BTX modification of Na channels in squid axons. I. State dependence of BTX action

The state dependence of Na channel modification by batrachotoxin (BTX) was investigated in voltage-clamped and internally perfused squid giant axons before (control axons) and after the pharmacological removal of the fast inactivation by pronase, chloramine-T, or NBA (pretreated axons). In control axons, in the presence of 2-5 microM BTX, a repetitive depolarization to open the channels was required to achieve a complete BTX modification, characterized by the suppression of the fast inactivation and a simultaneous 50-mV shift of the activation voltage dependence in the hyperpolarizing direction, whereas a single long-lasting (10 min) depolarization to +50 mV could promote the modification of only a small fraction of the channels, the noninactivating ones. In pretreated axons, such a single sustained depolarization as well as the repetitive depolarization could induce a complete modification, as evidenced by a similar shift of the activation voltage dependence. Therefore, the fast inactivated channels were not modified by BTX. We compared the rate of BTX modification of the open and slow inactivated channels in control and pretreated axons using different protocols: (a) During a repetitive depolarization with either 4- or 100-ms conditioning pulses to +80 mV, all the channels were modified in the open state in control axons as well as in pretreated axons, with a similar time constant of approximately 1.2 s. (b) In pronase-treated axons, when all the channels were in the slow inactivated state before BTX application, BTX could modify all the channels, but at a very slow rate, with a time constant of approximately 9.5 min. We conclude that at the macroscopic level BTX modification can occur through two different pathways: (a) via the open state, and (b) via the slow inactivated state of the channels that lack the fast inactivation, spontaneously or pharmacologically, but at a rate approximately 500-fold slower than through the main open channel pathway.     

Modulation of nerve membrane sodium channels by chemicals

1. Modulations of sodium channel kinetics by grayanotoxins and pyrethroids have been studied using voltage clamped, internally perfused giant axons from crayfish and squid. 2. Grayanotoxin I and alpha-dihydrograyanotoxin II greatly depolarize the nerve membrane through an increase in resting sodium channel permeability to sodium ions. 3. Grayanotoxins modify a fraction of sodium channel population to give rise to a slow conductance increase with little or no inactivation, and the slow conductance-membrane potential curve is shifted toward hyperpolarization. This accounts for the depolarization. 4. The tail current associated with step repolarization during the slow current in grayanotoxins decays with a dual exponential time course. 5. (+)-trans tetramethrin and (+)-trans allethrin also modify a fraction of sodium channel population in generating a slow current, which attains a maximum slowly and decays very slowly during a maintained depolarizing step. The membrane is depolarized only slightly. 6. The tail current associated with step repolarization during the slow current in the pyrethroids is very large in initial amplitude and decays very slowly. 7. The rate at which the sodium channel arrives at the modified open state in the presence of pyrethroids is expressed by a dual exponential function, and the slow phase disappears following removal of the sodium inactivation mechanism by internal perfusion of pronase. 8. A kinetic model is proposed to account for the actions of both grayanotoxins and pyrethroids on sodium channels. Both chemicals interact with the channel at both open and closed states to yield a modified open state which results in a slow sodium current.     

Kinetics of interaction of disopyramide with the cardiac sodium channel: Fast dissociation from open channels at normal rest potentials

Block of cardiac sodium channels is enhanced by repetitive depolarization. It is not clear whether the changes in drug binding result from a change in affinity that is dependent on voltage or on the actual state of the channel. This question was examined in rabbit ventricular myocytes by analyzing the kinetics of block of single sodium channel currents with normal gating kinetics or channels with inactivation and deactivation slowed by pyrethrin toxins. At −20 and −40 mV, disopyramide 100 μm blocked the unmodified channel. Mean open time decreased45 and34% at −20 and −40 mV during exposure to disopyramide. Exposure of cells to the pyrethrin toxins deltamethrin or fenvalrate caused at least a tenfold increase in mean open time, and prominent tail currents could be recorded at the normal resting potential. The association rate constant of disopyramide for the normal and modified channel at −20 mV was similar, ∼10×10⁶/m/sec. During exposure to disopyramide, changes in open and closed times and in open channel noise at −80 and −100 mV are consistent with fast block and unblocking events at these potentials. This contrasts with the slow unbinding of drug from resting channels at similar potentials. We conclude that the sodium channel state is a critical determinant of drug binding and unbinding kinetics.     

The action of sodium deoxycholate on Escherichia coli

Sodium deoxycholate is used in a number of bacteriological media for the isolation and classification of gram-negative bacteria from food and the environment. Initial experiments to study the effect of deoxycholate on the growth parameters of Escherichia coli showed an increase in the lag time constant and generation time and a decrease in the growth rate constant and total cell yield of this microorganism. Cell fractionation studies indicated that sodium deoxycholate at levels used in bacteriological media interferes with the incorporation of [U-14C]glucose into the cold-trichloroacetic acid-soluble, ethanol-soluble, and trypsin-soluble cellular fractions of E. coli. Finally, sodium deoxycholate interfered with the flagellation and motility of Proteus mirabilis and E. coli. It would appear then that further improvement of the deoxycholate medium may be in order.     

Na channel activation gate modulates slow recovery from use-dependent block by local anesthetics in squid giant axons.

The time course of recovery from use-dependent block of sodium channels caused by local anesthetics was studied in squid axons. In the presence of lidocaine or its quaternary derivatives, QX-222 and QX-314, or 9-aminoacridine (9-AA), recovery from use-dependent block occurred in two phases: a fast phase and a slow phase. Only the fast phase was observed in the presence of benzocaine. The fast phase had a time constant of several milliseconds and resembled recovery from the fast Na inactivation in the absence of drug. Depending on the drug present, the magnitude of the time constant of the slow phase varied (for example at -80 mV): lidocaine, 270 ms; QX-222, 4.4 s; QX-314, 17 s; and 9-AA, 14 s. The two phases differed in the voltage dependence of recovery time constants. When the membrane was hyperpolarized, the recovery time constant for the fast phase was decreased, whereas that for the slow phase was increased for QX-compounds and 9-AA or unchanged for lidocaine. The fast phase is interpreted as representing the unblocked channels recovering from the fast Na inactivation, and the slow phase as representing the bound and blocked channels recovering from the use-dependent block accumulated by repetitive depolarizing pulse. The voltage dependence of time constants for the slow recovery is consistent with the m-gate trapping hypothesis. According to this hypothesis, the drug molecule is trapped by the activation gate (the m-gate) of the channel. The cationic form of drug molecule leaves the channel through the hydrophilic pathway, when the channel is open. However, lidocaine, after losing its proton, may leave the closed channel rapidly through the hydrophobic pathway.     

Effects of yohimbine on squid axons.

Yohimbine, an indolealkylamine alkaloid, reduces the amplitude of the sodium current in the squid giant axon. For doses that reduce sodium current amplitude by up to 50%, there is no significant change in the kinetics or in any of the voltage-dependent parameters associated with sodium channels. The effective equilibrium constant for yohimbine binding to the sodium channel is 3 x 10(-4) M. Repetitive depolarizing pulses increase the inhibition of squid axon sodium current by yohimbine. This use-dependent inhibition is enhanced by increasing the concentration of yohimbine, by increasing the frequency of pulsing, and by increasing the magnitude or the duration of depolarization. It is reduced by hyperpolarizing prepulses. This behavior can be explained by a model wherein yohimbine binds more readily to open sodium channels than to closed sodium channels and wherein the Hodgkin-Huxley kinetic parameters are modified by the binding of the drug. This type of model may also explain the tonic and use-dependent inhibition previously described by others for local anesthetics.     

A molecular link between activation and inactivation of sodium channels

A pair of tyrosine residues, located on the cytoplasmic linker between the third and fourth domains of human heart sodium channels, plays a critical role in the kinetics and voltage dependence of inactivation. Substitution of these residues by glutamine (Y1494Y1495/QQ), but not phenylalanine, nearly eliminates the voltage dependence of the inactivation time constant measured from the decay of macroscopic current after a depolarization. The voltage dependence of steady state inactivation and recovery from inactivation is also decreased in YY/QQ channels. A characteristic feature of the coupling between activation and inactivation in sodium channels is a delay in development of inactivation after a depolarization. Such a delay is seen in wild-type but is abbreviated in YY/QQ channels at -30 mV. The macroscopic kinetics of activation are faster and less voltage dependent in the mutant at voltages more negative than -20 mV. Deactivation kinetics, by contrast, are not significantly different between mutant and wild-type channels at voltages more negative than -70 mV. Single-channel measurements show that the latencies for a channel to open after a depolarization are shorter and less voltage dependent in YY/QQ than in wild-type channels; however the peak open probability is not significantly affected in YY/QQ channels. These data demonstrate that rate constants involved in both activation and inactivation are altered in YY/QQ channels. These tyrosines are required for a normal coupling between activation voltage sensors and the inactivation gate. This coupling insures that the macroscopic inactivation rate is slow at negative voltages and accelerated at more positive voltages. Disruption of the coupling in YY/QQ alters the microscopic rates of both activation and inactivation.     

Slow sodium inactivation in nerve after exposure to sulhydryl blocking reagents

Exposure to N-ethylmaleimide (NEM), a reagent that binds covalently to protein sulfhydryl groups, results in a specific reduction in sodium conductance in crayfish axons. Resting potential, the delayed rise in potassium conductance, and the selectivity of the sodium channel are unaffected. Sodium currents are only slightly increased by hyperpolarizing prepulses of up to 50 ms duration, but can be restored to about 70% of their value before treatment if this duration is increased to 300-800 ms. The time to peak sodium current and the time constant of decay of sodium tail currents are unaffected by NEM, suggesting that the sodium activation system remains unaltered. Kinetic studies suggest that NEM reacts with a "slow" sodium inactivation system that is present in normal axons and that may be seen after depolarization produced by lowered the holding potential or increasing the external potassium concentration. NEM also perturbs the fast h inactivation system, and in a potential-dependent manner. At small depolarizations tauh is decreased, while at strong depolarizations it is increased over control values. Experiments with structural analogs of NEM suggest that sulfhydryl block is involved, but do not rule out an action similar to that of local anesthetics, p- Chloromercuriphenylsulfonic acid (PCMBS), another reagent with high specificity for SH groups, also blocks sodium currents, but restoration with prolonged hyperpolarizations is not possible.     

Tetrodotoxin block of sodium channels in rabbit Purkinje fibers. Interactions between toxin binding and channel gating

Tetrodotoxin (TTX) block of cardiac sodium channels was studied in rabbit Purkinje fibers using a two-microelectrode voltage clamp to measure sodium current. INa decreases with TTX as if one toxin molecule blocks one channel with a dissociation constant KD approximately equal to 1 microM. KD remains unchanged when INa is partially inactivated by steady depolarization. Thus, TTX binding and channel inactivation are independent at equilibrium. Interactions between toxin binding and gating were revealed, however, by kinetic behavior that depends on rates of equilibration. For example, frequent suprathreshold pulses produce extra use-dependent block beyond the tonic block seen with widely spaced stimuli. Such lingering aftereffects of depolarization were characterized by double-pulse experiments. The extra block decays slowly enough (tau approximately equal to 5 s) to be easily separated from normal recovery from inactivation (tau less than 0.2 s at 18 degrees C). The amount of extra block increases to a saturating level with conditioning depolarizations that produce inactivation without detectable activation. Stronger depolarizations that clearly open channels give the same final level of extra block, but its development includes a fast phase whose voltage- and time-dependence resemble channel activation. Thus, TTX block and channel gating are not independent, as believed for nerve. Kinetically, TTX resembles local anesthetics, but its affinity remains unchanged during maintained depolarization. On this last point, comparison of our INa results and earlier upstroke velocity (Vmax) measurements illustrates how much these approaches can differ.     

A simple Markov model of sodium channels with a dynamic threshold

Characteristics of action potential generation are important to understanding brain functioning and, thus, must be understood and modeled. It is still an open question what model can describe concurrently the phenomena of sharp spike shape, the spike threshold variability, and the divisive effect of shunting on the gain of frequency-current dependence. We reproduced these three effects experimentally by patch-clamp recordings in cortical slices, but we failed to simulate them by any of 11 known neuron models, including one- and multi-compartment, with Hodgkin-Huxley and Markov equation-based sodium channel approximations, and those taking into account sodium channel subtype heterogeneity. Basing on our voltage-clamp data characterizing the dependence of sodium channel activation threshold on history of depolarization, we propose a 3-state Markov model with a closed-to-open state transition threshold dependent on slow inactivation. This model reproduces the all three phenomena. As a reduction of this model, a leaky integrate-and-fire model with a dynamic threshold also shows the effect of gain reduction by shunt. These results argue for the mechanism of gain reduction through threshold dynamics determined by the slow inactivation of sodium channels.     

Inactivation viewed through single sodium channels

Recordings of the sodium current in tissue-cultured GH3 cells show that the rate of inactivation in whole cell and averaged single channel records is voltage dependent: tau h varied e-fold/approximately 26 mV. The source of this voltage dependence was investigated by examining the voltage dependence of individual rate constants, estimated by maximum likelihood analysis of single channel records, in a five-state kinetic model. The rate constant for inactivating from the open state, rather than closing, increased with depolarization, as did the probability that an open channel inactivates. The rate constant for closing from the open state had the opposite voltage dependence. Both rate constants contributed to the mean open time, which was not very voltage dependent. Both open time and burst duration were less than tau h for voltages up to -20 mV. The slowest time constant of activation, tau m, was measured from whole cell records, by fitting a single exponential either to tail currents or to activating currents in trypsin-treated cells, in which the inactivation was abolished. tau m was a bell-shaped function of voltage and had a voltage dependence similar to tau h at voltages more positive than -35 mV, but was smaller than tau h. At potentials more negative than about -10 mV, individual channels may open and close several times before inactivating. Therefore, averaged single channel records, which correspond with macroscopic current elicited by a depolarization, are best described by a convolution of the first latency density with the autocorrelation function rather than with 1 - (channel open time distribution). The voltage dependence of inactivation from the open state, in addition to that of the activation process, is a significant factor in determining the voltage dependence of macroscopic inactivation. Although the rates of activation and inactivation overlapped greatly, independent and coupled inactivation could not be statistically distinguished for two models examined. Although rates of activation affect the observed rate of inactivation at intermediate voltages, extrapolation of our estimates of rate constants suggests that at very depolarized voltages the activation process is so fast that it is an insignificant factor in the time course of inactivation. Prediction of gating currents shows that an inherently voltage-dependent inactivation process need not produce a conspicuous component in the gating current.     

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.     

Induction of a sodium-dependent depolarization by external calcium removal in human sperm

Removal of external calcium with EGTA (from 2.5 mm to nanomolar levels) caused a remarkable depolarization in human sperm. This depolarization was initially fast. It was followed by a slow phase that brought the Vm to values of over 0 mV in 1-2 min. The slow and sustained phase correlated with a sustained decrease in intracellular calcium. However, calcium removal still induced depolarization in sperm with enhanced intracellular calcium (induced by progesterone), indicating that the sustained depolarization was not caused by a sustained intracellular calcium decrease. The depolarization was reduced as the external sodium content was substituted with choline, indicating that it was due to a sodium current, and was observed in lithium but not in tetramethylammonium-containing medium. In low sodium medium, the addition of sodium after calcium removal induced depolarization to the extent of which slightly increased in 2 min. The depolarization was completely inhibited by external magnesium (Ki = 1.16 mm). The addition of calcium or magnesium to calcium removal-induced depolarized sperm induced hyperpolarization that was inhibited by ouabain and was also prevented in medium without potassium, suggesting that the activity of the electrogenic Na+,K+-ATPase was involved. The conductance activated by calcium removal might unveil the presence of a calcium channel that in the absence of external calcium allows sodium permeation and that in normal conditions might contribute to the resting intracellular calcium concentration.     

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.     

Pharmacological modifications of the sodium channels of frog nerve

Voltage clamp measurements on myelinated nerve fibers show that tetrodotoxin, saxitoxin, and DDT specifically affect the sodium channels of the membrane. Tetrodotoxin and saxitoxin render the sodium channels impermeable to Na ions and to Li ions and probably prevent the opening of individual sodium channels when one toxin molecule binds to a channel. The apparent dissociation constant of the inhibitory complex is about 1 nM for the cationic forms of both toxins. The zwitter ionic forms are much less potent. On the other hand, DDT causes a fraction of the sodium channels that open during a depolarization to remain open for a longer time than is normal. The effect cannot be described as a specific change in sodium inactivation or as a specific change in sodium activation, for both processes continue to govern the opening of the sodium channels and neither process is able to close the channels. The effects of DDT are very similar to those of veratrine.     

Native Pyroglutamation of Huwentoxin-IV: A Post-Translational Modification that Increases the Trapping Ability to the Sodium Channel

Huwentoxin-IV (HWTX-IV), a tetrodotoxin-sensitive (TTX-s) sodium channel antagonist, is found in the venom of the Chinese spider Ornithoctonus huwena. A naturally modified HWTX-IV (mHWTX-IV), having a molecular mass 18 Da lower than HWTX-IV, has also been isolated from the venom of the same spider. By a combination of enzymatic fragmentation and MS/MS de novo sequencing, mHWTX-IV has been shown to have the same amino acid sequence as that of HWTX-IV, except that the N-terminal glutamic acid replaced by pyroglutamic acid. mHWTX-IV inhibited tetrodotoxin-sensitive voltage-gated sodium channels of dorsal root ganglion neurons with an IC₅₀ nearly equal to native HWTX-IV. mHWTX-IV showed the same activation and inactivation kinetics seen for native HWTX-IV. In contrast with HWTX-IV, which dissociates at moderate voltage depolarization voltages (+50 mV, 180000 ms), mHWTX-IV inhibition of TTX-sensitive sodium channels is not reversed by strong depolarization voltages (+200 mV, 500 ms). Recovery of Nav1.7current was voltage-dependent and was induced by extreme depolarization in the presence of HWTX-IV, but no obvious current was elicited after application of mHWTX-IV. Our data indicate that the N-terminal modification of HWTX-IV gives the peptide toxin a greater ability to trap the voltage sensor in the sodium channel. Loss of a negative charge, caused by cyclization at the N-terminus, is a possible reason why the modified toxin binds much stronger. To our knowledge, this is the first report of a pyroglutamic acid residue in a spider toxin; this modification seems to increase the trapping ability of the voltage sensor in the sodium channel.     

Transcainide causes two modes of open-channel block with different voltage sensitivities in batrachotoxin-activated sodium channels.

Transcainide, a complex derivative of lidocaine, blocks the open state of BTX-activated sodium channels from bovine heart and rat skeletal muscle in two distinct ways. When applied to either side of the membrane, transcainide caused discrete blocking events a few hundred milliseconds in duration (slow block), and a concomitant reduction in apparent single-channel amplitude, presumably because of rapid block beyond the temporal resolution of our recordings (fast block). We quantitatively analyzed block from the cytoplasmic side. Both modes of block occurred via binding of the drug to the open channel, approximately followed 1:1 stoichiometry, and were similar for both channel subtypes. For slow block, the blocking rate increased, and the unblocking rate decreased with depolarization, yielding an overall enhancement of block at positive potentials, and suggesting a blocking site at an apparent electrical distance about 45% of the way from the cytoplasmic end of the channel (z delta approximately 0.45). In contrast, the fast blocking mode was only slightly enhanced by depolarization (z delta approximately 0.15). Phenomenologically, the bulky and complex transcainide molecule combines the almost voltage-insensitive blocking action of phenylhydrazine (Zamponi and French, 1994a (companion paper)) with a slow open-channel blocking action that shows a voltage dependence typical of simpler amines. Only the slower blocking mode was sensitive to the removal of external sodium ions, suggesting that the two types of block occur at distinct sites. Dose-response relations were also consistent with independent binding of transcainide to two separate sites on the channel.     

Pyrethroids Differentially Alter Voltage-Gated Sodium Channels from the Honeybee Central Olfactory Neurons

The sensitivity of neurons from the honey bee olfactory system to pyrethroid insecticides was studied using the patch-clamp technique on central ‘antennal lobe neurons’ (ALNs) in cell culture. In these neurons, the voltage-dependent sodium currents are characterized by negative potential for activation, fast kinetics of activation and inactivation, and the presence of cumulative inactivation during train of depolarizations. Perfusion of pyrethroids on these ALN neurons submitted to repetitive stimulations induced (1) an acceleration of cumulative inactivation, and (2) a marked slowing of the tail current recorded upon repolarization. Cypermethrin and permethrin accelerated cumulative inactivation of the sodium current peak in a similar manner and tetramethrin was even more effective. The slow-down of channel deactivation was markedly dependent on the type of pyrethroid. With cypermethrin, a progressive increase of the tail current amplitude along with successive stimulations reveals a traditionally described use-dependent recruitment of modified sodium channels. However, an unexpected decrease in this tail current was revealed with tetramethrin. If one considers the calculated percentage of modified channels as an index of pyrethroids effects, ALNs are significantly more susceptible to tetramethrin than to permethrin or cypermethrin for a single depolarization, but this difference attenuates with repetitive activity. Further comparison with peripheral neurons from antennae suggest that these modifications are neuron type specific. Modeling the sodium channel as a multi-state channel with fast and slow inactivation allows to underline the effects of pyrethroids on a set of rate constants connecting open and inactivated conformations, and give some insights to their specificity. Altogether, our results revealed a differential sensitivity of central olfactory neurons to pyrethroids that emphasize the ability for these compounds to impair detection and processing of information at several levels of the bees olfactory pathway.     

Dynamical characterization of inactivation path in voltage-gated Na+ ion channel by non-equilibrium response spectroscopy

Inactivation path of voltage gated sodium channel has been studied here under various voltage protocols as it is the main governing factor for the periodic occurrence and shape of the action potential. These voltage protocols actually serve as non-equilibrium response spectroscopic tools to study the ion channel in non-equilibrium environment. In contrast to a lot of effort in finding the crystal structure based molecular mechanism of closed-state(CSI) and open-state inactivation(OSI); here our approach is to understand the dynamical characterization of inactivation. The kinetic flux as well as energetic contribution of the closed and open- state inactivation path is compared here for voltage protocols, namely constant, pulsed and oscillating. The non-equilibrium thermodynamic quantities used in response to these voltage protocols serve as improved characterization tools for theoretical understanding which not only agrees with the previously known kinetic measurements but also predict the energetically optimum processes to sustain the auto-regulatory mechanism of action potential and the consequent inactivation steps needed. The time dependent voltage pattern governs the population of the conformational states which when couple with characteristic rate parameters, the CSI and OSI selectivity arise dynamically to control the inactivation path. Using constant, pulsed and continuous oscillating voltage protocols we have shown that during depolarization the OSI path is more favored path of inactivation however, in the hyper-polarized situation the CSI is favored. It is also shown that the re-factorisation of inactivated sodium channel to resting state occurs via CSI path. Here we have shown how the subtle energetic and entropic cost due to the change in the depolarization magnitude determines the optimum path of inactivation. It is shown that an efficient CSI and OSI dynamical profile in principle can characterize the open-state drug blocking phenomena.     

Inhibition of Muscarinic-Coupled Phosphoinositide Hydrolysis by N-Methyl-d-Aspartate Is Dependent on Depolarization via Channel Activation

The intent of this work was to elucidate the mechanism by which N-methyl-D-aspartate (NMDA) receptor agonists inhibit a second messenger system, namely, the stimulation of phosphoinositide (PI) hydrolysis activated by muscarinic cholinergic receptor agonists. NMDA inhibited cholinergic stimulation of PI hydrolysis in a dose- and time-dependent manner. NMDA exerts this effect indirectly through channel activation, because both MK-801 and N-[1-(2-thienyl)cyclohexyl]piperidine (TCP) prevented this action. Prevention of the NMDA effect by removal of sodium, but not calcium, from the incubation buffer suggested that depolarization may be the responsible mechanism. Depolarization alone proved sufficient to inhibit cholinergic activation of PI hydrolysis, because both veratridine and an elevated extracellular potassium level inhibited cholinergic stimulation of PI hydrolysis. The effect of NMDA appeared to require sodium flux through NMDA channels rather than through voltage-dependent sodium channels, because tetrodotoxin failed to inhibit the effect of NMDA. In correlative electrophysiologic experiments, NMDA profoundly inhibited evoked excitatory postsynaptic potentials and population action potentials of CA1 neurons, an effect almost certainly due to depolarization. The dose and time course of the electrophysiologic effects correlated well with the biochemical effects. Taken together, the data support the assertion that NMDA receptor activation inhibits PI hydrolysis by depolarization mediated by sodium flux through NMDA channels.