Caffeine-induced oscillations of the membrane potential inAplysia neurons

It has been found in culturedAplysia neurons, including L7 and L2–L6 neurons, that bath application of 40 mM caffeine evokes oscillations of the membrane potential (MP) with the amplitude of about 40 mV. The frequency of oscillations, on the crest of which action potentials (AP) arise, varied from 0.2 to 0.5 sec¹. The effect of caffeine was completely reversible. The MP waves demonstrated high sensitivity to membrane polarization: artificial depolarization increased the frequency of oscillations, while even subtle hyperpolarization resulted in a decrease in the frequency up to their complete disappearance. External application of CdCl₂ (1 mM), a nonspecific blocker of calcium channels, or ryanodine (50 μM, 20 min), release of Ca²⁻ from the intracellular stores, replacement of Ca²⁺ in the external medium by Mg²⁻, or Na⁺ by Li⁺, did not exert visible effect on the parameters of MP waves. It was concluded that Ca ions (changing of intracellular concentration of which is due to such processes as inward calcium current, ryanodine-sensitive caffeine-induced calcium release from the intracellular, stores, sodium-calcium exchange through the plasma membrane) do not play any significant part in generation of the MP waves. The most probable mechanism of caffeine-induced oscillations in the studied nerve cells is inhibition of voltage-activated outward potassium current and, as could be seen from our mathematical modeling, slowdown of inactivation of inward sodium current. It seems likely that these oscillations have a purely membrane origin. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 102–111, March–April, 2000.     

Extracellular calcium and microwave enhancement of membrane conductance in snail neurons

Summary Microwave irradiation has been shown to decrease the input resistance of snail neurons. In this study, we examined the role of extracellular calcium in triggering the microwave-induced enhancement of membrane conductance. Two sets of experiments were conducted. In the first set, nerve cells were superfused using Ringer solution with added Cd²⁺ (0.9 mM) which is a known blocker of calcium channels. In the second set, cells were superfused with low Ca²⁺ (0.7 mM) Ringer solution. Microwave irradiation was conducted at 2,450 MHz for 30 min with a specific absorption rate of 13 mW/g. It was found that 7 mM to 0.7 mM lowering of Ca²⁺ in bathing solution as well as blocking of calcium channels in neuronal membrane by means of Cd²⁺ did not influence the fall in membrane resistance induced by microwave radiation. In fact, the observed changed in membrane resistance in these experiments were nearly equal to those observed for neurons superfused by normal Ringer's. Thus, these results rule out the possible contribution of external Ca²⁺ in the observed microwave effect. Experiments with high Ca²⁺ solution also support this conclusion.     

Voltage dependence of acetylcholine-activated membrane conductance in sympathetic ganglion neurons

Acetylcholine-induced membrane conductance was investigated in superior cervical ganglion neurons using a patch-clamp technique. It was found that hyperpolarization and depolarization produce an increase and a reduction in acetylcholine (ACh) conductance. This reduction was unconnected with either reversal of the current induced by iontophoretic ACh application or the presence of Ca ions in the external solution. The time constant of relaxation (_r) of this current, produced by a jump in membrane potential, was found to increase e-fold when the membrane was hyperpolarized by 70 mV, matching the voltage dependence of ACh conductance. This led to the hypothesis that voltage-dependent ACh-induced conductance is entirely determined by the voltage dependence of nicotinic receptor channel gating kinetics.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 20, No. 2, pp. 167–171, March–April, 1988.     

Potassium ion accumulation in a periaxonal space and its effect on the measurement of membrane potassium ion conductance

Summary Potassium currents of various durations were obtained from squid giant axons voltage-clamped in artificial seawater solutions containing sufficient tetrodotoxin to block the sodium conductance completely. From instantaneous potassium current-voltage relations, the reversal potentials immediately at the end of these currents were determined. On the basis of these reversal potential measurements, the potassium ion concentration gradient across the membrane was shown to decrease as the potassium current duration increased. The kinetics of this change was shown to vary monotonically with the potassium ion efflux across the membrane estimated from the integral over time of the potassium current divided by the Faraday, and to be independent of both the external sodium ion concentration and the presence or absence of membrane series resistance compensation. It was assumed that during outward potassium current flow, potassium ions accumulated in a periaxonal space bounded by the membrane and an external diffusion barrier. A model system was used to describe this accumulation as a continuous function of the membrane currents. On this basis, the mean periaxonal space thickness and the permeability of the external barrier to K⁺ were found to be 357 Å and 3.21×10^–4 cm/sec, respectively. In hyperosmotic seawater, the value of the space thickness increased significantly even though the potassium currents were not changed significantly. Values of the resistance in series with the membrane were calculated from the values of the permeability of the external barrier and these values were shown to be roughly equivalent to series resistance values determined by current clamp measurements. Membrane potassium ion conductances were determined as a function of time and voltage. When these were determined from data corrected for the potassium current reversal potential changes, larger maximal potassium conductances were obtained than were obtained using a constant reversal potential. In addition, the potassium conductance turn-on with time at a variety of membrane potentials was shown to be slower when potassium conductance values were obtained using a variable reversal potential than when using a constant reversal potential.     

Relaxation studies on the interaction of hexamethonium with acetylcholine-receptor channels inAplysia neurons

The mode of action of the cholinergic antagonist hexamethonium on the excitatory responses of voltage-clamped Aplysia neurons to acetylcholine (ACh) has been examined by voltage- and concentration-jump relaxation analysis. At steady-state concentrations of ACh hyperpolarizing command steps induced inward current relaxations to a new steady-state level (Iss). The time constants of these inward relaxations, tau f, which approximate the mean single-channel lifetime, were increased both by increasing the membrane potential and by lowering the bath temperature (Q10 = 3) but were not affected by increasing the ACh concentration over the dose range employed. In the presence of hexamethonium hyperpolarizing command steps produced biphasic relaxations of the agonist-induced current. tau f was reduced in a voltage-dependent manner, the degree of reduction increasing with hyperpolarization. Slow, inverse relaxations were also triggered in the presence of hexamethonium. The time constant of this relaxation was reduced by increasing membrane potential and hexamethonium concentration. Both the estimated association (kf = 5 X 10(4) M-1 . sec-1) and the estimated dissociation (kb = 0.24-0.29 sec-1) rate constants derived from a three-state sequential model for block by hexamethonium were independent of the membrane potential. Similar rate constants were estimated from experiments with the concentration-jump technique, which were also independent of the membrane potential over the range -50 to -110 mV. It is suggested that the voltage-dependent actions of hexamethonium may originate either from an alteration of the channel opening and closing rate constants through an allosteric interaction with the ACh receptor, rather than through an influence of the transmembrane electric field on the rate of drug binding, or through a fast reaction which is rate-limited by voltage-independent diffusion.     

Effects of batrachotoxin on membrane potential and conductance of squid giant axons

The effects of batrachotoxin (BTX) on the membrane potential and conductances of squid giant axons have been studied by means of intracellular microelectrode recording, internal perfusion, and voltage clamp techniques. BTX (550–1100 nM) caused a marked and irreversible depolarization of the nerve membrane, the membrane potential being eventually reversed in polarity by as much as 15 mv. The depolarization progressed more rapidly with internal application than with external application of BTX to the axon. External application of tetrodotoxin (1000 nM) completely restored the BTX depolarization. Removal or drastic reduction of external sodium caused a hyperpolarization of the BTX-poisoned membrane. However, no change in the resting membrane potential occurred when BTX was applied in the absence of sodium ions in both external and internal phases. These observations demonstrate that BTX specifically increases the resting sodium permeability of the squid axon membrane. Despite such an increase in resting sodium permeability, the BTX-poisoned membrane was still capable of undergoing a large sodium permeability increase of normal magnitude upon depolarizing stimulation provided that the membrane potential was brought back to the original or higher level. The possibility that a single sodium channel is operative for both the resting sodium, permeability and the sodium permeability increase upon stimulation is discussed.     

Critical change of ion conductance and self-sustained potential oscillation in artificial membrane systems

A change in conductance of an artificial membrane at a threshold concentration of environmental salt solution was interpreted in terms of a change in adsorptive reaction rate on the interface which was derived from an autocatalytic reaction model. The model also accounted for a self-sustained potential oscillation which was observed when the salt concentration of one side of the membrane was higher than the threshold mentioned above and that of the other side lower.     

Reduction of the voltage-dependent calcium current inAplysia neurons by pentobarbital

Effects of pentobarbital on the calcium current of Aplysia neurons were investigated under current- and voltage-clamp conditions using the conventional two-microelectrode technique. Pentobarbital attenuated the progressive broadening of repeated action potentials of somata, suggesting a reduction in the calcium current. When calcium ion was replaced with barium ion in the perfusing solution, in which neither sodium nor potassium ions carried transmembrane currents, the barium current (IBa) which flowed through the calcium channel of the cell membrane was generated by depolarizing pulses of several hundred milliseconds applied every 1 min from a holding potential of -50 mV. The IBa was not affected by tetrodotoxin (30 microM). The current was decreased by pentobarbital (0.1-5 mM) in a dose-dependent manner. The inhibition was much greater at a lower pH of the perfusate, indicating that the uncharged form of the agent was responsible. The voltage-dependent inactivation of the IBa proceeded with two time constants [190 +/- 21 and 2020 +/- 146 msec (N = 4) at -10 mV], both of which were shortened by adding 1 mM pentobarbital [to 120 +/- 18 and 540 +/- 51 msec (N = 4), respectively]. The IBa recovered from the inactivation with two time constants [60 +/- 7 and 871 +/- 76 msec (N = 3) at -50 mV]. The anesthetic (1 mM) prolonged both of them, to 124 +/- 20 and 1480 +/- 172 msec (N = 3), respectively, resulting in a use-dependent depression of the current at 2-Hz stimulation. Pentobarbital reduced the IBa to a greater extent when the holding potential was more positive (-30 instead of -50 mV), indicating a higher affinity of the drug to the inactivated state of the channel. These findings suggest that the attenuation of the progressive broadening of successive spikes by pentobarbital is due to a decrease in the voltage- and time-dependent calcium current, ending in depression of transmitter release from the nerve terminal.     

ATP-activated ionic conductance in the somatic membrane of mammalian sensory ganglionic neurons

ATP receptors, activation of which leads to an increase in membrane conductance for monovalent cations and to the appearance of an inward ionic current at negative membrane potential values, have been found on the outer surface of the somatic membrane of sensory ganglionic neurons of rats and cats. The ATP-activated ionic channel is almost equally permeable for ions of the alkali metals and possesses marked permeability even for Tris and tetraethylammonium ions. Simultaneously with this, the channel demonstrates marked anomalous rectification. For the receptor to be activated, one ATP molecule must interact with it. Activation of the receptors is not connected with the use of energy of high-energy bonds. Affinity of the receptors for agonists is increased with a fall of membrane potential. Adenine nucleotides containing at least two phosphoric acid residues are agonists. The most active agonist is ATP. Adenosine monophosphate, adenosine, and puromycin competitively block these receptors. Pyrimidine nucleotides, and also purine nucleotides — guanosine triphosphate and inosine triphosphate — do not interact with ATP receptors. ATP-activated currents develop quickly and are then slowly desensitized. The desensitization time constant decreases with an increase in ATP concentration, to reach about 2 sec at a saturating concentration.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 16, No. 3, pp. 327–336, May–June, 1984.     

Effects of membrane lipids on ion channel structure and function

Biologic membranes are not simply inert physical barriers, but complex and dynamic environments that affect membrane protein structure and function. Residing within these environments, ion channels control the flux of ions across the membrane through conformational changes that allow transient ion flux through a central pore. These conformational changes may be modulated by changes in transmembrane electrochemical potential, the binding of small ligands or other proteins, or changes in the local lipid environment. Ion channels play fundamental roles in cellular function and, in higher eukaryotes, are the primary means of intercellular signaling, especially between excitable cells such as neurons. The focus of this review is to examine how the composition of the bilayer affects ion channel structure and function. This is an important consideration because the bilayer composition varies greatly in different cell types and in different organellar membranes. Even within a membrane, the lipid composition differs between the inner and outer leaflets, and the composition within a given leaflet is both heterogeneous and highly dynamic. Differential packing of lipids (and proteins) leads to the formation of microdomains, and lateral diffusion of these microdomains or "lipid rafts" serve as mobile platforms for the clustering and organization of bilayer constituents including ion channels. The structure and function of these channels are sensitive to specific chemical interactions with neighboring components of the membrane and also to the biophysical properties of their membrane microenvironment (e.g., fluidity, lateral pressure profile, and bilayer thickness). As specific examples, we have focused on the K+ ion channels and the ligand-gated nicotinicoid receptors, two classes of ion channels that have been well-characterized structurally and functionally. The responsiveness of these ion channels to changes in the lipid environment illustrate how ion channels, and more generally, any membrane protein, may be regulated via cellular control of membrane composition.     

Second messenger-controlled membrane conductance in locust (Locusta migratoria) olfactory neurons

The membrane conductance of olfactory neurons of Locusta migratoria was examined using the whole-cell configuration of the patch-clamp technique. Intracellular application of the second messenger inositol 1,4,5 trisphosphate (IP(3)) via a dual pipette technique elicited a clear increase in the membrane conductance. The IP(3)-induced conductance increased due to a rise in the extracellular concentration of calcium from 100&mgr;M to 4mM. Micromolar concentrations of ruthenium red partially blocked the IP(3)-induced increase in membrane conductance. Stimulating olfactory neurons with odour (hexenoic acid) resulted in an increase in the membrane conductance partially similar to that mediated IP(3). These findings suggest that stimulation with appropriate odours as well as intracellular application of IP(3) activate the same calcium-permeable ion channels in the plasma membrane of insect olfactory neurons.     

A novel action of quinine and quinidine on the membrane conductance of neurons from the vertebrate retina

The cinchona alkaloids quinine and quinidine have been shown to block a broad range of voltage-gated membrane conductances in a variety of excitable tissues. Using the whole-cell version of the patch clamp technique, we examined the effects of these compounds on voltage- dependent currents from horizontal cells dissociated enzymatically from the all-rod retina of the skate. We report here a novel and unexpected action of quinine and quinidine on isolated horizontal cells. In addition to blocking several of the voltage-activated currents of these cells, the introduction of the alkaloids evoked a large outward current when the cells were held at depolarized potentials. Using tail current analysis, the reversal potential of the outward current was close to O mV, and the current was markedly suppressed by extracellularly applied cobalt, acetate, and halothane. Depolarization in the presence of quinine also permitted entry into the cells of extracellularly applied Lucifer yellow (MW = 443 D), whereas a 3-kD fluorescein-dextran complex was excluded. These findings suggest that the large, apparently nonselective conductance induced by quinine and quinidine results from the opening of hemi-gap junctional channels.     

Effects of hyperbaric gases on membrane nanostructure and function in neurons

This mini-review summarizes current ideas of how hyperbaric gases (>1-10 atmospheres absolute) affect neuronal mechanisms of excitability through molecular interaction with membrane components. The dynamic nature of the lipid bilayer, its resident proteins, and the underlying cytoskeleton make each respective nanostructure a potential target for modulation by hyperbaric gases. Depending on the composition of the gas mixture, the relative concentrations of O(2) and inert gas, and total barometric pressure, the net effect of a particular gas on the cell membrane will be determined by the gas' 1) lipid solubility, 2) ability to oxidize lipids and proteins (O(2)), and 3) capacity, in the compressed state, to generate localized shear and strain forces between various nanostructures. A change in the properties of any one membrane component is anticipated to change conductance of membrane-spanning ion channels and thus neuronal function.