Inhibition of sodium currents in frog ranvier node treated with local anesthetics Role of slow sodium inactivation

Effects of different local anesthetics of sodium permeability were studied in single nerve fibres of frog by the method of voltage clamp. Inhibition of sodium current by externally applied tertiary anesthetics, procaine and trimecaine, was the sum of a potentially independent block (reduced $\text{P}{}_{\mathrm{rmNa}}$) and slow sodium inactivation with time constants ranging from tens to hundreds of ms depending on membrane potential (at room temperature). Externally applied uncharged benzocaine produced a potentially independent block only. According to dose-response curves both processes are one-to-one reactions. In the case of trimecaine equilibrium constant the reaction responsible for reduction of $\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ is about 0.3 mM, while that for slow inactivation is more than ten times less (0.02 mM). Increasing pH from 5.6 to 8.5 markedly accelerated the slow inactivation process at all potential values. Divalent cations Ca²⁺ and Ni²⁺ shifted the steady-state slow inactivation curve along the potential axis and simultaneously reduced slow inactivation at the saturation level. Permanently charged quaternary trimecaine was ineffective when applied externally. Internally applied tertiary anesthetics and quaternary trimecaine as well as externally applied quaternary derivative of lidocaine QX-572 produced a progressively irreversible block enhanced by depolarization and inhibition reversibly increased by repetitive short-term depolarization (frequency-dependent inhibition). Inhibition of sodium currents by repetitive stimulation observed also in the case of externally applied tertiary anesthetics is due mainly to slow inactivation. The data suggests the existence of several types of receptor sites through which local anesthetics exert their blocking action on sodium permeability.     

Effects of strychnine on the potassium conductance of the frog node of Ranvier

The nature of the block of potassium conductance by strychnine in frog node of Ranvier was investigated. The block is voltage-dependent and reaches a steady level with a relaxation time of 1 to several ms. Block is increased by depolarization or a reduction in [K+]O as well as by increasing strychnine concentration. A quaternary derivative of strychnine produces a similar block only when applied intracellularly. In general and in detail, strychnine block resembles that produced by intracellular application of the substituted tetraethylammonium compounds extensively studied by C.M. Armstrong (1969. J. Gen Physiol. 54:553-575. 1971. J. Gen. Physiol. 58:413-437). The kinetics, voltage dependence, and dependence on [K+]O of strychnine block are of the same form. It is concluded that tertiary strychnine must cross the axon membrane and block from the axoplasmic side in the same fashion as these quaternary amines.     

Effects of KC 3791 on sodium and potassium channels in frog node of Ranvier

Effects of a new antiarrhytmic compound KC 3791 on sodium (INa) and potassium (IK) currents were studied in frog myelinated nerve fibres under voltage clamp conditions. When applied externally to the node of Ranvier, KC 3791 (KC) at concentrations of 10(-5)-10(-4) mol.l-1 produced both tonic and cumulative (use-dependent) inhibition of INa. An analysis of the frequency-, voltage- and time dependence of cumulative block by KC suggested that this block resulted from a voltage-dependent interaction of the drug with open Na channels. The progressive decrease in INa during repetitive pulsing was due to accumulation of Na channels in the resting-blocked state: closing of the activation gate after the end of each depolarizing pulse stabilized the KC-"receptor" complex. To unblock these channels a prolonged washing of the node had to be combined with a subsequent repetitive stimulation of the membrane; this suggested that channel could not become cleared of the blocker unless the activation gate has opened. KC also proved to be capable of blocking open K channels at outwardly directed potassium currents (IK). This block increased during membrane depolarization. Unblocking of K channels after the end of a depolarizing pulse proceeded much faster than unblocking of Na channels under identical conditions. Cumulative inhibition of outward IK during high-frequency membrane stimulation was therefore readily reversible upon a decrease in pulsing frequency.     

Alamethicin channels incorporated into frog node of ranvier: calcium-induced inactivation and membrane surface charges

Alamethicin, a peptide antibiotic, partitions into artificial lipid bilayer membranes and into frog myelinated nerve membranes, inducing a voltage-dependent conductance. Discrete changes in conductance representing single-channel events with multiple open states can be detected in either frog node or lipid bilayer membranes. In 120 mM salt solution, the average conductance of a single channel is approximately 600 pS. The channel lifetimes are roughly two times longer in the node membrane than in a phosphatidylethanolamine bilayer at the same membrane potential. With 2 or 20 mM external Ca and internal CsCl, the alamethicin-induced conductance of frog nodal membrane inactivates. Inactivation is abolished by internal EGTA, suggesting that internal accumulation of calcium ions is responsible for the inactivation, through binding of Ca to negative internal surface charges. As a probe for both external and internal surface charges, alamethicin indicates a surface potential difference of approximately -20 to -30 mV, with the inner surface more negative. This surface charge asymmetry is opposite to the surface potential distribution near sodium channels.     

Single potassium channel conductance in the frog node of Ranvier.

The single K+-channel conductance was calculated from the variance of the spontaneous potassium noise currents in voltage clamped frog node. Essential for this calculation is the mean potassium conductance during the noise measurement. So far this quantity has been underestimated, apparently due to K+-ion accumulation. With the proper values, the single K+-channel conductance is an increasing function of membrane voltage.     

Effect of conditioning potential on potassium current kinetics in the frog node.

The kinetics of potassium conductance changes were determined in the voltage clamped frog node (Rana esculenta), as a function of conditioning prepotential. The conditioning potential duration varied from 1 to 50 ms and the amplitude between -60 and +130 mV (relative to rest). The conductance kinetics were determined at a single test potential of +20 mV (depolarization) by means of the slope of log [ninfinity - nt] vs. time relationship which defines the time constant of the process (tau). The values of tau, after conditioning hyperpolarizations, were around 5 ms, up to 10 times greater than values obtained following a strong depolarization. The tau vs. pre-potential curve was sigmoid in shape. These differences were only slightly dependent on [K+]0 or conditioning pulse duration. The steady-state current values were also found to be a function of conditioning potential. After conditioning hyperpolarizations, the log [ninfinity - nt] vs. time curve could not be fitted by a single exponent regardless of the power of n chosen. The prepotential dependency of potassium current kinetics is inconsistent with the Hodgkin-Huxley axon model where the conductance parameters are assumed to be in either one of two possible states, and where the rate of transfer from one state to the other follows first order kinetics. In contrast the described kinetics may be consistent with complex multistate potassium "channel" models or membranes consisting of a number of types of channels.     

Effect of procaine on electrical activity of the ranvier node in solutions with high and low pH

The effect of procaine on generation of the action potential and its derivative in solutions with different pH values was studied in experiments on single Ranvier nodes. Minimal concentrations of procaine depressing the action potential were increased in solutions with low pH and reduced in solutions with high pH. The calculated concentrations of the basic and cationic forms of procaine changed regularly: With an increase in pH of the medium the basic decreased and the cationic increased. Excitability of the membrane (the number of sodium channels capable of excitation) did not change regularly in accordance with a change in pH of the medium: It fell on both a decrease and an increase in the pH of the solution. It was concluded from the results that the two forms of procaine interact with the membrane, but with different effectiveness.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 161–167, March–April, 1974.     

Displacement currents in sodium channels of the ranvier node membrane

The displacement current was recorded in the Ranvier node membrane ofRana ridibunda. This current was shown to be due to conversion of charges from the initial state in which they were when a high negative voltage was present on the membrane into the final state. The dependence of the displacement charge on the membrane potential and state of activation of the sodium channels suggests that the displacement current is connected with activation of the m gates of the sodium channels. Considering the density of the displaced charges, the density of the sodium channels can be estimated to be 5000 channels/µ².A. A. Ukhtomskii Institute of Physiology, A. A. Zhdanov Leningrad State University. Translated from Neirofiziologiya, Vol. 8, No. 4, pp. 410–417, July–August, 1976.     

Blocking calcium-dependent potassium in permeability of skeletal muscle fibers in the frog by a sodium-free medium

In voltage-clamped frog muscle fibres, the total or partial substitution of NaCl by LiCl does not alter the calcium-dependent slow outward potassium current. In contrast, an equimolar substitution of NaCl by choline-Cl (+atropine) or trisCl induces a rapid and reversible blockage of the current.     

Effects of valinomycin on lymphocytes independent of potassium permeability

10^?7 M valinomycin affects human lymphocytes in the following manner: (1) it is non-toxic; (2) it inhibits mitogenesis; (3) it causes a reduction in cell ATP; and (4) it causes a marked increase in steady-state Na⁺ exchange. However, it has a minimal effect on cell ion (K⁺, Na⁺, Ca²⁺, Mg²⁺) contents and no effect whatever on K⁺ exchange. Neither the fast nor the slow fraction of steady-state K⁺ exchange is affected by 10^?7 M valinomycin. The various reported effects of valinomycin on lymphocyte functions cannot be assumed to be due to changes in plasma membrane K⁺ permeability. The mechanism of the increase in steady-state Na⁺ exchange, and whether or not it is related to inhibition of mitogenesis, are unsettled issues.     

Two components of calcium-dependent potassium permeability coexist in the membrane of frog skeletal muscle

Two components of Ca-dependent K permeabilities were demonstrated in the membrane of voltage-clamped muscles fibres by raising the internal calcium concentration with 3 mM caffeine or 0.1 microM Bay K 8644. One, which is TEA-sensitive, is enhanced by an increase of intracellular calcium and the other, which is apamin-sensitive, is inhibited by high levels of internal calcium concentration.     

Effects of potassium,sodium, and azide on the ionic movements that accompany activity in frog nerves

Stimulation of intact or desheathed frog sciatic nerves produced an increase in the sodium content and a decrease in the potassium content of this tissue. In desheathed preparations the magnitudes of the changes in ionic contents decreased as the concentration of the potassium in the bathing solution was increased, while changing the external sodium concentration produced small effects on the ionic shifts. During tetanization, the rate of decline of the compound action potential also decreased as the external potassium concentration increased. Eliminating the activity respiration with 0.2 mM azide did not greatly modify the changes in sodium and potassium distribution that accompanied activity in either intact or desheathed nerves.     

Probenecid: Effects on water permeability in frog urinary bladder

The action of probenecid (probenecid is an agent known to inhibit the release of cyclic AMP by pigeon erythrocytes) on the urinary bladder of Rana esculenta has been investigated.     

Effect of aconitine on some properties of the sodium channels of the ranvier node membrane

Aconitine causes the appearance of two types of modified channels in the Ranvier node membrane. Channels of the first type are activated at high negative potentials and are inactivated only partly or not at all; their selectivity is sharply reduced: The mean ratio of potassium to sodium permeability is 0.72. The properties of these channels are stable with time. The second type of modified channels lose their conductivity during the action of aconitine, and their kinetic characteristics and region of activation are similar to those of normal sodium channels. They are less selective than normally: the ratio of potassium to sodium permeability is 0.22. The sensitivity of the modified channels to tetrodotoxin is at the same level as that of normal sodium channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 8, No. 2, pp. 152–160, March–April, 1976.     

Threshold for repetitive activity for a slow stimulus ramp: a memory effect and its dependence on fluctuations

We have obtained new insights into the behavior of a class of excitable systems when a stimulus, or parameter, is slowly tuned through a threshold value. Such systems do not accommodate no matter how slowly a stimulus ramp is applied, and the stimulus value at onset of repetitive activity shows a curious, nonmonotonic dependence on ramp speed. (Jakobsson, E. and R. Guttman. Biophys. J. 1980. 31:293-298.) demonstrated this for squid axon and for the Hodgkin-Huxley (HH) model. Furthermore, they showed theoretically that for moderately slow ramps the threshold increases as the ramp speed decreases, but for much slower ramp speeds threshold decreases as the ramp speed decreases. This latter feature was found surprising and it was suggested that the HH model, and squid axon in low calcium, exhibits reverse accommodation. We have found that reverse accommodation reflects the influence of persistent random fluctuations, and is a feature of all such excitable systems. We have derived an analytic condition which yields an approximation for threshold in the case of a slow ramp when the effect of fluctuations are negligible. This condition predicts, and numerical calculations confirm, that the onset of oscillations occurs beyond the critical stimulus value which is predicted by treating the stimulus intensity as a static parameter, i.e., the dynamic aspect of a ramp leads to a delay in the onset. The condition further demonstrates a memory effect, i.e., firing threshold is dependent on the initial state of the system. For very slow ramps then, fluctuations diminish both the delay and memory effects.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition modifies the effects of slow calcium-activated potassium channels on epileptiform activity in a neuronal network model

Generation of epileptiform activity typically results from a change in the balance between network excitation and inhibition. Experimental evidence indicates that alterations of either synaptic activity or intrinsic membrane properties can produce increased network excitation. The slow Ca²⁺-activated K⁺ currents (sI_AHP) are important modulators of neuronal firing rate and excitability and have important established and potential roles in epileptogenesis. While the effects of changes in sI_AHP on individual neuronal excitability are readily studied and well established, the effects of such changes on network behavior are less well known. The experiments here utilize a defined small network model of multicompartment pyramidal cells and an inhibitory interneuron to study the effects of changes in sI_AHP on network behavior. The benefits of this model system include the ability to observe activity in all cells in a network and the effects of interactions of multiple simultaneous influences. In the model with no inhibitory interneuron, increasing sI_AHP results in progressively decreasing burst activity. Adding an inhibitory interneuron changes the observed effects; at modest inhibitory strengths, increasing sI_AHP in all network neurons actually results in increased network bursting (except at very high values). The duration of the burst activity is influenced by the length of delay in a feedback loop, with longer loops resulting in more prolonged bursting. These observations illustrate that the study of potential antiepileptogenic membrane effects must be extended to realistic networks. Network inhibition can dramatically alter the observations seen in pure excitatory networks.