, and iii), the structure of NaK mutated to mimic cyclic nucleotide-gated channels conducts Na⁺ and K⁺. These results support the hypothesis that the filter of cation selective channels can adopt at least two different structures: a conductive one, represented by the x-ray structures of the NaK-CNG chimeras, and a closed one, represented by the x-ray structures of the WT NaK.     

Gating of inward-rectifier K+ channels by intracellular pH.

Inward rectifier K+ channels of the Kir1.1 (ROMK) and Kir4.1 subtype are predominantly expressed in epithelial cells where they are responsible for K+ transport across the plasma membrane. Uniquely among the members of the Kir family, these channels are gated by intracellular pH in the physiological range. pH-gating involves structural rearrangements in cytoplasmic domains and the P-loop of the Kir protein. The energy for the gating transition is delivered by protonation of a lysine residue that is located prior to the first transmembrane segment and serves as a 'pH sensor'. The anomalous titration required for lysine operating in the neutral pH range results from its close interaction with two positively charged arginines from the distant N- and C-termini termed the R/K/R triad. Disturbance of this triad as results from a number of point mutations found in patients with hyperprostaglandin E syndrome (HPS) increases the pKa of the pH sensor and results in channels being permanently inactivated under physiological conditions. This article will focus on the mechanism of pH-gating, its implications for the tertiary structure of Kir proteins and on its significance for the pathogenesis of HPS.     

Inactivation of calcium channels

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.     

Gating of Na channels in the rat cortical collecting tubule: effects of voltage and membrane stretch

The gating kinetics of apical membrane Na channels in the rat cortical collecting tubule were assessed in cell-attached and inside-out excised patches from split-open tubules using the patch-clamp technique. In patches containing a single channel the open probability (Po) was variable, ranging from 0.05 to 0.9. The average Po was 0.5. However, the individual values were not distributed normally, but were mainly < or = 0.25 or > or = 0.75. Mean open times and mean closed times were correlated directly and inversely, respectively, with Po. In patches where a sufficient number of events could be recorded, two time constants were required to describe the open-time and closed-time distributions. In most patches in which basal Po was < 0.3 the channels could be activated by hyperpolarization of the apical membrane. In five such patches containing a single channel hyperpolarization by 40 mV increased Po by 10-fold, from 0.055 +/- 0.023 to 0.58 +/- 0.07. This change reflected an increase in the mean open time of the channels from 52 +/- 17 to 494 +/- 175 ms and a decrease in the mean closed time from 1,940 +/- 350 to 336 +/- 100 ms. These responses, however, could not be described by a simple voltage dependence of the opening and closing rates. In many cases significant delays in both the activation by hyperpolarization and deactivation by depolarization were observed. These delays ranged from several seconds to several tens of seconds. Similar effects of voltage were seen in cell-attached and excised patches, arguing against a voltage-dependent chemical modification of the channel, such as a phosphorylation. Rather, the channels appeared to switch between gating modes. These switches could be spontaneous but were strongly influenced by changes in membrane voltage. Voltage dependence of channel gating was also observed under whole-cell clamp conditions. To see if mechanical perturbations could also influence channel kinetics or gating mode, negative pressures of 10-60 mm Hg were applied to the patch pipette. In most cases (15 out of 22), this maneuver had no significant effect on channel behavior. In 6 out of 22 patches, however, there was a rapid and reversible increase in Po when the pressure was applied. In one patch, there was a reversible decrease. While no consistent effects of pressure could be documented, membrane deformation could contribute to the variation in Po under some conditions.