Asymmetrical displacement current and its relation with the activation of sodium current in the membrane of frog myelinated nerve

1. Sodium currents (INa) and asymmetrical displacement currents (ID) were measured in the same nerve fibres from Rana esculenta under similar conditions. 2. For exploring possible kinetic and steady state relations between INa and ID the following quantities were compared: (i) the activation of the sodium channels and (ii) the charge displacement of ID. 3. The delay of sodium activation increased after hyperpolarization. A corresponding effect on the displacement of charge was not observed. 4. Upon a small depolarization sodium activation rose slower than the displacement of charge, whereas at large depolarizations sodium activation reached a steady level before the charge displacement. 5. Upon repolarization to various levels between -52 and 12 mV relative to the resting potential, the ratio between the time constants of charge displacement and of sodium tail current varied between 3 and 1. 6. In the steady state the sodium activation was one half at about the same potential as the charge displacement but exhibited a clearly steeper voltage dependence. 7. Blocage of sodium channels with tetrodotoxin did not affect the asymmetrical displacement current. Replacing a part of external Na by tris did not alter the sodijm activation process. 8. The results indicate that the asymmetrical displacement of charge may reflect states of the gating mechanism in sodium channels but cannot be considered as a correlate of the Hodgkin Huxley m variable.     

Lack of excitability in the internodal membrane of myelinated nerve fiber in frog

Monophasic action potentials of about 70 and 10 mV were recorded by inserting a microelectrode into the axon and the myelin sheath of an intact myelinated fiber, respectively. When the intra-axonal or the intra-myelinic microelectrode was used for both stimulation and recording, only the anodal current was effective in inducing action potentials. The inter-nodal membrane was, therefore, intrinsically inexitable.     

Block of gating currents by ultraviolet radiation in the membrane of myelinated nerve

Summary The effect of ultraviolet radiation on the asymmetrical displacement currents in the membrane of the node of Ranvier was measured and compared with the ultraviolet blocking of the sodium current. Ultraviolet radiation irreversibly reduced the peak sodium current and the charge displaced during a depolarizing test pulse, the relative reduction being independent of potential. The ratio of the ultraviolet sensitivities of the sodium and the asymmetrical displacement currents is 2.3±0.2. This result suggests two independent identical gating particles per sodium channel in the membrane of myelinated nerve.Supported by Deutsche Forschungsgemeinschaft SFB 38 Membranforschung-Bonn-Bad Godesberg     

Differential effects of tetracaine on delayed potassium channels and displacement currents in frog skeletal muscle.

1. Delayed K+-currents and displacement currents were studied with a voltage-clamp technique. 2. In normal fibres, the conductance of the delayed channel grows e-fold per 3 millivolts at sufficiently negative potentials and reaches a limiting value of 2-10 m-mho/cm2 (mean 5-8 m-mho/cm2) at positive potentials. Adding tetracaine (2 mM) reduces the limiting conductance, shifts the voltage-dependence of the delayed channel to +25 mV more positive potentials and slows the kinetics fourfold. 3. By contrast, the displacement currents are virtually unaltered by 2 mM tetracaine. Their voltage-dependence is shifted by less than 5 mV and their kinetics are unaffected. 4. Tetraethylammonium ions (TEA) are known to slow the kinetics of delayed K+-channels fivefold but fail, like tetracaine, to change the kinetics of the displacement currents. 5. Both tetracaine and TEA have thus large effects on the 'gating' of the delayed channel, yet little or none on the displacement currents. This suggests that the displacement currents in skeletal muscle are for the most part unrelated to the opening and closing of delayed channels. It is estimated that 'gating' the delayed channel in muscle may require no more than 1 or 2% of the observed charge displacement.     

Oscillating repolarization in action potentials of frog sensory myelinated nerve fibres

Current and voltage clamp experiments were performed in single myelinated sensory nerve fibres of Rana esculenta. The K current was blocked by external tetraethylammonium-chloride and internal CsCl. Negative prepotentials led to the formation of a plateau in the repolarization phase of the action potential, and further regenerative depolarizations emerged from this plateau. A three-state model for Na inactivation based on voltage clamp data was sufficient to simulate these observations.     

A barbiturate-induced potassium permeability increase in the myelinated nerve membrane

Effects of the highly lipid soluble barbiturates methohexital and thiopental on potential clamped myelinated nerve fibres were analyzed. Both were found to affect the potassium as well as sodium transport system. The methohexital effect on the potassium system was complex. At low potential values it reduced the permeability, while at high values it increased the permeability as well as slowed down the kinetics. It antagonized the rectification of IK at high potential values caused by increased axoplasmic [Na+]. The effect may be described as a combination of a decreased permeability constant (PK) and a methohexital induced potassium permeability with a potential and time dependence different to the ordinary potassium system. Thiopental reduced PK only. The effect on the sodium system was similar for the studied barbiturates and similar to that described for other barbiturates: a decrease of the permeability constant (PNa) and a shift of the h infinity-U relation in negative direction along the potential axis.     

Blocking and modifying actions of octanol on Na channels in frog myelinated nerve

The actions of externally applied n-octanol on Na channels in myelinated frog nerve fibres were studied under voltage clamp conditions. Upon octanol application peak Na inward currents declined in two phases: 90% of the reduction occurred in less than 2 min but a steady-state was reached only after 15 min. During washout the currents came to a stable level within 10 min. The reduction of Na inward currents by octanol was dependent on the amplitude and duration of prepotentials. At the resting potential (VH = 0 mV) 0.4 mM octanol reduced peak Na inward currents at V = 60 mV by 50%. After a prepulse of -60 mV and 50 ms duration Na currents decreased only by 20%. At a hyperpolarizing holding potential of VH = -28 mV 0.7 mM octanol reduced peak inward Na currents to one half. Octanol depressed Na currents at all potentials by approximately the same factor. The Na reversal potential VNa remained unchanged. 0.7 mM external octanol shifted the Na activation curve m infinity (V) by 5 mV to more positive and the inactivation curve h infinity (V) by 14 mV to more negative potentials. The midpoint slopes of both curves were reduced. The time constants of Na activation and inactivation at small depolarizations were decreased. The conductance gamma of a single Na channel and the number No of conducting Na channels per node were determined from nonstationary Na current fluctuations. 0.7 mM octanol increased gamma by a factor of 1.6 and reduced No by a factor of 0.34. It is concluded that octanol blocks some Na channels and modifies the remaining unblocked channels.     

Acetylcholine-induced currents at plasma membrane of the frog oocyte

Although with remarkable variability, membrane permeability in Rana oocytes can be modified by application of acetylcholine. The experiments were carried out in voltage-clamp conditions. Like in Xenopus, the responses proved to be related to activation of muscarinic receptors operating membrane channels probably selective for Cl-. At differences with Xenopus, the net acetylcholine-induced current showed remarkable deviation from linearity, displaying outward-going rectification. Application of acetylcholine typically produced opening of membrane channels, while in late spring, we observed the opposite effect in several batches of oocytes.     

The effect of temperature on Na currents in rat myelinated nerve fibres

Voltage clamp experiments were performed in single myelinated nerve fibres of the rat and the effect of temperature on Na currents was investigated between 0 degrees C and 40 degrees C. The amplitude of the peak Na current changed with a Q10 = 1.1 between 40 degrees and 20 degrees C and with a Q10 = 1.3 between 20 degrees and 10 degrees C. Below 10 degrees C the peak Na current changed with a Q10 = 1.9. The temperature coefficient for time-to-peak (tp), the measure for Na activation, and tau h1 and tau h2, the time constants for Na inactivation changed throughout the temperature range. Q10 for all of these kinetic parameters increased from 1.8-2.1 between 40 degrees and 20 degrees C to 2.6-2.7 between 20 degrees and 10 degrees C. Below 10 degrees C Q10 increased to 3.7 for tau h1 and tp, and to 2.9 for tau h2. When the series resistance artifacts were minimized by addition of 6 nM TTX, the Q10's at T less than 10 degrees C were 2.9-3.0. When the temperature was decreased from 20 degrees to 0 degrees C, both the curve relating Na permeability to potential, PNa(V), and the steady state Na inactivation curve, h infinity (V), were reversibly shifted towards more negative potentials by 6 mV and 11 mV, respectively. When the temperature was increased from 20 degrees to 37 degrees C no shifts occurred. The Hodgkin-Huxley rate constants alpha h(V) and beta h(V) were calculated from h infinity (V) and tau h (or tau h1) at 20 degrees and 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

The response of the myelinated nerve fiber to short duration biphasic stimulating currents

The response of a single Node of Ranvier to short duration biphasic stimulating currents was studied. Such stimulus patterns are known to be less likely to induce tissue damage than are longer monophasic stimuli. The presence of the anodic phase of a pulse pair was found to abolish excitation in cases where the cathodic phase alone was near threshold. In addition, peak twitch force from the tibialis anterior muscle of cat was found to be reduced by the use of biphasic motor nerve stimulation in place of monophasic stimulation. The abolition phenomenon could be eliminated by the introduction of a delay of 100 μs between the phases of a biphasic stimulus waveform. This research was supported by the National Institutes of Health, Grant No. NINCDS NO1-NS-2-2314 (Neural Prosthesis Program) and GM 01090-15 Training Grant.     

The effect of membrane polarization on the time course of the end-plate current in frog sartorius muscle

1. Intracellular recordings of the end-plate current at different membrane potentials were made, using a voltage-clamp technique.2. When the membrane potential is varied between about +40 and -120 mV, the half-decline time of the end-plate current increases from about 0.6 msec to about 1.6 msec.3. It is suggested that the stability of receptor-mediator complex is affected by the membrane potential.     

Block of Na channels in the membrane of myelinated nerve by benzocaine

The actions of the neutral local anesthetic benzocaine on Na channels were studied in voltage-clamp experiments on single myelinated nerve fibres of the frog by measurements of sodium currents, asymmetry currents, and sodium current fluctuations. 2. 1 mM benzocaine reduced the peak Na currents during various depolarizations V between 20 and 120 nV to 63% of their control values but did not change the time constant of Na activation. 3. 1 mM benzocaine altered asymmetry currents during 1 ms pulses V between 20 and 120 mV in the same was as the early Na currents: It reduced the amplitude to 64% but did not affect the kinetics of the currents. 4. The charge displacement of the asymmetry current during the pulse (Qon) was compared with the charge displacement after the pulse (Qoff). Without benzocaine the relative charge Qoff/Qon Declined to a constant level (0.42 at V = 40mV, 0.25 at V = 100 mV) with increasing pulse durations. In the presence of 1 mM benzocaine the charges Qoff after pulses to V = 40 or 100 mV are almost independent of pulse duration and approximately equal to the control Qoff values after 5.6 ms pulses. Thus, the immobilizations caused by Na inactivation and benzocaine are not additive. 5. Na currents and Na-current fluctuations were recorded during depolarizations V between 24 and 48 mV in the presence of 0.1 mM benzocaine and 7 microM Anemonia toxin II. A lower limit of 8.6 pS was derived for the conductance of a single Na channel. The value agrees with other estimates of the conductance of Na channels which had not been treated by local anesthetics. This suggests an "all-or-none blocking" of Na channels by benzocaine.     

The association of the supernormal period and the depolarizing afterpotential in myelinated frog and rat sciatic nerve

Excitability properties of isolated frog and rat sciatic nerve fibers were examined using intra-axonal and sucrose-gap recording techniques. Paired stimulation experiments on rat myelinated fibers indicate that a small proportion (11%; n = 84) of these axons demonstrate decreased threshold indicative of a supernormal period. In contrast, 81% (n = 23) of frog axons displayed a supernormal period. A depolarizing afterpotential was observed in most of the rat and frog fibers having a supernormal period and the depolarizing afterpotential increased in magnitude and duration during hyperpolarization. In addition to whole nerve stimulation, a supernormal period could be induced by stimulation of a single axon via current passage through the recording microelectrode. Brief (2-5 ms) subthreshold depolarizing pulses were followed by a slowly decaying depolarization and a period of increased excitability that mimicked the supernormal period. A supernormal period was also observed in the whole nerve preparation using a sucrose-gap technique. The magnitude and duration of the supernormal period, as measured in the sucrose-gap, were greater for frog nerve than for rat nerve. Additionally, a larger postspike negativity, the extracellular equivalent of the intra-axonally observed depolarizing afterpotential, was present in sucrose-gap recordings for frog nerve than for rat nerve. The results indicate that the depolarizing afterpotential is an important determinant of the supernormal period, and that both the depolarizing afterpotential and supernormal period are more prominent in frog than in rat sciatic nerve.