The kinetics of local anesthetic blockade of end-plate channels

The effect of the local anesthetic QX222 on the kinetics of miniature end-plate currents (MEPC)and acetylcholine-induced end-plate current fluctuations was studied in voltage-clamped frog cutaneous pectoris neuromuscular junctions. The rate constants for a kinetic scheme of local anesthetic blockage of end-plate channels were calculated from the MEPC decay parameters. At 18 degrees C the blocking rate constant was 1.1 +/- 0.3 x 10(7) exp (-0.009 +/- 0.003 x V)s -1M-1, and the unblocking rate constant was 5.7 +/- 0.6 exp (0.011 +/- 0.002 x V)s -1. The dissociation constant was close to 10 microM at -80 mV. End-plate fluctuations indicated that the local anesthetic QX222 lowered the effective single-channel conductance, suggesting a finite blocked state conductance that was calculated to be 1.6 pS. The apparent differences between QX222 interaction with end-plate and extrajunctional channels are discussed.     

Quantitative simulation of endplate currents at neuromuscular junctions based on the reaction of acetylcholine with acetylcholine receptor and acetylcholinesterase.

Two kinetic models are introduced which predict amplitudes and time-courses of endplate currents and miniature endplate currents at neuromuscular junctions, at both normal and acetylcholinesterase-inhibited endplates. Appropriate differential rate equations reflecting interactions of acetylcholine with acetylcholine receptor and with esterase, diffusion of acetylcholine both within and from the synaptic cleft, and cooperativity between receptor site occupancy and ion channel opening are solved. Acetylcholine release into the cleft is assumed to be instantaneous. The simpler homogeneous reaction space model accurately predicts decay phase time constants are inaccurate. The two-reaction space model predicts amplitudes and time constants within a factor of two of those observed experimentally. The simulations indicate that the amplitudes and time-courses are primarily determined by the chemical reaction rates that characterize acetylcholine interactions with receptor and esterase and that these interactions occur under nonequilibrium conditions. Approximately 50% of the total ion channels in the initial reaction space are predicted to be opened at the peak endplate current. The cooperative opening of ion channels by acetylcholine requires that acetylcholine be introduced into the cleft in discrete, concentrated elements. Virtually all the open channels are confined to the initial reaction space, although acetylcholine-bound receptor sites can be much more widely distributed.     

The rising phase of the miniature endplate current at the frog neuromuscular junction

(1) The rising phase of minature endplate currets was recorded at the frog's neuromuscular junction using both the two electrode voltage clamp and a single external electrode, or Strickholm, voltage clamp. (2) The $\text{Q}$(10) of the miniature endplate current rising phase was 2.3 in a variety of solutions selected to alter presynaptic behavior. (3) Increasing the solution's viscosity by an amount sufficient to slow the diffusion coefficient of acetylcholine by a third has no effect on the duration of the rising or the decay phase. This solution does seem to further slow the miniature endplate current decay phase, but not the rising phase, after inhibition of the acetylcholinesterase. (4) As the membrane potential is made more positive, the miniature endplate current rising phase is prolonged, with an $\text{e-}\text{fold}$ slowing per 170 mV change. (5) It is concluded that neither presynaptic nor subsynaptic events determine the rising phase of miniature endplate currents at the frog neuromuscular junction. Rather, the limiting step occurs within the membrane and is most likely a change in the binding constant of the receptor for the acetylcholine molecule.     

Effects of ammonium ions on endplate channels

Miniature endplate currents, recorded from voltage-clamped toad sartorius muscle fibers in solutions containing ammonium ions substituted for sodium ions, were increased in amplitude and decayed exponentially with a slower time constant than in control (Na) solution. The peak conductance of miniature endplate currents was also greater in ammonium solutions. The acetylcholine null potential was - 2.8 +/- 0.8 mV in control solution, and shifted to 0.9 +/- 1.6 mV in solutions in which NH4Cl replaced half the NaCl. In solutions containing NH4Cl substituted for all the NaCl, the null potential was 6.5 +/- 1.3 mV. Single channel conductance and average channel lifetime were both increased in solutions containing ammonium ions. The exponential relationship between the time constant of decay of miniature endplate currents or channel lifetime and membrane potential was unchanged in ammonium solutions. A slight but consistent increase in peak conductance during miniature endplate currents and single channel conductance was seen as membrane potential became more positive (depolarized) in both control and ammonium solutions. Net charge transfer was greater in ammonium solutions than in control solution, whether measured during a miniature endplate current or through a single channel. The results presented here are consistent with an endplate channel model containing high field strength, neutral sites.     

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.     

Endplate channel block by guanidine derivatives

The effects of the n-alkyl derivatives of guanidine on the frog neuromuscular junction were studied using the two-microelectrode voltage clamp and other electrophysiological techniques. Methyl-, ethyl- , and propylguanidine stimulated the nerve-evoked release of transmitter. However, amyl-and octylguanidine had no apparent presynaptic action. All of the derivatives blocked the postsynaptic response to acetylcholine, the potency sequence being octyl-greater than amyl-greater than propyl-, methyl-greater than ethylguanidine. Methyl- and octylguanidine did not protect the receptor from alpha- bungarotoxin block, suggesting that these compounds do not bind to the receptor but probably block the ionic channel. Methyl-, ethyl-, and propylguanidine shortened inward endplate currents but prolonged outward currents. Amylguanidine prolonged both inward and outward endplate currents, and the currents became biphasic at negative membrane potentials. Octylguanidine increased the rate of decay of endplate currents at all potentials. All of the derivatives blocked inward endplate currents more markedly than outward currents, resulting in a highly nonlinear current-voltage relation. Methyl-, ethyl-, and propylguanidine reversed the voltage dependence of endplate current decay, while amyl-and octylguanidine reduced the voltage dependence of endplate current decay. Octylguanidine appears to block the ionic channel in both the open and the closed state. The block of the open channel follows pseudo-first-order kinetics with a forward rate constant of 4-6 X 10(7) M-1 s-1.     

Acetylcholine receptor kinetics at slow fiber neuromuscular junctions

Acetylcholine receptors in slow fiber neuromuscular junctions of garter snake (sp. Thamnophis) produced synaptic responses that were more complicated than those observed from twitch fibers. Although the slow fiber miniature end plate currents decayed monoexponentially with time, both the current fluctuations spectrum and the voltage jump end plate current required two temporal components for good theoretical fits. This behavior was accurately accounted for by a generalized version of the three-state kinetic model by del Castillo and Katz. Application of the model allowed not only the rate of channel closing to be estimated, but also the rate of channel opening (from the closed state with acetylcholine bound) and the apparent rate of acetylcholine unbinding from the receptor. The results suggest that at the peak of the miniature end plate current local receptor saturation occurs.     

Dependence of Miniature Endplate Current on Kinetic Parameters of Acetylcholine Receptors Activation: A Model Study

Mathematical modeling was applied to study the dependence of miniature endplate current (MEPC) amplitude and temporal parameters on the values of the rate constants of acetylcholine binding to receptors (k₊) when cholinesterase was either active or inactive. The simulation was performed under two different sets of parameters describing acetylcholine receptor activation–one with high and another with low probability (Po_high and Po_low) of receptor transition into the open state after double ligand binding. The dependence of model MEPC amplitudes, rise times, and decay times on k₊ differs for set Po_low and set Po_high. The main outcome is that for set Po_high, the rise time is significantly longer at low values of k₊ because of the prolongation of ACh diffusion time to the receptor. For the set Polow, the rise time is shorter at low values of k1, which can be explained by the small probability of AChR forward isomerization after ACh binding and faster MEPC's peak formation.     

Charged local anesthetics block ionic conduction in the sheep cardiac sarcoplasmic reticulum calcium release channel.

We have examined the effect of the charged local anesthetics QX314, QX222, and Procaine on monovalent cation conduction in the Ca2+ release channel of the sheep cardiac sarcoplasmic reticulum. All three blockers only affect cation conductance when present at the cytoplasmic face of the channel. QX222 and Procaine act as voltage-dependent blockers. With 500 Hz filtering, this is manifest as a relatively smooth reduction in single-channel current amplitude most prominent at positive holding potentials. Quantitative analysis gives an effective valence of approximately 0.9 for both ions and Kb(0)s of 9.2 and 15.8 mM for QX222 and Procaine, respectively. Analysis of the concentration dependence of block suggests that QX222 is binding to a single site with a Km of 491 microM at a holding potential of 60 mV. The use of amplitude distribution analysis, with the data filtered at 1 to 2 kHz, reveals that the voltage and concentration dependence of QX222 block occurs largely because of changes in the blocker on rate. The addition of QX314 has a different effect, leading to the production of a substate with an amplitude of approximately one-third that of the control. The substate's occurrence is dependent on holding potential and QX314 concentration. Quantitative analysis reveals that the effect is highly voltage dependent, with a valence of approximately 1.5 caused by approximately equal changes in the on and off rates. Kinetic analysis of the concentration dependence of the substate occurrence reveals positive cooperativity with at least two QX314s binding to the conduction pathway, and this is largely accounted for by changes in the on rate. A paradoxical increase in the off rate at high positive holding potentials and with increasing QX314 concentration at 80 mV suggests the existence of a further QX314-dependent reaction that is both voltage and concentration dependent. The substate block is interpreted physically as a form of partial occlusion in the vestibule of the conduction pathway giving a reduction in single-channel current by electrostatic means.     

Voltage- and time-dependent action of histrionicotoxin on the endplate current of the frog muscle

Histrionicotoxin, a toxin isolated from skin secretions of a Colombian arrow poison frog, Dendrobates histrionicus, decreased the amplitude and time-course of the endplate current, and altered the voltage dependence of the half-decay time. In addition, the toxin produced a characteristic nonlinearity in the current-voltage relationship of the endplate current when 3-s voltage conditioning steps were used. Reduction in time of the conditioning steps to 10 ms made the current-voltage relationship linear. The decrease in peak amplitude of the endplate current (epc) produced by histrionicotoxin measured during long hyperpolarizing conditioning steps was fitted by a single exponential function. The calculated rate constants ranged from 0.03 to 0.14 s-1 and varied with membrane potential at hyperpolarizing levels. The voltage- and time-dependent action of histrionicotoxin does not require an initial activation of receptors by acetylcholine (ACh). The characteristic of the current-voltage relationship can be accounted for by the observed voltage and time dependency of the attenuation of the endplate current amplitude in the presence of histrionicotoxin during long conditioning steps. These effects of histrionicotoxin on the peak amplitude, and on the voltage and time dependence of the epc were concentration-dependent and slowly reversible upon washing out the toxin. Thus, the voltage- and time-dependent action of histrionicotoxin at the endplate is related to an increase in the affinity between the toxin and the ACh receptor-ionic channel complex. This increase in affinity is postulated to be due to a conformational change of the macromolecule in the presence of histrionicotoxin which is demonstrated to be relatively slow, i.e., on the order of tens of seconds.     

A re-examination of curare action at the motor endplate.

Recent evidence indicates that curare, in addition to its competitive' interference with endplate receptors, can block open ionic channels by a 'non-competitive' action on the activated acetylcholine-receptor complex. These findings called for further study of the kinetic behaviour of endplate channels and their modification by curare. Examining impulse-evoked endplate currents and acetylcholine-induced current fluctuations, it is found that the lifetime of the open channel is shortened by relatively high concentrations of curare (greater than 5 micrometer), an effect which shows up most strikingly at hyperpolarized levels of membrane potential (-130 mV and above). No shortening of this kind is observed when a neuromuscular block of equal or greater intensity is produced by a dose of alpha-bungarotoxin. Two other neuromuscular blocking agents, gallamine and pancuronium are shown to have an action on channel kinetics which cannot be explained by competitive receptor binding, but conforms to the hypothesis of rapidly repeated blocking and unblocking of individual ion channels, which had been proposed originally to account for the endplate action of local anaesthetics.     

Different sensitivity of miniature endplate currents of the rat extensor digitorum longus, soleus and diaphragm muscles to a novel acetylcholinesterase inhibitor C-547

A novel derivative of 6-methyluracil, C-547, increased the amplitude and prolonged the duration of miniature endplate currents (MEPCs) which is typical for acetylcholinesterase inhibition. In the soleus and extensor digitorum longus significant potentiation was detected at nanomolar concentrations. In contrast, in the diaphragm muscle, the increase in the amplitudes of the MEPCs and the decay time constant appeared only when the concentration of C-547 was elevated to 1 x 10(-7) M. Possible consequences for the exploitation of this drug, which can selectively inhibit AChE in particular synapses, are discussed.     

Current-dependent block of endplate channels by guanidine derivatives

Methyl- and ethylguanidine block the endplate current in frog muscle. Both derivatives blocked inward-going endplate currents without affecting outward endplate currents. Repetitive stimulation that evoked several inward endplate currents enhanced the block, which suggests that these agents interact with open endplate channels. The relative conductance vs. potential curve exhibited a transition from a low to a high value near the reversal potential for the endplate current, both in normal and in 50% Na solution. In the latter solution, the reversal potential for endplate current was shifted by a mean value of 16 mV in the direction of hyperpolarization. The results suggest that methyl- and ethylguanidine block open endplate channels in a manner dependent on the direction of current flow rather than on the membrane potential.     

Changes in synaptic potential properties during acetylcholine receptor accumulation and neurospecific interactions in Xenopus nerve-muscle cell culture

Acetylcholine receptors in the muscle cell membrane accumulate at the nerve contact area in Xenopus cell cultures. The correlation between spontaneous synaptic potential properties and extent of acetylcholine receptor accumulation was studied. Small and infrequent miniature endplate potentials were measured before acetylcholine receptor accumulation which was observed with fluorescence microscopy using tetramethylrhodamine-conjugated α-bungarotoxin. As acetylcholine receptors accumulate at the nerve contact area, these synaptic potentials become larger and their frequency increases dramatically. In nerve-contacted muscle cells where spontaneous synaptic activity could not be detected, extensive acetylcholine receptor accumulation was not found at sites of nerve contact. Furthermore, muscle cells which exhibited extensive acetylcholine receptor accumulation along the nerve always produced miniature endplate potentials. Thus acetylcholine receptor accumulation and the presence of miniature endplate potentials were strongly correlated. Noncholinergic neurons from dorsal root ganglia did not form functional synaptic contacts with muscle cells nor acetylcholine receptor accumulation along the path of contact. Furthermore, explants from tadpole spinal cord formed functional synaptic contacts with muscle cells but rarely caused AChR localization. These data are discussed in terms of developmental processes during neuromuscular junction formation.     

The permeability of the endplate channel to organic cations in frog muscle

The relative permeability of endplate channels to many organic cations was determined by reversal-potential criteria. Endplate currents induced by iontophoretic "puffs" of acetylcholine were studied by a Vaseline gap, voltage clamp method in cut muscle fibers. Reversal potential changes were measured as the NaCl of the bathing medium was replaced by salts of organic cations, and permeability ratios relative to Na+ ions were calculated from the Goldman-Hodgkin-Katz equation. 40 small monovalent organic cations had permeability ratios larger than 0.1. The most permeant including NH4+, hydroxylamine, hydrazine, methylamine, guanidine, and several relatives of guanidine had permeability ratios in the range 1.3--2.0. However, even cations such as imidazole, choline, tris(hydroxymethyl)aminomethane, triethylamine, and glycine methylester were appreciably permeant with permeability ratios of 0.13--0.95. Four compounds with two charged nitrogen groups were also permeant. Molecular models of the permeant ions suggest that the smallest cross-section of the open pore must be at least as large as a square, 6.5 A x 6.5 A. Specific chemical factors seem to be less important than access or friction in determining the ionic selectivity of the endplate channel.     

The rise times of miniature endplate currents suggest that acetylcholine may be released over a period of time.

Models of miniature endplate currents predict 20-80% rise times of 100 microseconds or less. These predictions are substantially less than most of the rise times recorded in the literature. New measurements were made of rise times at the frog neuromuscular junction using extracellular recording. The mean 20-80% rise time was 250 microseconds. Rise times were variable; at 20 degrees C, 95% of them fell in a range from 140 to 460 microseconds. The most questionable assumption in the models is that the acetylcholine (ACh) is released instantaneously. Modifying the model, so that ACh diffuses from the vesicle through a pore, lengthens the rise time to observed levels. It has been proposed that ACh is released from the vesicle in exchange for Na+. However, the rise times of miniature endplate currents recorded in solutions in which the Na+ is replaced by sucrose are in the normal range. The Q10 for the rise of miniature endplate currents is approximately 2, which is consistent with the models and with temperature effects on pore formation in mast cells.     

Flickering of a nicotinic ion channel to a subconductance state.

Nicotinic acetylcholine channels show bursts of activity where open channel currents are separated from each other by short closed periods called flickers. These flickers presumably represent transitions from the open state to the state preceding the first opening of a burst (doubly liganded, closed state). Using tissue cultured chick pectoral muscle, we have examined the amplitude distribution of flickers. Of those events sufficiently long to permit accurate measurement of the amplitude (approximately 25% of all flickers), approximately two-thirds had a mean current equal to 10% of the fully open channel. The remaining one-third did appear to close completely. The subconducting flicker state is not a requisite step preceding channel opening. We conclude that there are three types of flicker events: a short event (time constant approximately 0.1 ms) whose current distribution is uncertain and two longer events (time constant approximately 1 ms), one of which has a current approximately 10% of the main open state and the other of which has a current indistinguishable from zero. In contrast, the amplitude of flickers induced by the local anesthetic QX-222 is indistinguishable from zero.     

Effects of hyaluronidase on miniature endplate potentials and currents at the frog neuromuscular junction

The effects of 0.1% testicular hyaluronidase on miniature endplate potentials and currents (MEPP and MEPC) were investigated in frog pectorocutaneous muscle. The action of hyaluronidase on preparations with armine-induced blockade of acetylcholinesterase was associated with decreased amplitude and duration of MEPP and MEPC half-decay time and rising phase. The correlation between amplitude and half-decay time of MEPP and MEPC declined at the same time, while MEPC decay remained exponential. Treating preparations having intact acetylcholinesterase with hyaluronidase increased the length of MEPC halfdecay, with duration of the rising phase and amplitude remaining constant. It is suggested that enzymatic breakdown of a proportion of the glycocalix of cells forming the neuromuscular junction and a portion of the extracellular matrix at the synaptic cleft leads to attenuation of nonspectific acetylcholine binding, thus facilitating acetylcholine diffusion into the synaptic cleft.A. A. Zhdanov State University, Leningrad. Translated from Neirofiziologiya, Vol. 20, No. 1, pp. 113–119, January–February, 1988.     

Effects of "non-quantal" acetylcholine on miniature endplate currents in mice

The possibility of postsynaptic potentiation (PSP) and desensitization developing due to nonquantal acetylcholine (ACh) secretion was investigated in mouse diaphragm with reference to time-amplitude relationships of miniature endplate currents (MEPC). The H effect (which characterizes nonquantal secretion (NS) of ACh) fell to zero over 3 h under the action of armine-induced inhibition of acetylcholinesterase (AChE) at a temperature of 20°C. A decline in the decay time constant () of MEPC unaccompanied by observable alteration in MEPC amplitude occurred at the same time. This accelerated decay of MEPC was not observed in the absence of NS (the early stages of denervation). Start of NS did not show any effect on maximum retardation of MEPC decay due to AChE inhibition, indicating that no PSP sets in under the effects of non-quantal secretion. The effect of decline in accelerated with a rise in temperature; it could be reproduced with neostigmine replacing armine, while remained unchanged in the time spells investigated with AChE in its active state. Non-quantal ACh is not thought to produce substantial retardation of MEPC decay, although it does bring about desensitization, signs of which may be partially masked owing to concurrent onset of PSP.S. V. Kurashov Medical Institute, Kazan'. Translated from Neirofiziologiya, Vol. 22, No. 4, pp. 507–513, July–August, 1990.     

Cation selectivity of acetylcholine-activated ionic channel of frog endplate

Ionic selectivity of the acetylcholine-activated ionic channel of frog endplate membranes to various organic cations has been studied. The ratio of test cation permeability (PX) to sodium permeability (PNa) was estimated by two methods, one based on the measurements in test cation solutions of the amplitude of transient depolarization induced by iontophoretic application of acetylcholine, and the other on the measurements of the reversal potential for the membrane current induced by iontophoretic application of acetylcholine under voltage-clamp conditions. The endplate channel is relatively nonselective to various test cations. The permeabilities relative to Na are ammonium (1.71), formamidine (1.49), methylamine (1.39), hydrazine (1.35), and Li (0.76), as measured from the reversal potential for acetylcholine currents, and guanidine (0.74), aminoguanidine (0.20), methylguanidine (0), and choline (0) as measured from the amplitude of acetylcholine potential. Methylguanidine and aminoguanidine block the endplate channel with the apparent dissociation constants of 0.5 and 15 mM, respectively. Based on these data, the dimensions of selectivity filter of acetylcholine-activated channel appear to be slightly larger than those of the sodium channel of frog nodes and smaller than those of the epithelial membrane of gallbladder of frogs and rabbits.