Cation selectivity of the resting membrane of squid axon

Summary Permeability constant ratios among monovalent cations were studied in the resting membrane of a giant axon of a Pacific squid,Loligo opalescens, by observing the relationship between the membrane potential and the ion concentration.The average permeability ratios are: Tl, 1.8; K, 1.0; Rb, 0.72; Cs, 0.16; Na, <0.08; Li, <0.08. These permeability ratios suggest that neither valinomycin nor nonactin are adequate models for the sites producing the resting permeability in the axonal membrane.Cyclic polyetherbis(t-butyl cyclohexyl) 18-crown-6 does not increase the permeability ratioP _Cs/P _K except when applied at concentrations (5×10^–5 m) at which the surfactant properties of this molecule may become significant.     

Nerve membrane electrical characteristics near the resting state

Summary An experimental analysis of the squid axon membrane impedance in the vicinity of the resting state and as a function of frequency is presented. Particular attention was devoted to the measurement of theresonance frequency, for which the absolute magnitude of the impedance attains its maximum value, in different, extracellular solutions, at various temperatures and in the presence of constant depolarizations or hyperpolarizations.The variations in the concentration of sodium, potassium and divalent ions and the addition of tetrodotoxin, changed markedly the maximum impedance but had little effect, at a fixed temperature, on the resonance frequency, whose temperature dependance is described by aQ ₁₀ variable from 3.7 (around 4 °C) to 1.9 (around 15 °C). Substitution of heavy water decreased the resonance frequency by a factor 1.25, fairly independent of temperature. Steady depolarizations or hyperpolarizations produced large variations of the resonance frequency, with strong temperature dependance.The results indicate that the resonance frequency is directly related to the membrane permeability changes which take place quite independently of the composition of the extra cellular solution and are governed by the electric field existing within the membrane structure rather than by the total membrane potential, to which membrane-solution boundary potentials can give a large contribution.     

Structural complexes in the squid giant axon membrane sensitive to ionic concentrations and cardiac glycosides

Giant nerve fibers of squid Sepioteuthis sepioidea were incubated for 10 min in artificial sea water (ASW) under control conditions, in the absence of various ions, and in the presence of cardiac glycosides. The nerve fibers were fixed in OsO4 and embedded in Epon, and structural complexes along the axolemma were studied.     

Electrical properties of squid axon membrane. II. Effect of partial degradation by phospholipase A and pronase on electrical characteristics

Passive electrical characteristics of perfused squid axon membrane are investigated. In a previous publication, we reported that the capacitance of intact squid axon membrane is partly frequency dependent. We extended the same measurement to perfused axons. We found that the electrical characteristics of perfused axon membrane are essentially the same as those of intact axons. In this work, we investigated the effects of phospholipase A and pronase on the membrane capacitance. Phospholipase A is known to block the sodium activation and pronase to eliminate the sodium inactivation. Phospholipase A is found to increase the frequency dependent as well as the frequency independent capacitances. Our tentative conclusion is that this enzyme perturbs the lipid structure and decreases its thickness. Pronase is found to increase the frequency dependent capacitance slightly while the capacitance of the lipid layer remains unaltered. Although voltage clamp data indicate that the pronase disrupts the excitatory mechanism extensively, this enzyme has relatively little effect on the overall membrane capacitance.     

Electrical properties of squid axon membrane. II. Effect of partial degradation by phospholipase A and pronase on electrical characteristics.

Passive electrical characteristics of perfused squid axon membrane are investigated. In a previous publication, we reported that the capacitance of intact squid axon membrane is partly frequency dependent. We extended the same measurement to perfused axons. We found that the electrical characteristics of perfused axon membrane are essentially the same as those of intact axons. In this work, we investigated the effects of phospholipase A and pronase on the membrane capacitance. Phospholipase A is known to block the sodium activation and pronase to eliminate the sodium inactivation. Phospholipase A is found to increase the frequency dependent as well as the frequency independent capacitances. Our tentative conclusion is that this enzyme perturbs the lipid structure and decreases its thickness. Pronase is found to increase the frequency dependent capacitance slightly while the capacitance of the lipid layer remains unaltered. Although voltage clamp data indicate that the pronase disrupts the excitatory mechanism extensively, this enzyme has relatively little effect on the overall membrane capacitance.     

Electrical characteristics of stomatal guard cells: The ionic basis of the membrane potential and the consequence of potassium chlorides leakage from microelectrodes

The membrane electrical characteristics of stomatal guard cells in epidermal strips from Vicia faba L. and Commelina communis L. were explored using conventional electrophysiological methods, but with double-barrelled microelectrodes containing dilute electrolyte solutions. When electrodes were filled with the customary 1–3 M KCl solutions, membrane potentials and resistances were low, typically decaying over 2–5 min to near-30 mV and <0.2 k·cm² in cells bathed in 0.1 mM KCl and 1 mM Ca²⁺, pH 7.4. By contrast, cells impaled with electrodes containing 50 or 200 mM K⁺-acetate gave values of-182±7 mV and 16±2 k·cm² (input resistances 0.8–3.1 G, n=54). Potentials as high as (-) 282 mV (inside negative) were recorded, and impalement were held for up to 2 h without appreciable decline in either membrane parameter. Comparison of results obtained with several electrolytes indicated that Cl^- leakage from the microelectrode was primarily responsible for the decline in potential and resistance recorded with the molar KCl electrolytes. Guard cells loaded with salt from the electrodes also acquired marked potential and conductance responses to external Ca²⁺, which are tentatively ascribed to a K⁺ conductance (channel) at the guard cell plasma membrane.Measurements using dilute K⁺-acetate-filled electrodes revealed, in the guard cells, electrical properties common to plant and fungal cell membranes. The cells showed a high selectivity for K⁺ over Na⁺ (permeability ratio P_Na/P_K=0.006) and a near-Nernstian potential response to external pH over the range 4.5–7.4 (apparent P_H/P_K=500–600). Little response to external Ca²⁺ was observed, and the cells were virtually insensitive to CO₂. These results are discussed in the context of primary, charge-carrying transport at the guard cell plasma membrane, and with reference to possible mechanisms for K⁺ transport during stomatal movements. They discount previous notions of Ca²⁺-and CO₂-mediated transport control. It is argued, also, that passive (diffusional) mechanisms are unlikely to contribute to K⁺ uptake during stomatal opening, despite membrane potentials which, under certain, well-defined conditions, lie negative of the potassium equilibrium potential likely prevailing.Abbreviations and symbols EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Hepes 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - Mes 2-(N-morpholino) propanesulfornic acid - E equilibrium potential - G_m membrane conductance - R_in input resistance - V_m membrane potential     

Ionic channels through the axon membrane (a review)

Ionic channels are discrete sites at which the passive movement of ions takes place during nervous excitation. Three types of channels are distinguished. 1. Leakage channels that are permanently open to various cations. 2. Na channels that open promptly on depolarization but slowly close again (inactivate) on sustained depolarization and that are predominantly permeable to Na⁺ ions. 3. K channels that on depolarization open after some delay but stay open and that are mainly passed by K⁺ ions. The selectivity sequence of the Na channels of the squid axon (or frog nerve) is as follows: Na⁺ ≈ Li⁺>(T1+)>NH⁺ ₄?K⁺> Rb⁺, Cs⁺; that of K channels is: (T1+)>K⁺>Rb⁺>NH⁺ ₄?Na⁺, Cs⁺, Na channels are selectively blocked by tetrodotoxin (TTX) or saxitoxin (STX), K channels by tetraethylammonium ions (TEA). Either channel type is reversibly blocked when one drug molecule binds to one site per channel, the equilibrium dissociation constant of these reactions being about 3×10^?9 MTTX (or STX) and 4×10^?4 M TEA, respectively. Because of their specificity and high affinity, TTX and STX are used to “titrate” the Na channels whose density appears to be of the order of 100/Μm². The “gates” of the channels operate as a function of potential and time but independent of the permeating ion species. Drugs (e.g. veratridine) and enzymes (e.g. pronase, applied intraaxonally) cause profound changes in the gating function of the Na channels without influencing their selectivity. This points to separate structures for gating and ion discrimination. The latter is thought to be, in part, brought about by a “selectivity filter” of which detailed structural ideas exist. Recent experiments suggest that the gates of the Na channels are controlled by charged particles moving within the membrane under the influence of the electrical field.     

Study of ionic currents across a model membrane channel using Brownian dynamics.

Brownian dynamics simulations have been carried out to study ionic currents flowing across a model membrane channel under various conditions. The model channel we use has a cylindrical transmembrane segment that is joined to a catenary vestibule at each side. Two cylindrical reservoirs connected to the channel contain a fixed number of sodium and chloride ions. Under a driving force of 100 mV, the channel is virtually impermeable to sodium ions, owing to the repulsive dielectric force presented to ions by the vestibular wall. When two rings of dipoles, with their negative poles facing the pore lumen, are placed just above and below the constricted channel segment, sodium ions cross the channel. The conductance increases with increasing dipole strength and reaches its maximum rapidly; a further increase in dipole strength does not increase the channel conductance further. When only those ions that acquire a kinetic energy large enough to surmount a barrier are allowed to enter the narrow transmembrane segment, the channel conductance decreases monotonically with the barrier height. This barrier represents those interactions between an ion, water molecules, and the protein wall in the transmembrane segment that are not treated explicitly in the simulation. The conductance obtained from simulations closely matches that obtained from ACh channels when a step potential barrier of 2-3 kTr is placed at the channel neck. The current-voltage relationship obtained with symmetrical solutions is ohmic in the absence of a barrier. The current-voltage curve becomes nonlinear when the 3 kTr barrier is in place. With asymmetrical solutions, the relationship approximates the Goldman equation, with the reversal potential close to that predicted by the Nernst equation. The conductance first increases linearly with concentration and then begins to rise at a slower rate with higher ionic concentration. We discuss the implications of these findings for the transport of ions across the membrane and the structure of ion channels.     

Phosphatidylethanolamine-phosphatidylglycerol bilayer as a model of the inner bacterial membrane

Phosphatidylethanolamine (PE) and phosphatidylglycerol (PG) are the main lipid components of the inner bacterial membrane. A computer model for such a membrane was built of palmitoyloleoyl PE (POPE) and palmitoyloleoyl PG (POPG) in the proportion 3:1, and sodium ions (Na+) to neutralize the net negative charge on each POPG (POPE-POPG bilayer). The bilayer was simulated for 25 ns. A final 10-ns trajectory fragment was used for analyses. In the bilayer interfacial region, POPEs and POPGs interact readily with one another via intermolecular hydrogen (H) bonds and water bridges. POPE is the main H-bond donor in either PEPE or PEPG H-bonds; PGPG H-bonds are rarely formed. Almost all POPEs are H-bonded and/or water bridged to either POPE or POPG but PE-PG links are favored. In effect, the atom packing in the near-the-interface regions of the bilayer core is tight. Na+ does not bind readily to lipids, and interlipid links via Na+ are not numerous. Although POPG and POPE comprise one bilayer, their bilayer properties differ. The average surface area per POPG is larger and the average vertical location of the POPG phosphate group is lower than those of POPE. Also, the alkyl chains of POPG are more ordered and less densely packed than the POPE chains. The main conclusion of this study is that in the PE-PG bilayer PE interacts more strongly with PG than with PE. This is a likely molecular-level event behind a regulating mechanism developed by the bacteria to control its membrane permeability and stability consisting in changes of the relative PG/PE concentration in the membrane.     

Electrical coupling and dye transfer between axon segments in the medium giant axon of the earthworm

Electrical coupling between axon segments has been studied in the medium giant axon, which had been partially isolated from the ventral nerve cord of the earthworm. Evidence has been obtained that in addition to the coupling structures in the septum there are diffusion- and current-pathways through the pseudo-myelin which seem to be more permeable to fluorescein than the channels in the septum. These findings offer an explanation for the discrepancy of the experimentally determined specific resistance of the septum and the expected values based on nexus density and channel size in the septum.Based on material presented at the Symposium Intercellular Communication Stuttgart, September 16–17, 1982     

Generation of resting membrane potential

This brief review is intended to serve as a refresher on the ideas associated with teaching students the physiological basis of the resting membrane potential. The presentation is targeted toward first-year medical students, first-year graduate students, or senior undergraduates. The emphasis is on general concepts associated with generation of the electrical potential difference that exists across the plasma membrane of every animal cell. The intention is to provide students a general view of the quantitative relationship that exists between 1) transmembrane gradients for K(+) and Na(+) and 2) the relative channel-mediated permeability of the membrane to these ions.     

Electrical characteristics in an excitable element of lipid membrane.

Electrical characteristics in a membrane constructed from a porous filter adsorbed with a lipid analogue, dioleoyl phosphate (DOPH), were investigated in a situation interposed between 100 mM NaCl + 3 mM CaCl2 and 100 mM KCl. Calcium ions affected significantly the membrane characteristics. The membrane potential was negative on the KCl side, which implies the higher permeability to K+ than Na+; this tendency was increased by a tiny amount of Ca2+. While the membrane showed a low electrical resistance of several k omega . cm2 under K+/Na+ gradient, it showed several M omega . cm2 by Ca2+. The surface structure of the membrane exhibited many voids in the low-resistance state, but the surface was covered by oil droplets in the high-resistance state. Oscillations of the membrane potential appeared spontaneously with application of the electrical current from the KCl side to the NaCl + CaCl2 side. The frequency was increased with the electrical current. All these results were explained comprehensively using an electrochemical kinetic model taking account of the Ca2+ binding effect, where DOPH assemblies make a phase transition between oil droplets due to Ca2+ and multi-bilayers with excess K+. The oscillation arises from coupling of the phase transition to accumulation and release of K+ or Ca2+. This membrane can be used as an excitable element regulated by Ca2+ in neuro-computer devices.     

Light scattering spectroscopy of the squid axon membrane

Light scattering studies on the giant squid axon were done using the technique of optical mixing spectroscopy. This experimental approach is based on the use of laser light to detect the fluctuations of membrane macromolecules which are associated with conductance fluctuations. The light scattering spectra were similar to the Lorentzian-like behavior of conductance fluctuations, possibly reflecting an underlying conformational change in the specific membrane sites responsible for the potassium ion conductance. The amplitude of the spectra measured, increased when the membrane was depolarized and decreased on hyperpolarization. The spectra were fit to the sum of two terms, a (1/fcomponent and a simple Lorentzian term. Spectra from deteriorating axons did not show sensitivity to membrane potential changes. It is shown theoretically that fluctuations due to the voltage-dependent variable, n, of the Hodgkin-Huxley formalism are identical to the voltage fluctuations. The derived power spectrum is that of a second order system, capable of showing resonance peaking only if the voltage dependence of the potassium rate constants is included in the analysis. The lack of resonance peaking in the observed light scattering spectra, indicates that the data are best described by a damped second order system.