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.     

Kinetics of interaction of scorpion toxin with sodium channels in the Ranvier node membrane

The kinetics of binding the toxin ofButhus eupeus venom with sodium channels with a holding potential of –120 mV and subsequent dissociation of the toxin-channel complex during a shift of membrane potential (V_M) to between –60 and +120 mV were investigated by the voltage clamping method on the Ranvier node membrane. The rate of dissociation was shown to increase if V_M was shifted toward more positive values, exponentially with an e-fold increase every 32.3 mV. The results are in agreement with the hypothesis that dissociation of the toxin-channel complex during depolarization is determined by the difference between electrical energies of the inactivated states of normal and toxin-modified channels.Institute of Cytology, Academy of Sciences of the USSR, Leningrad. Translated from Neirofiziologiya, Vol. 12, No. 6, pp. 619–626, November–December, 1980.     

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.     

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.     

Blockade of sodium and potassium channels in the node of Ranvier by ajmaline and N-propyl ajmaline

The inhibition of sodium and potassium currents in frog myelinated fibres by ajmaline (AM) and its quaternary derivative, N-propyl ajmaline (NPA), depends on voltage-clamp pulses and the state of channel gating mechanisms. The permanently charged NPA and protonated AM interact only (or mainly) with open channels, while unprotonated AM affects preferently inactivated Na channels. Inhibition of Na currents by NPA and AM does not depend on the current direction and Na ion concentration in external or internal media. In contrast only the outward potassium currents can be blocked by NPA and AM; the inward potassium currents in high K+ ions external media are resistant to the blocking action of these drugs. The voltage dependence of ionic current inhibition by charged drugs suggests the location of their binding sites in the inner mouths of Na and K channels. Judging by the kinetics of current restoration after cessation of pulsing, the drug-binding site complex is much more stable in Na than in potassium channels. Batrachotoxin and aconitine, unlike veratridine and sea anemone toxin, decrease greatly the affinity of Na channel binding sites to NPA and AM. The effects of NPA and AM are compared with those of local anesthetics and other amine blocking drugs.     

Kinetic model of the gating system of the sodium channel in a Ranvier node membrane

A kinetic model of the sodium channel gating system consisting of four subunits with three states--closed (X), open (Y) and inactivated (Z)--is proposed. For the channel to conduct, all the four subunits must be in the open state. The transitions between states X and Y are independent, while those between states X and Z are coupled, so that for the particle considered transition of one of two neighbouring particles into state Z increases the activation energy of the step by kT. The model fits rather well to the experimental data.     

Stimulus-dependent blockade of sodium channels of the Ranvier node membrane by the phenothiazine derivative ethmozine

The action of the antiarrhythmic drug ethmozine on sodium channels of the membrane was studied in experiments on single from Ranvier nodes by the voltage clamp method. Application of ethmozine to both the outer and the inner side of the membrane reduced the amplitude of the sodium current INa; the kinetics of this current and steady-state inactivation of the sodium channels were unchanged. Tonic and phasic (transient, stimulus-dependent) components can be distinguished in the ethmozine block of the sodium current. Tonic blockage of the sodium current develops slowly and can be potentiated by high-frequency stimulation of the membrane. The possible nature of the tonic block is discussed. The stimulus-dependent blockade of the sodium current deepens with an increase in the frequency and amplitude of depolarizing stimuli. Prolonged membrane depolarization does not evoke any additional blocking of the sodium current. It is concluded that the stimulus-dependent blockade is due to interaction between ethomizine and open sodium channels. Modification of the channels by batrachotoxin (preventing inactivation of the sodium channels) makes them insensitive to ethmozine. Increasing the potassium ion concentration on the outer side of the membrane was found to reduce the tonic effect of ethmozine and to potentiate the stimulus-dependent blockade. The action of ethmozine was compared with the effects of tertiary and quaternary local anesthetics.A. V. Vishnevskii Institute of Surgery, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 13, No. 4, pp. 380–389, July–August, 1981.     

Ruminal starch digestion characteristics in vitro of barley cultivars with varying amylose content

Three experiments were conducted to study effects of amylose/amylopectin ratios and starch particle size on ruminal digestion characteristics of barley starch using an automated in vitro gas production system. In Experiment 1, starch digestion characteristics were measured in 12 barley cultivars with different amylose/amylopectin ratios, both as milled grain and as purified starch isolated from the original grain samples. The same 12 cultivars, harvested 1 year later from the same locations, were used in Experiment 2. Gas production was measured in milled samples, and in neutral detergent fibre (NDF) extracted from the same samples. The objective of this approach was to estimate gas production from neutral detergent solubles (NDS) as an approximation of starch. This was done by subtracting the NDF gas curve from the total gas production curve. In Experiment 3, starch digestion characteristics were measured for large and small starch granules from nine of the original cultivars used in Experiment 1. The gas curves obtained were fitted to a three-pool Gompertz model, and the effective rate of digestion (kd) was estimated with a two-compartmental rumen model. In Experiment 1, the effective starch kd for milled barley and purified starch were 0.122 and 0.118/h, respectively. Barley cultivars with low amylose (LA) had a higher effective kd (0.148/h) compared with cultivars with normal amylose (NA) (0.115/h) and high amylose (HA) (0.102/h) (P=0.010). Results obtained with milled barley were supported by the purified barley starch sample results, but differences were smaller and only numerically different. In Experiment 2, the ranking of the amylose groups was consistent with those in Experiment 1 (i.e., LA > NA > HA) (P=0.096). However, these differences were not reflected in the effective kd for the NDS fraction (P=0.366). Thus, factors other than those related to starch per se, or other structural features, are apparently important. Barley cultivars in the LA group had a higher effective kd for aNDF (0.098/h) than did NA and HA barley (0.060 and 0.055/h, respectively). Thus, the effect of the amylose group on the effective kd for aNDF corresponded well with the milled barley results. The NDF fraction, directly or indirectly, has a clear impact on the ruminal digestion rate of barley starch. There was no difference in the effective kd for starch between the small (0.126/h) and large (0.129/h) starch granules.     

Basolateral potassium membrane permeability of A6 cells and cell volume regulation

The K⁺ permeabilities (⁸⁶Rb(K) transport) of the basolateral membranes (J_bK) of a renal cell line (A6) were compared under isosmotic and hypo-osmotic conditions (serosal side) to identify the various components involved in cell volume regulation.Changing the serosal solution to a hypo-osmotic one (165 mOsm) induced a fast transient increase in Ca _i (max <1 min) and cell swelling (max at 3–5 min) followed by a regulatory volume decrease (5–30 min) and rise in the SCC (stabilization at 30 min). In isosmotic conditions (247 mOsm), the ⁸⁶Rb(K) transport and the SCC were partially blocked by Ba²⁺, quinidine, TEA and glibenclamide, the latter being the least effective. Changing the osmolarity from isosmotic to hypo-osmotic resulted in an immediate (within the first 3–6 min) stimulation of the ⁸⁶Rb(K) transport followed by a progressive decline to a stable value higher than that found in isosmotic conditions. A serosal Ca²⁺-free media or quinidine addition did not affect the initial osmotic stimulation of J_bK but prevented its secondary regulation, whereas TEA, glibenclamide and DIDS completely blocked the initial J_bK increase. Under hypo-osmotic conditions, the initial J_bK increase was enhanced by the presence of 1 mm of barium and delayed with higher concentrations (5 mm). In addition, cell volume regulation was fully blocked by quinidine, DIDS, NPPB and glibenclamide, while partly inhibited by TEA and calcium-free media.We propose that a TEA- and glibenclamide-sensitive but quinidine-insensitive increase in K⁺ permeability is involved in the very first phase of volume regulation of A6 cells submitted to hypo-osmotic media. In achieving cell volume regulation, it would play a complementary role to the quinidine-sensitive K⁺ permeability mediated by the observed calcium rise.This work was supported by grants from the Commissariat à l'Energie Atomique and the Centre National de Recherche Scientifique URA 638.     

Loss of membrane cholesterol influences lysosomal permeability to potassium ions and protons

Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K⁺ and H⁺, and the organelle stability. Through the measurements of lysosomal β-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-β-cyclodextrin (MβCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K⁺/H⁺ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MβCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K⁺ and H⁺. Loss of this membrane sterol is critical for the organelle acidification and stability.     

Conduction of a nerve impulse along a myelineated fiber while varying the membrane properties of nodes of Ranvier

The mathematical model of a frog myelinated axon [1, 3] has been used to study the dependence of the conduction velocity (theta) on the parameters of the nodal membrane: its capacity (CN), leakage conductance (gl), sodium and potassium maximum conductances (gNa, gK). Calculations have shown that theta practically does not depend on gK:theta raises only by 3% when gK is diminished to zero. The increase of theta with reducing of gl or CN can be described by formulae: theta (m/s) = 19-300.gl (muS) or theta = 16-1.87.CN(pF) (Fig 1,2). Theta depends strongly on gNa:theta approximately equal to (gNa)7/8 (Fig 3). Due to the capacity of Na channels (determined by the gating charge movement) there is a maximum in the relation between the number of Na channels per node and the theta (Fig 4). A clear-cut maximum does exist also in the curve relating theta to the nodal membrane area (Fig. 5a). The position of the maximum and the shape of this curve depend on gl and CN but not on gNa (Fig. 5b).     

Temperature- and structure-dependent interaction of pyrethroids with the sodium channels in frog node of Ranvier

(1) The interaction of a series of pyrethroid insecticides with the Na+ channels in myelinated nerve fibres of the clawed frog, Xenopus laevis, was investigated using the voltage clamp technique. (2) Out of 11 pyrethroids 9 insecticidally active compounds induce a slowly decaying Na+ tail current on termination of a step depolarization, whereas the Na+ current during depolarization was hardly affected. These tail currents are most readily explained by a selective reduction of the rate of closing of the activation gate in a fraction of the Na+ channels that have opened during depolarization. (3) The rate of decay of the Na+ tail current varies considerably with pyrethroid structure. After alpha-cyano pyrethroids the decay is at least one order of magnitude slower than after non-cyano pyrethroids. The decay always follows a single-exponential time course and is reversibly slowed when the temperature is lowered from 25 to 0 degrees C. Arrhenius plots in this temperature range are linear. (4) These results indicate that the relaxation of the activation gate in pyrethroid-affected Na+ channels is governed by an apparent first order, unimolecular process and that the rate of relaxation is limited by a single energy barrier. Application of transition state theory shows that after alpha-cyano pyrethroids this energy barrier is 9.6 kJ/mol higher than after non-cyano pyrethroids. (5) Differences in rate of decay of the Na+ tail current account for the reported differences in repetitive nerve activity induced by various pyrethroids. In addition, the effect of temperature on the rate of decay explains the increase in repetitive activity with cooling.