Characterization of the gating conformational changes in the felbamate binding site in NMDA channels

The anticonvulsant effect of felbamate (FBM) is ascribable to inhibition of N-methyl-d-aspartate (NMDA) currents. Using electrophysiological studies in rat hippocampal neurons to examine the kinetics of FBM binding to and unbinding from the NMDA channel, we show that FBM modifies NMDA channel gating via a one-to-one binding stoichiometry and has quantitatively the same enhancement effect on NMDA and glycine binding to the NMDA channel. Moreover, the binding rates of FBM to the closed and the open/desensitized NMDA channels are 187.5 and 4.6 x 10(4) M(-1) s(-1), respectively. The unbinding rates of FBM from the closed and the open/desensitized NMDA channels are approximately 6.2 x 10(-2) and approximately 3.1 s(-1), respectively. From the binding and unbinding rate constants, apparent dissociation constants of approximately 300 and approximately 70 microM could be calculated for FBM binding to the closed and the open/desensitized NMDA channels, respectively. The slight (approximately fourfold) difference in FBM binding affinity to the closed and to the open/desensitized NMDA channels thus is composed of much larger differences in the binding and unbinding kinetics (approximately 250- and approximately 60-fold difference, respectively). These findings suggest that the effects of NMDA and glycine binding coalesce or are interrelated before or at the actual activation gate, and FBM binding seems to modulate NMDA channel gating at or after this coalescing point. Moreover, the entrance zone of the FBM binding site very likely undergoes a much larger conformational change along the gating process than that in the binding region(s) of the binding site. In other words, the FBM binding site becomes much more accessible to FBM with NMDA channel activation, although the spatial configurations of the binding ligand(s) for FBM themselves are not altered so much along the gating process.     

Two blocking sites of amino-adamantane derivatives in open N-methyl-D-aspartate channels.

Using whole-cell patch-clamp techniques, we studied the blockade of open N-methyl-D-aspartate (NMDA) channels by amino-adamantane derivatives (AADs) in rat hippocampal neurons acutely isolated by the vibrodissociation method. The rapid concentration-jump technique was used to replace superfusion solutions. A kinetic analysis of the interaction of AAD with open NMDA channels revealed fast and slow components of their blockade and recovery. Mathematical modeling showed that these kinetic components are evidence for two distinct blocking sites of AADs in open NMDA channels. A comparative analysis of different simplest models led us to conclude that these AAD blocking sites can be simultaneously occupied by two blocker molecules. The voltage dependence of the AAD block suggested that both sites were located deep in the channel pore.     

Open channel block and alteration of N-methyl-D-aspartic acid receptor gating by an analog of phencyclidine.

We investigated inhibition of the N-methyl-D-aspartic acid (NMDA) receptor-channel complex by N-ethyl-1,4,9, 9alpha-tetrahydro-4alphaR-cis-4alphaH-fluoren-++ +4alpha-amine (NEFA), a structural analog of phencyclidine (PCP). Using the whole-cell recording technique, we demonstrated that NEFA inhibits NMDA responses with an IC50 of 0.51 microM at -66 mV. We determined that NEFA binds to the open channel, and subsequently the channel can close and trap the blocker. Once the channel has closed, NEFA is unable to dissociate until the channel reopens. Single-channel recordings revealed that NEFA reduces the mean open time of single NMDA-activated channels in a concentration-dependent manner with a forward blocking rate (k+) of 39.9 microM-1 s-1. A computational model of antagonism by NEFA was developed and constrained using kinetic measurements of single-channel data. By multiple criteria, only models in which blocker binding in the channel causes a change in receptor operation adequately fit or predicted whole-cell data. By comparing model predictions and experimental measurements of NEFA action at a high NMDA concentration, we determined that NEFA affects receptor operation through an influence on channel gating. We conclude that inhibition of NMDA receptors by PCP-like blockers involves a modification of channel gating as well as block of current flow through the open channel.     

Gating Models of the Anomalous Mole-Fraction Effect of Single-Channel Current in <Emphasis Type="Italic">Chara</Emphasis>

The dependence of single-channel current on the Tl+/K+ mole fraction exhibiting a minimum at [Tl+]/[K+] of about 1:15 is proportional to open probability in bursts. Five models are suggested to explain modulation of gating by the Tl+/K+ ratio. Three models start from a channel with 4 identical subunits, each with an allosteric binding site for K+ or Tl+. In the first model, ion binding is directly observable as a transition from one Markov state to another. This model can explain the dependence of the apparent single-channel current on Tl+ concentrations. However, the predicted linear dependence on ion concentrations of the apparent rate constants was not observed in measurements in 25 or 250 mM KNO3 and 250 mM Tl NO3. The second model can overcome this problem by introducing saturation kinetics for ion binding. In the third model, gating is caused by inherent vibrations of the protein, and the rate constants of the related transitions depend on the occupation of the allosteric sites. The fourth model is based on the foot-in-the-door approach with the essential feature that two K+ ions in the selectivity filter are necessary to keep the pore radius suitable for K+ ions. The fifth model is also a foot-in-the-door model, but non-Markovian because, similar to model 3, it is assumed that the conformation of the protein (and thus the rate constants of the Markov model of the time series) depends on the force exerted by the temporal average over the states of a Markov model of ion occupation. These ions may reside in the pore itself or outside.     

Conserved structural and functional control of N-methyl-D-aspartate receptor gating by transmembrane domain M3

The molecular events controlling glutamate receptor ion channel gating are complex. The movement of transmembrane domain M3 within N-methyl-d-aspartate (NMDA) receptor subunits has been suggested to be one structural determinant linking agonist binding to channel gating. Here we report that covalent modification of NR1-A652C or the analogous mutation in NR2A, -2B, -2C, or -2D by methanethiosulfonate ethylammonium (MT-SEA) occurs only in the presence of glutamate and glycine, and that modification potentiates recombinant NMDA receptor currents. The modified channels remain open even after removing glutamate and glycine from the external solution. The degree of potentiation depends on the identity of the NR2 subunit (NR2A < NR2B < NR2C,D) inversely correlating with previous measurements of channel open probability. MTSEA-induced modification of channels is associated with increased glutamate potency, increased mean single-channel open time, and slightly decreased channel conductance. Modified channels are insensitive to the competitive antagonists D-2-amino-5-phosphonovaleric acid (APV) and 7-Cl-kynurenic acid, as well as allosteric modulators of gating (extracellular protons and Zn(2+)). However, channels remain fully sensitive to Mg(2+) blockade and partially sensitive to pore block by (+)MK-801, (-)MK-801, ketamine, memantine, amantadine, and dextrorphan. The partial sensitivity to (+)MK-801 may reflect its ability to stimulate agonist unbinding from MT-SEA-modified receptors. In summary, these data suggest that the SYTANLAAF motif within M3 is a conserved and critical determinant of channel gating in all NMDA receptors.     

Kinetic analysis of phasic inhibition of neuronal sodium currents by lidocaine and bupivacaine.

Phasic ("use-dependent") inhibition of sodium currents by the tertiary amine local anesthetics, lidocaine and bupivacaine, was observed in voltage-clamped node of Ranvier of the toad, Bufo marinus. Local anesthetics were assumed to inhibit sodium channels through occupation of a binding site with 1:1 stoichiometry. A three-parameter empirical model for state-dependent anesthetic binding to the Na channel is presented: this model includes two discrete parameters that represent the time integrals of binding and unbinding reactions during a depolarizing pulse, and one continuous parameter that represents the rate of unbinding of drug between pulses. The change in magnitude of peak sodium current during a train of depolarizing pulses to 0 mV was used as an assay of the extent of anesthetic binding at discrete intervals; estimates of model parameters were made by applying a nonlinear least-squares algorithm to the inhibition of currents obtained at two or more depolarizing pulse rates. Increasing the concentration of drug increased the rate of binding but had little or no effect on unbinding, as expected for a simple bimolecular reaction. The dependence of the model parameters on pulse duration was assessed for both drugs: as the duration of depolarizing pulses was increased, the fraction of channels binding drug during each pulse became significantly larger, whereas the fraction of occupied channels unbinding drug remained relatively constant. The rate of recovery from block between pulses was unaffected by pulse duration or magnitude. The separate contributions of open (O) and inactivated (I) channel binding of drug to the net increase in block per pulse were assessed at 0 mV: for lidocaine, the forward binding rate ko was 1.3 x 10(5) M-1 s-1, kl was 2.4 x 10(4) M-1 s-1; for bupivacaine, ko was 2.5 x 10(5) M-1 s-1, kl was 4.4 x 10(4) M-1 s-1. These binding rates were similar to those derived from time-dependent block of maintained Na currents in nodes where inactivation was incomplete due to treatment with chloramine-T. The dependence of model parameters on the potential between pulses (holding potential) was examined. All three parameters were found to be nearly independent of holding potential from -70 to -100 mV. These results are discussed with respect to established models of dynamic local anesthetic-Na channel interactions.     

Blockade of GABAA receptor channels by niflumic acid prevents agonist dissociation

The modulation by the nonsteroidal anti-inflammatory drug niflumic acid (NFA) of the GABAA receptor-mediated currents was studied in acutely isolated cerebellar Purkinje cells using the whole-cell recording and fast drug application system. At concentrations of 3–300 μM NFA potentiated GABA (2 μM)-activated currents, and at concentrations of 1–3 mM NFA blocked these responses. The NFA-induced block was strongly voltage-dependent. Analysis of the voltage dependence of the block suggests that the blocking action of NFA is a result of NFA binding at the site located within GABAA channel pore. The termination of GABA and NFA application was followed by a transient increase of the inward current — “tail” current. These data suggest that NFA acts as a sequential open channel blocker, which prevents dissociation of agonist while the channel is blocked. The tail current develops because, prior to dissociation of agonist, the channels that are in the blocked state must return to the close state via the open state. The tail currents were compared in the presence and absence of gabazine, a competitive antagonist that also allosterically inhibits GABA_A receptors. Application of gabazine only during development of tail current did not change neither amplitude nor time course of this current, while noncompetitive antagonists picrotoxin and penicillin blocked it. Protection of tail current from gabazine block indicates that GABA cannot dissociate from the open-blocked state and the agonist was trapped on the receptor while the channel was open. Trapping was specific for the agonist, because the positive allosteric modulator zolpidem (benzodiazepine site agonist) was able to potentiate the tail current in the absence of GABA in the external solution. Our observations provide a model-independent functional support of the hypothesis that open channel block of GABAA channels by NFA prevents an escape of the agonist from its binding sites.     

Molecular size and hydrophobicity as factors which determine the efficacy of the blocking action of amino-adamantane derivatives on NMDA channels

Neurons isolated from the CA-1 region of rat hippocampal slices by the "vibrodissociation" method were voltage-clamped in the whole cell configuration. The currents through NMDA channels were recorded in response to rapid application (solution exchange time <30 ms) of 100 microM aspartate (ASP) in a Mg2+-free solution in the presence of 3 microM glycine. When added to the ASP solution, amantadine as well as other amino-adamantane derivatives (AAD) produced an open-channel blockade of NMDA channels. Membrane hyperpolarization enhanced the AAD block. The affinity between NMDA channels and AAD was different for various AAD. The analysis of the experimental data led us to conclude that this affinity depended both on the molecular size of the blocker (calculated using HyperChem molecular modeling program) and on the blocker's hydrophobicity (calculated according to Hansch and Leo, 1979). The affinity between NMDA channels and AAD diminished with an increase in molecular size and raised with an increase in blocker's hydrophobicity. We propose an empirical equation which describes the dependence of affinity on the size and hydrophobicity of the blocker. The estimated critical diameter of the NMDA channel pore where the AAD blocking site is located proved to be about 17 A.     

hERG K+ channel blockade by the antipsychotic drug thioridazine: An obligatory role for the S6 helix residue F656

The phenothiazine antipsychotic agent thioridazine has been linked with prolongation of the QT interval on the electrocardiogram, ventricular arrhythmias, and sudden death. Although thioridazine is known to inhibit cardiac hERG K(+) channels there is little mechanistic information on this action. We have investigated in detail hERG K(+) channel current (I(hERG)) blockade by thioridazine and identified a key molecular determinant of blockade. Whole-cell I(hERG) measurements were made at 37 degrees C from human embryonic kidney (HEK-293) cells expressing wild-type and mutant hERG channels. Thioridazine inhibited I(hERG) tails at -40mV following a 2s depolarization to +20mV with an IC(50) value of 80nM. Comparable levels of I(hERG) inhibition were seen with physiological command waveforms (ventricular and Purkinje fibre action potentials). Thioridazine block of I(hERG) was only weakly voltage-dependent, though the time dependence of I(hERG) inhibition indicated contingency of blockade upon channel gating. The S6 helix point mutation F656A almost completely abolished, and the Y652A mutation partially attenuated, I(hERG) inhibition by thioridazine. In summary, thioridazine is one of the most potent hERG K(+) channel blockers amongst antipsychotics, exhibiting characteristics of a preferential open/activated channel blocker and binding at a high affinity site in the hERG channel pore.     

Comparison of the effects of internal TEA+ and Cs+ on potassium current in squid giant axons.

Internal tetraethylammonium (TEA) and cesium ions block outward potassium current in nerve membrane in a voltage-dependent manner. Blockade with Cs+ occurs virtually instantaneously after membrane depolarization, whereas blockade with TEA+ occurs after a delay. The latter result suggested to Armstrong (1966, J. Gen. Physiol., 50:279-293; 1969, J. Gen. Physiol., 54:553-575) that potassium channels must open before TEA+ blockade can occur, which is in contrast to Cs+ blockade, which appears to be independent of channel gating. The results in this study concerning the effect of TEA+ on inward (tail) current argue against the Armstrong model. Specifically, TEA+ (partially) blocks inward current without altering the tail current time constant. This result indicates that TEA+ can occupy its binding site within the channel whether or not the channel gates are open. This alternative hypothesis can describe both the steady-state and time-dependent components of TEA+ blockade.     

Study functional architecture of NMDA receptor channels with their blockers

Blockade of ionic currents through NMDA receptor channels in acutely isolated rat hippocampal neurons by tetraalkylammonium compounds, 9-aminoacridine and Mg2+ was studied using whole-cell patch-clamp technique. The currents through NMDA channels were elicited by 100 microM aspartate application in a Mg(2+)-free 3 microM glycine-containing solution. An analysis of the kinetics, charge transfer and dependencies of the stationary current inhibition on the membrane potential and the agonist and the blocker concentrations showed that the blockers affect NMDA channel closure, desensitization and the agonist dissociation in different ways. The size of the blocker proved to be the determinant of the blocker action on the NMDA channel gating machinery: large blockers prevented the channel closure and/or desensitization, smaller ones only partly affected these processes, while the smallest did not affect at all. It was shown that the apparent blocker affinity to the channel, 1/IC50, depended not only on the microscopic dissociation constant, Kd, but also on the number of the blocker binding sites, their mutual dependence, and, which is much more important, on the blocker interaction with the channel gating machinery. Based upon the data obtained, there was advanced hypotheses on the NMDA channel geometry and the structure of its gating machinery. The diameter of the channel at the level of the activation gate was estimated as 11 A.     

Stereoselective block of a human cardiac potassium channel (Kv1.5) by bupivacaine enantiomers.

Stereoselective drug-channel interactions may help to elucidate the molecular basis of voltage-gated potassium channel block by local anesthetic drugs. We studied the effects of the enantiomers of bupivacaine on a cloned human cardiac potassium channel (hKv1.5). This channel was stably expressed in a mouse Ltk- cell line and studied using the whole-cell configuration of the patch-clamp technique. Both enantiomers modified the time course of this delayed rectifier current. Exposure to 20 microM of either S(-)-bupivacaine or R(+)-bupivacaine did not modify the activation time constant of the current, but reduced the peak outward current and induced a subsequent exponential decline of current with time constants of 18.7 +/- 1.1 and 10.0 +/- 0.9 ms, respectively. Steady-state levels of block (assessed with 250-ms depolarizing pulses to +60 mV) averaged 30.8 +/- 2.5% (n = 6) and 79.5 +/- 3.2% (n = 6) (p < 0.001), for S(-)- and R(+)-bupivacaine, respectively. The concentration dependence of hKv1.5 inhibition revealed apparent KD values of 27.3 +/- 2.8 and 4.1 +/- 0.7 microM for S(-)-bupivacaine and R(+)-bupivacaine, respectively, with Hill coefficients close to unity, suggesting that binding of one enantiomer molecule per channel was sufficient to block potassium permeation. Analysis of the rate constants of association (k) and dissociation (l) yielded similar values for l (24.9 s-1 vs. 23.6 s-1 for S(-)- and R(+)-bupivacaine, respectively) but different association rate constants (1.0 x 10(6) vs. 4.7 x 10(6) M-1 s-1 for S(-)- and R(+)-bupivacaine, respectively). Block induced by either enantiomer displayed a shallow voltage dependence in the voltage range positive to 0 mV, i.e., where the channel is fully open, consistent with an equivalent electrical distance delta of 0.16 +/- 0.01. This suggested that at the binding site, both enantiomers of bupivacaine experienced 16% of the applied transmembrane electrical field, referenced to the inner surface. Both bupivacaine enantiomers reduced the tail current amplitude recorded on return to -40 mV and slowed their time course relative to control, resulting in a "crossover" phenomenon.(ABSTRACT TRUNCATED AT 400 WORDS)     

Blocker state dependence and trapping in hyperpolarization-activated cation channels: evidence for an intracellular activation gate

Hyperpolarization-activated cation currents (I(h)) are key determinants of repetitive electrical activity in heart and nerve cells. The bradycardic agent ZD7288 is a selective blocker of these currents. We studied the mechanism for ZD7288 blockade of cloned I(h) channels in excised inside-out patches. ZD7288 blockade of the mammalian mHCN1 channel appeared to require opening of the channel, but strong hyperpolarization disfavored blockade. The steepness of this voltage-dependent effect (an apparent valence of approximately 4) makes it unlikely to arise solely from a direct effect of voltage on blocker binding. Instead, it probably indicates a differential affinity of the blocker for different channel conformations. Similar properties were seen for ZD7288 blockade of the sea urchin homologue of I(h) channels (SPIH), but some of the blockade was irreversible. To explore the molecular basis for the difference in reversibility, we constructed chimeric channels from mHCN1 and SPIH and localized the structural determinant for the reversibility to three residues in the S6 region likely to line the pore. Using a triple point mutant in S6, we also revealed the trapping of ZD7288 by the closing of the channel. Overall, the observations led us to hypothesize that the residues responsible for ZD7288 block of I(h) channels are located in the pore lining, and are guarded by an intracellular activation gate of the channel.     

Evaluation and comparison of ion permeation and agonist selectivities for N-methyl-d-aspartate receptor channels with different subunit compositions in bilayer lipid membranes based on integrated single-channel currents

To quantify the ion-permeation ability of the recombinant epsilon1-4/zeta1 channel activated by agonists, the magnitude of agonist-induced integrated single-channel currents for the epsilon2-4/zeta1 N-methyl-d-aspartate (NMDA) channels in bilayer lipid membranes (BLMs) was evaluated electrochemically based on the single-channel recordings. The recombinant epsilon2-4/zeta1 channels were purified from Chinese hamster ovary cells expressing each channel and incorporated in BLMs formed by the tip-dip method. Three typical agonists, l-glutamate, NMDA, and (2S, 3R, 4S) isomer of 2-(carboxycyclopropyl)glycine (l-CCG-IV), were investigated at a concentration of 50 microM. The magnitude of l-glutamate-induced integrated current was found to depend on the epsilon-subunit composition and to increase in the order of epsilon2/zeta1 > epsilon1/zeta1 approximately epsilon4/zeta1 > epsilon3/zeta1, which differs from that of the reported binding affinities (EC(50)) between l-glutamate and each channel type. On the other hand, the magnitude of the integrated currents induced by NMDA and l-CCG-IV did not vary among the four channel types. The order of agonist selectivity toward the epsilon2-4/zeta1 channels in terms of the magnitude of the integrated current was l-glutamate > l-CCG-IV approximately NMDA for the epsilon2/zeta1 channel, l-CCG-IV > NMDA > l-glutamate for the epsilon3/zeta1 channel, and l-CCG-IV approximately l-glutamate > NMDA for the epsilon4/zeta1 channel, suggesting that the agonist selectivity also depends on the epsilon-subunit composition. The present study shows that each epsilon1-4/zeta1 channel has its own ability of ion permeation, i.e., its own signal transduction ability, which is not parallel to its binding ability.     

Modulation of 4-AP block of a mammalian A-type K channel clone by channel gating and membrane voltage.

We examined the state-, voltage-, and time dependences of interaction between 4-AP and a mammalian A-type K channel clone (rKv1.4) expressed in Xenopus oocytes using whole-cell and single-channel recordings. 4-AP blocked rKv1.4 from the cytoplasmic side of the membrane. The development of block required channel opening. Block was potentiated by removing the fast inactivation gate of the channel (deletion mutant termed "Del A"). A short-pulse train that activated rKv1.4 without inactivation induced more block by 4-AP than a long pulse that activated and then inactivated the channel. These observations suggest that both activation and inactivation gates limit the binding of 4-AP to the channel. Unblock of 4-AP also occurred during channel opening, because unblock required depolarization and was accelerated by more frequent or longer depolarization pulses (use-dependent unblock). Analysis of the concentration dependence of rate of block development indicated that 4-AP blocked rKv1.4 with slow kinetics (at -20 mV, binding and unbinding rate constants were 3.2 mM-1 s-1 and 4.3 s-1). This was consistent with single-channel recordings: 4-AP induced little or no changes in the fast kinetics of opening and closing within bursts, but shortened the mean burst duration and, more importantly, reduced the probability of channel opening by depolarization. Depolarization might decrease the affinity of 4-AP binding site in the open channel, because stronger depolarization reduced the degree of steady-state block by 4-AP. Furthermore, after 4-AP block had been established at a depolarized holding voltage, further depolarization induced a time-dependent unblock. Our data suggest that 4-AP binds to and unbinds from open rKv1.4 channels with slow kinetics, with the binding site accessibility controlled by the channel gating apparatus and binding site affinity modulated by membrane voltage.     

Dependence of the concentration of the demi-maximal action of a channel blocker on the agonist concentration

Based on the analysis of kinetic scheme of blocking of open channels at any number of blocker binding sites, the dependence of current on blocker concentration was found. A variant of this dependence for a trapping blocker was also found. The restrictions of the applicability of the Hill equation and the necessity of taking into account the dependence of the concentration of demi-maximal blocker action (IC50) on the concentration of agonist were shown.     

Probing an open CFTR pore with organic anion blockers

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ion channel that conducts Cl- current. We explored the CFTR pore by studying voltage-dependent blockade of the channel by two organic anions: glibenclamide and isethionate. To simplify the kinetic analysis, a CFTR mutant, K1250A-CFTR, was used because this mutant channel, once opened, can remain open for minutes. Dose-response relationships of both blockers follow a simple Michaelis-Menten function with K(d) values that differ by three orders of magnitude. Glibenclamide blocks CFTR from the intracellular side of the membrane with slow kinetics. Both the on and off rates of glibenclamide block are voltage dependent. Removing external Cl- increases affinity of glibenclamide due to a decrease of the off rate and an increase of the on rate, suggesting the presence of a Cl- binding site external to the glibenclamide binding site. Isethionate blocks the channel from the cytoplasmic side with fast kinetics, but has no measurable effect when applied extracellularly. Increasing the internal Cl- concentration reduces isethionate block without affecting its voltage dependence, suggesting that Cl- and isethionate compete for a binding site in the pore. The voltage dependence and external Cl- concentration dependence of isethionate block are nearly identical to those of glibenclamide block, suggesting that these two blockers may bind to a common binding site, an idea further supported by kinetic studies of blocking with glibenclamide/isethionate mixtures. By comparing the physical and chemical natures of these two blockers, we propose that CFTR channel has an asymmetric pore with a wide internal entrance and a deeply embedded blocker binding site where local charges as well as hydrophobic components determine the affinity of the blockers.     

Gating of Shaker K+ channels: I. Ionic and gating currents.

Ionic and gating currents from noninactivating Shaker B K+ channels were studied with the cut-open oocyte voltage clamp technique and compared with the macropatch clamp technique. The performance of the cut-open oocyte voltage clamp technique was evaluated from the electrical properties of the clamped upper domus membrane, K+ tail current measurements, and the time course of K+ currents after partial blockade. It was concluded that membrane currents less than 20 microA were spatially clamped with a time resolution of at least 50 microseconds. Subtracted, unsubtracted gating currents with the cut-open oocyte voltage clamp technique and gating currents recorded in cell attached macropatches had similar properties and time course, and the charge movement properties directly obtained from capacity measurements agreed with measurements of charge movement from subtracted records. An accurate estimate of the normalized open probability Po(V) was obtained from tail current measurements as a function of the prepulse V in high external K+. The Po(V) was zero at potentials more negative than -40 mV and increased sharply at this potential, then increased continuously until -20 mV, and finally slowly increased with voltages more positive than 0 mV. Deactivation tail currents decayed with two time constants and external potassium slowed down the faster component without affecting the slower component that is probably associated with the return between two of the closed states near the open state. In correlating gating currents and channel opening, Cole-Moore type experiments showed that charge moving in the negative region of voltage (-100 to -40 mV) is involved in the delay of the conductance activation but not in channel opening. The charge moving in the more positive voltage range (-40 to -10 mV) has a similar voltage dependence to the open probability of the channel, but it does not show the gradual increase with voltage seen in the Po(V).     

Preferential closed channel blockade of HERG potassium currents by chemically synthesised BeKm-1 scorpion toxin

The scorpion toxin peptide BeKm-1 was synthesised by fluorenylmethoxycarbonyl solid phase chemistry and folded by air oxidation. The peptide's effects on heterologous human ether-a-go-go-related gene potassium current (I(HERG)) in HEK293 cells were assessed using 'whole-cell' patch clamp. Blockade of I(HERG) by BeKm-1 was concentration-dependent, temperature-dependent, and rapid in onset and reversibility. Blockade also exhibited inverse voltage dependence, inverse dependence on duration of depolarisation, and reverse use- and frequency-dependence. Blockade by BeKm-1 and recombinant ergtoxin, another scorpion toxin known to block HERG, differed in their recovery from HERG current inactivation elicited by strong depolarisation and in their ability to block HERG when the channels were already activated. We conclude that synthetic BeKm-1 toxin blocks HERG preferentially through a closed (resting) state channel blockade mechanism, although some open channel blockade also occurs.     

Effect of ajmaline on transient outward current in rat ventricular myocytes

The mechanism of ajmaline-induced inhibition of the transient outward current (I(to)) has been investigated in right ventricular myocytes of rat using the whole cell patch clamp technique. Ajmaline decreased the amplitude and the time integral of I(to) in a concentration-dependent, but frequency- and use-independent manner. In contrast to the single exponential time course of I(to)-inactivation in control conditions (tau(i) = 37.1 +/- 2.7 ms), the apparent inactivation was fitted by a sum of two exponentials under the effect of ajmaline with concentration-dependent fast and slow components (tau(f) = 11.7 +/- 0.8 ms, tau(s) = 57.6 +/- 2.7 ms at 10 micromol/l) suggesting block development primarily in the open channel state. An improved expression enabling to calculate the association and dissociation rate constants from the concentration dependence of tau(f) and tau(s) was derived and resulted in k(on) = 4.57 x 10(6) +/- 0.32 x 10(6) mol(-1).l.s(-1) and k(off) = 20.12 +/- 5.99 s(-1). The value of K(d) = 4.4 micromol/l calculated as k(off) / k(on) was considerably lower than IC(50) = 25.9 +/- 2.9 micromol/l evaluated from the concentration dependence of the integrals of I(to). Simulations on a simple model combining Hodgkin-Huxley type gating kinetics and drug-channel interaction entirely in open channel state agreed well with the experimental data including the difference between the K(d) and IC(50). According to the model, the fraction of blocked channels increases upon depolarization and declines if depolarization is prolonged. The repolarizing step induces recovery from block with time constant of 52 ms. We conclude that in the rat right ventricular myocytes, ajmaline is an open channel blocker with fast recovery from the block at resting voltage.