Mechanism of 4-aminopyridine action on voltage-gated potassium channels in lymphocytes

The mechanism by which 4-aminopyridine (4-AP) blocks the delayed rectifier type potassium (K+) channels present on lipopolysaccharide-activated murine B lymphocytes was investigated using whole-cell and single channel patch-clamp recordings. 4-AP (1 microM-5 mM) was superfused for 3-4 min before applying depolarizing pulses to activate the channel. During the first pulse after application of 4-AP above 50 microM, the current inactivated faster, as compared with the control, but its peak was only reduced at high concentrations of 4-AP (Kd = 3.1 mM). During subsequent pulses, the peak current was decreased (Kd = 120 microM), but the inactivation rate was slower than in the control, a feature that could be explained by a slow unblocking process. After washing out the drug, the current elicited by the first voltage step was still markedly reduced, as compared with the control one, and displayed very slow activation and inactivation kinetics; this suggests that the K+ channels move from a blocked to an unblocked state slowly during the depolarizing pulse. These results show that 4-AP blocks K+ channels in their open state and that the drug remains trapped in the channel once it is closed. On the basis of the analysis of the current kinetics during unblocking, we suggest that two pathways lead from the blocked to the unblocked states. Computer simulations were used to investigate the mechanism of action of 4-AP. The simulations suggest that 4-AP must bind to both an open and a nonconducting state of the channel. It is postulated that the latter is either the inactivated channel or a site on closed channels only accessible to the drug once the cell has been depolarized. Using inside- and outside-out patch recordings, we found that 4-AP only blocks channels from the intracellular side of the membrane and acts by reducing the mean burst time. 4-AP is a weak base (pK = 9), and thus exists in ionized or nonionized form. Since the Kd of channel block depends on both internal and external pH, we suggest that 4-AP crosses the membrane in its nonionized form and acts from inside the cell in its ionized form.     

A model for 4-aminopyridine action on K channels: similarities to tetraethylammonium ion action.

We present a model for the action of 4-aminopyridine (4AP) on K channels. The model is closely based on the gating current studies of the preceding paper and has been extended to account for ionic current data in the literature. We propose that 4AP, like tetraethylammonium ion and other quaternary ammonium ions, enters and leaves the channel only when the activation gate is open, a proposal that is strongly supported by the literature. Once in the open channel, 4AP's major action is to bias the activation gate toward the closed conformation by approximately the energy of a hydrogen bond. S4 segment movement, as reflected in gating currents, is almost normal for a 4AP-occupied channel; only the final opening transition is affected. The model is qualitatively the same as the one used for many years to explain the action of quaternary ammonium ions.     

Differences in presynaptic action of 4-aminopyridine and tetraethylammonium at frog neuromuscular junction

4-Aminopyridine markedly potentiates transmitter release at the frog pectoris neuromuscular junction by increasing the quantal content even when applied at low concentrations (5-20 microM). This enhancement of transmitter release is associated with greater minimum synaptic latency, but the dispersion of the synaptic latencies does not appear much affected. This is in contrast with the action of tetraethylammonium (0.2-0.5 mM) in which case similar enhancement of transmitter release results not only in larger minimum synaptic latency but also in greater dispersion of the synaptic latencies. The time course of transmitter release associated with enhanced transmitter output is hence much more prolonged in the presence of tetraethylammonium than 4-aminopyridine, at least for low concentrations of 4-aminopyridine (5-20 microM). This indicates that their presynaptic actions differ significantly. This conclusion is further strengthened by the finding that unlike tetraethylammonium, 4-aminopyridine induces bursts of release, presumably by producing multiple action potentials in the nerve terminal. Tetraethylammonium probably acts by blocking the delayed potassium conductance, but the blockade of Ca2+-activated K+ conductance cannot be excluded. 4-Aminopyridine, however, probably blocks the fast inactivating (IA) K+ current, but it also may be acting directly on the voltage-dependent Ca2+ conductance or on the intracellular Ca2+ buffering.     

Wiping reflex in the intact and spinal frogs

Studying the rostral wiping reflex, RWR, in intact grass frogs allowed us to identify two of its forms: flexor and extensor reflexes (FWR and EWR, respectively). Reflex fields of FWR and EWR considerably overlap; when the common zones are stimulated, both reflex forms can be observed. Mixed forms of the reflex are also possible. The probability of initiation of a certain RWR form depends on the stimulus position. Both FWR and EWR are preserved after spinalization, but their receptive fields are somewhat displaced. The hindlimb movement was divided into three phases: positioning (P), lowering, or orientation (L), and brushing aside (BA). In the intact frog, each phase is of approximately similar duration. The reflex phases and their temporal relations are preserved after spinalization. Total movement duration became longer in spinal animals, as compared with that in intact ones; in addition, this duration began to depend on the stimulus position. In the intact frogs, the position of a working limb shows the linear dependence on the stimulus localization; this dependence is preserved after spinalization. Our results allow us to conclude that the central generator of wiping reflex includes several subsystems determining the reflex form in a probabilistic manner.Neirofiziologiya/Neurophysiology, Vol. 27, No. 4, pp. 278–287, July–August, 1995.     

Effects of 4-aminopyridine on the action potential and the after-hyperpolarization of cat spinal motoneurons

In cats under pentobarbital anaesthesia, intramotoneuronal administrations of 4-aminopyridine significantly prolong the falling phase of the antidromic action potential but have much less effect on the orthodromic action potential. 4-aminopyridine probably blocks the fast K channels involved in the repolarization of the membrane and indirectly activates ionic channels through enhancement of synaptic transmission, also suggested by the potentiation of excitatory postsynaptic potentials. In many cells, 4-aminopyridine depresses the amplitude and prolongs the time course of the after-hyperpolarization; therefore 4-aminopyridine may also partly block Ca2+-activated K+ channels.     

延髓腹外侧区在降压反射中的作和

在各种心血管反射中,降压反射是最主要的,延髓腹外侧区在降压反射中起重要作用,目前认为降压反射中枢通路中至少有四种成分是最基本的：（１）孤束核中的神经元;（２）延髓腹外侧头端的交感前运动细胞;（３）延髓腹外侧尾端;（４）疑核或／和迷走神经背运动核。此外,兴奋性与抑制性氨基酸受体和抑制性神经元也是中枢通路的关键成分,下丘脑视上核与室旁核的升压素分泌细胞也有一定作用。     

Synaptic transmission in the sixth ganglion of the cockroach: action of 4-aminopyridine.

1. Study was made of the action of 4-aminopyridine (5 X 10(-5) M) on synaptic transmission in the last abdominal ganglion of Periplaneta americana. The 'oil-gap' technique was used to record postsynaptic events in a single giant axon. 2. 4-AP quickly increased the 'background' of postsynaptic activity, which consisted of 'spontaneous' unitary EPSPs and IPSPs. Postsynaptic spikes were also propagated. 3. Both evoked EPSPs (stimulation of cercal nerve XI) and evoked IPSPs (stimulation of cercal nerve X) were greatly increased in amplitude although their duration (half-time) was unaltered. 4. 4-AP triggered presynaptic action potentials in the cercal nerves (recorded with external electrodes). These 'antidromic' potentials appeared singly or sometimes repetitively, especially after electrical stimulation of the cercal nerves. They were often in monosynaptic correlation with unitary EPSPs. 5. Neither the resting potential nor the postsynaptic membrane resistance was modified. 6. There were no changes in the equilibrium potentials of the ions involved in postsynaptic events. 7. The results may be essentially explained by an increase in transmitter release after 4-AP treatment, which may be partly the result of a rise in presynaptic terminal excitability, and partly the result of a lengthening of the presynaptic action potentials.     

Determination of 4-aminopyridine in plasma

Gas-liquid chromatographic procedures for the determination of 4-aminopyridine in human and animal plasma are reported. The procedures involve the addition of an internal standard 3-methyl-4-aminopyridine to plasma followed by extraction into methylene chloride/isopropranol under alkaline conditions. The isolated amines are converted to their pentafluoropropionyl or monochlorodifluoracetyl derivatives and are quantified after chromatographic separation using either electron-capture or nitrogen-phosphorus detectors. The sensitivity of these procedures is such that nanogram amounts of 4-aminopyridine can be readily determined.     

Responses of auditory medullary units to sinusoidally amplitude-modulated tones in frogs

Spike discharges of medullary units ofRana ridibunda in response to tones of optimal frequency for the neuron, with sinusoidal amplitude modulation, was studied. Reproduction of sound modulation in unit activity was assessed by the use of phase histograms of responses corresponding to the period of modulation. Amplitude modulation was reproduced in the firing pattern of neurons of the dorsal nucleus over a wide range of modulation frequencies and carrier levels. Accentuation of small changes of amplitude for modulation frequencies of 70–150 Hz was observed in many neurons of the superior olives. The phase of the response was a linear function of modulation frequency both in the dorsal nucleus and in the superior olives. The greatest enhancement of amplitude changes corresponded to low modulation indices.Academician N. N. Andreev Acoustics Institute, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 3, pp. 390–396, May–June, 1985.     

Extracellular action potentials of skeletal muscle fibre affected by 4-aminopyridine: a model study

A new specially designed analytical function approximating the intracellular action potentials (ICAPs) for calculation of the extracellular potentials (ECAPs) at various radial and axial distances from the active fibre is proposed. 4-Aminopyridine (4-AP) was used to obtain ICAPs with a prolonged repolarization phase in order to investigate the influence of changes in ICAP shape on the ECAPs. From the experimentally recorded ICAPs before and after treatment of frog skeletal muscle fibres with 4-AP, approximated by the new function, the ECAPs were calculated applying the linesource model in a finite fibre. Using this function allowed calculation of the ECAPs at distances not accessible for the experimental recordings. The total ionic current (I _i) during the action potential was calculated using the cable equation. Our results showed that the ratio of the first positive to the negative phases of the ECAPs of treated fibres increased at large radial distances (3000 m and more) and the terminal positive phase was asymmetric with an abrupt initial deflection followed by a slow inverse deflection. The calculated ECAPs at various axial distances from the fibre end (cylindrical and conical part) and at radial distances from the fibre membrane ranging from 0 to 5000 m, corresponded in shape to the experimentally recorded potentials of untreated and 4-AP-treated muscle fibres.