Electrical excitability of artificial enzyme membranes: III. Hysteresis and oscillations observed with immobilized acetylcholinesterase membranes

Experimental evidence for memory and oscillations in artificial acetylcholinesterase membranes is presented. When acetylcholine is injected on one side of an artificial proteinic membrane bearing acetylcholinesterase, a potential difference is recorded as a function of time. The steady-state potential due to the enzyme activity for increasing and decreasing substrate concentrations exhibits a hysteresis loop. The non-linearity of the enzyme reaction coupled with the diffusion constraints cause also some instabilities, such as oscillations of the membrane potential.     

Monoclonal Antibodies Against the Voltage-Sensitive Sodium Channel from Rat Skeletal Muscle: Mapping Antibody Binding Sites

Twenty-one monoclonal antibodies specific for the rat skeletal muscle voltage-sensitive sodium channel have been characterized according to subunit reactivity, recognition of carbohydrates, and mutual binding interactions. All antibodies recognize the 260-kDa alpha-subunit of the sodium channel on immunoblots. N-Acetylneuraminic acid inhibited the binding of five antibodies in a concentration-dependent manner, but five other monosaccharides known to be components of the channel had no effect on antibody binding. Competition studies using biosynthetically labeled antibodies separated these 21 antibodies into groups recognizing at least nine distinct domains. Through common interactions between domains, these could in turn be associated into two larger topologically related regions. One region encompasses seven interacting domains and 16 antibodies. This region is probably extracellular by virtue of the interaction of one subgroup with N-acetylneuraminic acid, and may represent a particularly immunogenic region on this channel protein.     

Electrical excitability of artificial enzyme membranes: II. Electrochemical and enzyme properties of immobilized acetylcholinesterase membranes

This paper deals with aspects of the reciprocal interaction between enzyme activity and the microenvironment or the potential difference in artificial proteinaceous membranes bearing cross-linked acetylcholinesterase. The potential difference resulting from asymmetric substrate injection into the system is recorded as a function of time. The influence of the membrane charge density on both enzyme activity and potential difference is studied by varying the external solution pH. The enzyme specific potential is initiated by local change of pH at the membrane level and the dependence on the buffer strength is studied. The recorded potential difference appears to be the result of the reciprocal interaction between enzyme reaction and the diffusion of substrate or products.     

Excitability changes in the crustacean motor axon following activity

It has been shown experimentally that the crustacean motor axon is supernormally excitable following a train of action potentials (Zucker 1974). Such a phenomenon can lead to recruitment of terminals which are unexcited at low rates of stimulation. Although currents underlying the crustacean motor axon have been characterized (Connor et al. 1977), it is not known whether this membrane model accounts for a supernormal period, what might cause superexcitablity in this model, or how excitability might change during repetitive stimulation. In present study, it is demonstrated that the crustacean motor axon model does predict a supernormal period, that the supernormal period results from slow recovery from inactivation of the transient potassium, or A, current, and that supernormal excitability is enhanced by repetitive stimulation.     

Atrial Rate And Rhythm Abnormalities In A Patient With Hyperkalemia

A 67 year old man presented with a serum potassium of 7.7 mEq/L and slow atrial flutter with variable A-V block and peaked T waves. Initial treatment for hyperkalemia was followed by an increase in the atrial flutter rate to 300 beats per minute. After hemodialysis the rhythm converted to sinus.     

Membrane Potential Dependent Duration of Action Potentials in Cultured Rat Hippocampal Neurons

(1) Fluctuations of the membrane potential states are essential for the brain functions from the response of individual neurons to the cognitive function of the brain. It has been reported in slice preparations that the action potential duration is dependent on the membrane potential states. (2) In order to examine whether dependence of action potential duration on the membrane potential could happen in isolated individual neurons that have no network connections, we studied the membrane potential dependence of the action potential duration by artificially setting the membrane potentials to different states in individual cultured rat hippocampal neurons using patch-clamp technique. (3) We showed that the action potential of individual neurons generated from depolarized membrane potentials had broader durations than those generated from hyperpolarized membrane potentials. (4) Furthermore, the membrane potential dependence of the action potential duration was significantly reduced in the presence of voltage-gated K⁺ channel blockers, TEA, and 4-AP, suggesting involvement of both delayed rectifier I _K and transient I _A current in the membrane potential dependence of the action potential duration. (5) These results indicated that the dependence of action potential duration on the membrane potential states could be an intrinsic property of individual neurons. Bo Gong and Mingna Liu contributed equally to this work.     

Modulation of membrane potential and ionic currents by the AT1 and AT2 receptors of angiotensin II

Angiotensin II, the principal effector of the renin-angiotensin system, modulates various ionic currents. Its effects on potassium currents, including outward transient potassium current, the inward or outward rectifiers, as well as Ca²⁺-activated potassium currents, is well described. Other ionic currents, such as voltage-dependent calcium currents, cationic or chloride currents, are also altered by the hormone. All these effects provoke changes in membrane potential, such as modulation of action potential firing or resting membrane potential and control intracellular calcium concentration. Summarized here are the results obtained on these membrane electrical properties using electrophysiological recordings.     

Antiphase oscillations of endothelium and smooth muscle [Ca2+]i in vasomotion of rat mesenteric small arteries

The mechanisms leading to vasomotion in the presence of noradrenaline and inhibitors of the sarcoplasmic/endoplasmic reticulum calcium ATPase were investigated in isolated rat mesenteric small arteries. Isobaric diameter and isometric force were measured together with membrane potential in endothelial cells and smooth muscle cells (SMC). Calcium in the endothelial cells and SMC was imaged with confocal microscopy. In the presence of noradrenaline and cyclopiazonic acid, ryanodine-insensitive oscillations in tone were produced. The frequency was about 1 min(-1) and amplitude about 70% of the maximal tone. The amplitude was reduced by indomethacin and increased with L-NAME. Vasomotion was inhibited by nifedipine and by 40 mM potassium. The frequency was increased and amplitude decreased by removal of the endothelium and by application of charybdotoxin and apamin. The vasomotion was associated with in-phase oscillations of membrane potential in endothelial cells and SMC and oscillations of [Ca2+]i that were in near anti-phase. We suggest a working model for the generation of oscillation based on a membrane oscillator where ion channels in both endothelial cells and SMC interact via a current running between the two cell types through myoendothelial gap junctions, which sets up a near anti-phase oscillation of [Ca2+]i in the two cell types.     

The Bacterial Translocon SecYEG Opens upon Ribosome Binding

In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist.     

Excitable cell made of thermal proteinoids

Such phenomena as electrical polarization across the membrane, electrical discharges, current-voltage characteristics, negative resistance and some light characteristics are described for synthetic cells made of thermal proteinoids. The thermal proteinoid cell is considered as a structural and functional model of the excitable natural cell.     

Na<Superscript>+</Superscript> Modulates Anion Permeation and Block of P2X<Subscript>7</Subscript> Receptors from Mouse Parotid Glands

Experiments were conducted to test the hypothesis that aliphatic hydrocarbons bind to pockets/crevices of sodium (Na⁺) channels to cause action potential (AP) block. Aliphatic solutes exhibiting successively greater octanol/water partitition coefficients (K _ow) were studied. Each solute blocked Na⁺ channels. The 50% effective concentration (EC₅₀) to block APs could be mathematically predicted as a function of the solute’s properties. The solutes studied were methyl ethyl ketone (MEK), cyclohexanone, dichloromethane, chloroform and triethylamine (TriEA); the K _ow increased from MEK to TriEA. APs were recorded from frog nerves, and test solutes were added to Ringer’s solution bathing the nerve. When combined with EC₅₀s for solutes with log K _ows < 0.29 obtained previously, the solute EC₅₀s could be predicted as a function of the fractional molar volume (dV/dm = [dV/dn]/100), polarity (P) and the hydrogen bond acceptor basicity (β) by the following equation: Fluidity changes cannot explain the EC₅₀s. Each of the solutes blocks Na⁺ channels with little or no change in kinetics. Na⁺ channel block explains much of the EC₅₀ data. EC₅₀s are produced by a combination of effects including ion channel block, fluidity changes and osmotically induced structural changes. As the solute log K _ow increases to values near 1 or greater, Na⁺ channel block dominates in determining the EC₅₀. The results are consistent with the hypothesis that the solutes bind to channel crevices to cause Na⁺ channel and AP block.     

Self-excitation in a porous membrane doped with sorbitan monooleate (Span-80) induced by an Na+/K+ concentration gradient

The electrical potential across a fine-pore membrane doped with sorbitan monooleate (Span-80) imposed between aqueous solutions of NaCl and KCl was studied. It was found that this system showed rhythmic and sustained oscillations of electrical potential between the two aqueous solutions. These oscillations were attributed to the change of permeability of Na+ and K+ across the membrane, which originated from the phase transition of Span-80 molecules within the fine pores. Impedance measurement across the membrane also suggested a change in permeability. It was found that this membrane exhibited the property of differential negative resistance. In relation to this, it was shown that Na+ and K+ have different effects on the aggregation of Span-80 molecules. The mechanism of oscillation is discussed in relation to the ability of Span-80 molecules to behave as a dynamic channel through the membrane. This oscillatory phenomenon is interesting because in biological nervous membranes a difference between the concentrations of Na+ and K+ across the membranes is essential for excitability.     

低氧对大鼠颈动脉体培养细胞膜电位及输入阻抗的影响

本文采用离体培养的大鼠颈动脉体主细胞(glomus cell)的细胞群体和单细胞的标本,观察了不同程度的低氧对细胞膜电位(MP)和输入阻抗(Ri)的影响。在常氧(20％O_2,5％CO_2,75％N_2)条件下培养的细胞(常氧细胞)暴露在常氧中测得的MP和Ri值为对照值。当常氧细胞暴露在低氧(10％O_2,5％CO_2,85％N_2)时,MP幅度有的增加(超极化),有的减少(去极化),MP值增加和减少的细胞数各占细胞总数的百分比大体相同,当暴露在100％N_2中时,MP增加的细胞百分数明显高于MP减少的细胞百分数。在低氧条件下培养的细胞(低氧细胞),暴露在常氧中测得的MP和Ri值与对照值比较无显著性差别,但是,当低氧细胞暴露在低氧中时MP和Ri的值均明显增加(MP:P<0.01,Ri:P<0.05)。结果提示:颈动脉体的glomus细胞可能在感受pO_2变化中起重要作用。     

Positive inotropic action of NMDA receptor antagonist (+)-MK801 in rat heart

(+)-MK801, a noncompetitive NMDA receptor antagonist, was reported to exhibit anticonvulsive and neuroprotective activities during the postischemic period. Intravenous administration of (+)-MK801 produced tachycardia in rats, but bradycardia in pigs. We examined the mechanical and electrophysiological effects of (+)-MK801 on rat cardiac tissues. (+)-MK801 dose-dependently increased (3–100 µM) twitch tension in rat atria and ventricular strips. The spontaneous beating rate in rat right atria, however, was dose-dependently decreased by (+)-MK801. The inotropic effect of (+)-MK801 was affected neither by ₁-antagonist (1 µM prazosin) nor by ₁-adrenoceptor antagonist (3 µM atenolol), but significantly by a transient outward K⁺ channel blocker (3 mM 4-aminopyridine). (+)-MK801 did not cause any significant change of intracellular cAMP content. Electrophysiological study in rat ventricular cells revealed that (+)-MK801 concentration-dependently prolonged the action potential duration with a concomitant decrease in the maximum rate of the action potential upstroke (V_max) and an increase in the recovery time constant of V_max. Voltage clamp study showed that (+)-MK801 (3 µM) reduced inward Na⁺ current (I_Na), along with a slowing of its recovery from inactivation and a slight negative shift of its voltage-dependent steady-state inactivation curves. At a much higher concentration (30 µM), (+)-MK801 slightly reduced the amplitude of L-type calcium inward current (I_Ca), although the voltage dependence of its steady-state inactivation was unaffected. For the potassium currents in rat ventricular cells, 3 µM of (+)-MK801 reduced the peak transient outward current (I_to), steady-state outward current (I_ss) and inward current through K₁ channels. The inhibition of I_to was associated with a prominent negative shift in the voltage dependence of its steady-state inactivation curve. The outward current through K₁ channels was unaffected. These results indicate that (+)-MK801 may be a strong I_Na and I_to blocker with some I_Ca blocking activity. The inhibition of I_to and other K⁺ efflux would prolong action potential duration, produce positive inotropic action and contribute to the negative chronotropic effect of (+)-MK801.     

Protein- and Lipid-Reactive Agents Alter Outer Hair Cell Lateral Membrane Motor Charge Movement

Outer hair cells from the mamma*lian cochlea are mechanically active cells that rely on charged voltage sensors within their lateral plasma membrane to gate the integral membrane motor protein, prestin, into one of two area states. Here we use protein and lipid reactive reagents to probe the influence of these bilayer components on motor-induced nonlinear membrane capacitance. Of the protein-reactive reagents tested, cross-linking and sulfhydryl reagents were most effective in altering steady state and time-varying motor activity. Of the lipid-altering agents, chloroform and HePC were most effective. Chloroform, in particular, drastically modified the susceptibility of the motor to prior voltage (initial conditions). Our data suggest that outer hair cell motor activity derives substantially from interactions with its lipid environment.This revised version was published online in June 2005 with a corrected cover date.     

The problem of nonstationary ion fluxes in excitable membranes

Time-dependent electrodiffusion through a membrane is analysed within a simple model treating the boundary-layers in a consistent manner. It is shown that time-independent reversal potentials for the ion fluxes exist only under steady-state conditions. We argue that this result holds very generally. Therefore nonstationary effects like ion storage and depletion inside the membrane should not contribute to the phenomena of excitability.Glossary of Symbols A _mv [V] functional cf. Equation (3) - C membrane capacitance - d one half the thickness of the membrane - F[V] functional cf. Equation (1) - g _i electrochemical potential inside membrane - g _i electrochemical potentials outside membrane at x ±d, respectively - i (index) refers to i-th ionic species - J electric current across membrane - j = j ^} = j ^< current density measured by external electrodes - j _i (x) current density inside membrane in x-direction - j _i ^inst(x) instantaneous current density - J _i ^stat steady-state current density - k Boltzmann constant - m (index) is used in Sec. 2 to denote the independent diffusion currents - n ^< ionic strength of electrolyte at x = - - n _i density of ions inside membrane - n _i density of ions outside membrane at x = ±, respectively - Q charge per unit area of boundary layers at x ± d, respectively - Q ₀ fixed charge per unit area of membrane - q elementary charge - q _i ionic charges - T temperature - it time - V membrane potential (= (-)-()) - V _i Nernst potential - V potential drops inside boundary layers (can be neglected, see Appendix II) - V ^± potential steps at x = ± d, cf. Equation (29) - V ₀ = V ^–-V ⁺ - w _i activation energy inside membrane - x spatial coordinate perpendicular to membrane - y, z spatial coordinates parallel to membrane - dielecric constant - ₀ dielectric constant of electrolyte solution ( 80) - _m dielectric constant of membrane ( 5) - (x) electrostatic potential - charge density of boundary layers - ⁰ fixed charge density inside membrane - spatial average, cf. Equation (12)