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.     

Dual-wavelength ratiometric fluorescence measurement of the membrane dipole potential.

The electrostatic potentials associated with cell membranes include the transmembrane potential (delta psi), the surface potential (psi s), and the dipole potential (psi D). psi D, which originates from oriented dipoles at the surface of the membrane, rises steeply just within the membrane to approximately 300 mV. Here we show that the potential-sensitive fluorescent dye 1-(3-sulfonatopropyl)-4-[beta[2-(di-n-octylamino)-6- naphthyl]vinyl]pyridinium betaine (di-8-ANEPPS) can be used to measure changes in the intramembrane dipole potential. Increasing the content of cholesterol and 6-ketocholestanol (KC), which are known to increase psi D in the bilayer, results in an increase in the ratio, R, of the dye fluorescence excited at 440 nm to that excited at 530 nm in a lipid vesicle suspension; increasing the content of phloretin, which lowers psi D, decreases R. Control experiments show that the ratio is insensitive to changes in the membrane's microviscosity. The lack of an isosbestic point in the fluorescence excitation and emission spectra of the dye at various concentrations of KC and phloretin argues against 1:1 chemical complexation between the dye and KC or phloretin. The macromolecular nonionic surfactant Pluronic F127 catalyzes the insertion of KC and phloretin into lipid vesicle and cell membranes, permitting convenient and controlled modulation of dipole potential. The sensitivity of R to psi D is 10-fold larger than to delta psi, whereas it is insensitive to changes in psi S. This can be understood in terms of the location of the dye chromophore with respect to the electric field profile associated with each of these potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optical measurement of conduction in single demyelinated axons

Demyelination was initiated in Xenopus sciatic nerves by an intraneural injection of lysolecithin over a 2-3-mm region. During the next week macrophages and Schwann cells removed all remaining damaged myelin by phagocytosis. Proliferating Schwann cells then began to remyelinate the axons, with the first few lamellae appearing 13 d after surgery. Action potentials were recorded optically through the use of a potential-sensitive dye. Signals could be detected both at normal nodes of Ranvier and within demyelinated segments. Before remyelination, conduction through the lesion occurred in only a small fraction of the fibers. However, in these particular cases we could demonstrate continuous (nonsaltatory) conduction at very low velocities over long (greater than one internode) lengths of demyelinated axons. We have previously found through loose patch clamp experiments that the internodal axolemma contains voltage-dependent Na+ channels at a density approximately 4% of that at the nodes. These channels alone, however, are insufficient for successful conduction past the transition point between myelinated and demyelinated regions. Small improvements in the passive cable properties of the axon, adequate for propagation at this site, can be realized through the close apposition of macrophages and Schwann cells. As the initial lamellae of myelin appear, the probability of success at the transition zone increases rapidly, though the conduction velocity through the demyelinated segment is not appreciably changed. A detailed computational model is used to test the relative roles of the internodal Na+ channels and the new extracellular layer. The results suggest a possible mechanism that may contribute to the spontaneous recovery of function often seen in demyelinating disease.     

The influence of external cations and membrane potential on Ca- activated Na efflux in Myxicola giant axons

In microinjected Myxicola giant axons with elevated [Na]i, Na efflux was sensitive to Cao under some conditions. In Li seawater, sensitivity to Cao was high whereas in Na seawater, sensitivity to Cao was observed only upon elevation of [Ca]o above the normal value. In choline seawater, the sensitivity of Na efflux to Cao was less than that observed in Li seawater whereas Mg seawater failed to support any detectable Cao-sensitive Na efflux. Addition of Na to Li seawater was inhibitory to Cao-sensitive Na efflux, the extent of inhibition increasing with rising values of [Na]o. The presence of 20 mM K in Li seawater resulted in about a threefold increase in the Cao-activated Na efflux. Experiments in which the membrane potential, Vm, was varied or held constant when [K]o was changed showed that the augmentation of Ca- activated Na efflux by Ko was not due to changes in Vm but resulted from a direct action of K on activation by Ca. The same experimental conditions that favored a large component of Cao-activated Na efflux also caused a large increase in Ca influx. Measurements of Ca influx in the presence of 20 mM K and comparison with values of Ca-activated Na efflux suggest that the Na:Ca coupling ratio may be altered by increasing external [K]o. Overall, the results suggest that the Cao- activated Na efflux in Myxicola giant axons requires the presence of an external monovalent cation and that the order of effectiveness at a total monovalent cation concentration of 430 mM is K + Li greater than Li greater than Choline greater than Na.     

Imaging system for morphometric assessment of absorption or fluorescence in stained cells

An image acquisition and processing system has been developed for quantitative microscopy of absorption or fluorescence in stained cells. Three different light transducers are used in the system to exploit the best characteristics of these sensors for different biological measurements. A digital scanner, in the form of a linear array charge-coupled device (CCD), acquires data with high spatial and photometric resolution. A color (RGB) camera is employed when spectral information is required for the segmentation of cellular subcomponents. An image-intensified charged-injection device (CID) camera provides for very low light intensity measurements, primarily for fluorescence-labeled cells. Properties of these transducers, such as contrast transfer function, linearity, and photo-response nonuniformity, have been measured. Two dedicated image processing units were incorporated into the system. The front-end processor, based on a digital signal processor, provides functions such as object detection, raw image calibration, compression, artifact removal, and filtering. The second image processor is associated with the frame memory and includes a histogram processor, a dedicated arithmetic logic unit for image processing functions, and a graphics module for one-bit overlay functions. An interactive program was developed to acquire cell images and to experiment with a range of segmentation algorithms, feature extractions, and other image processing functions. The results of any image operation are displayed on the video monitor. Once a desired processing sequence is determined, the sequence may be stored to become part of a command library and can be executed thereafter as a single instruction.     

Internal cations, membrane current, and sodium inactivation gate closure in Myxicola giant axons.

Steady state to peak Na current ratio (INa,/INapeak) in Myxicola is greater, under some conditions, in internal Cs than in K, indicating less steady state inactivation in Csi. Csi effects are selective for steady state inactivation, with negligible effects on single-pulse inactivation time constants (Th). Mean Th ratios (Csi to Ki) were 1.04 and 1.02 at 0 and 10 mV. Two pulse inactivation time constants were also little affected. Inactivation is blocked in an all or none manner. Ki has little effect on steady state inactivation in the presence of inward INa, with INa/INapeak often declining to zero at positive potentials and independent of external Na concentration from 1/4 to 2/3 artificial sea water (ASW). Cs also has little effect at more negative potentials, but more with either more positive potentials or Na reduction, both reducing inward INa. K effects are evident when Na channel current is outward. A site in the current pathway when occupied selectively blocks inactivation gate closure. As occupancy does not depend significantly on potential, the site must not be very deep into the membrane field. Inactivation gates may associate with these sites on closure. The inactivated state may consist of a positively-charged structure occluding the inner channel mouth.     

Tracer and nontracer potassium fluxes in squid giant axons and the effects of changes in external potassium concentration and membrane potential

The efflux of labeled and unlabeled potassium ions from the squid giant axon has been measured under a variety of experimental conditions. Axons soaked in sea water containing 42K ions lost radioactivity when placed in inactive sea water according to kinetics which indicate the presence of at least two cellular compartments. A rapidly equilibrating superficial compartment, probably the Schwann cell, was observed to elevate the specific activity of 42K lost from such axons to K-free sea water for a period of hours. The extra radioactive potassium loss from such axons during stimulation, however, was shown to have a specific activity identical within error to that measured in the axoplasm at the end of the experiment. The same was shown for the extra potassium loss occurring during passage of a steady depolarizing current. Axons placed in sea water with an elevated potassium ion concentration (50 mM) showed an increased potassium efflux that was in general agreement with the accompanying increase in membrane conductance. The efflux of potassium ions observed in 50 mM K sea water at different membrane potentials did not support the theory that the potassium fluxes obey the independence principle.     

Reconstitution of myoglobin from apoprotein and heme, monitored by stopped-flow absorption, fluorescence and circular dichroism

The reconstitution reaction of ferric cyanomyoglobin from apomyoglobin and hemin dicyanide was investigated with a stopped-flow apparatus by the use of five kinds of probes; (a) Soret absorption, (b) fluorescence quenching of tryptophan, (c) far-ultraviolet CD, (d) near-ultraviolet CD, and (e) Soret CD. After mixing of apomyoglobulin with equimolar amounts of hemin dicyanide, the Soret absorption band was shifted to longer wavelengths within 10 ms. The shifted band kept its shape for a few seconds, and then gradually shifted to shorter wavelengths. A rate constant of the slow reaction was 1.1 x 10(-2) s-1. Time courses of fluorescence quenching followed a second-order reaction with a rate constant of 9 x 10(7) M-1 s-1. Far-ultraviolet CD recovered to the level of native state within the response time of an apparatus (= 64 ms). Near-ultraviolet CD and Soret CD changed with first-order rate constants of 5-30 s-1 and 5 x 10(-3) s-1 respectively. On the basis of the kinetic results we propose the following reconstitution pathway of myoglobin. Apomyoglobin has essentially a highly folded structure similar to myoglobin, but there are some differences in the secondary structure between them. In the first step, heme enters the pocket-like site of apomyoglobin and interacts with surrounding hydrophobic residues in the pocket, and then the interaction may give a complete ordered structure to the protein. Second, the tertiary structure of the heme pocket is partly constructed. Third, the iron-proximal His bond occurs, followed by the attainment of the final conformation. This sequence of the events shows that the polypeptide chain is entirely folded before the completion of three-dimensional structure of the heme pocket. The reconstitution pathway is fairly different from that of the alpha subunit of hemoglobin reported by Leutzinger and Beychok [Proc. Natl Acad. Sci. USA (1981) 78, 780-784], which described how a drastic recovery in helicity was observed on the heme-binding, and that the recovery is introduced by the formation of the heme pocket structure. The difference in the results found for the alpha subunit and myoglobin suggests a difference in conformation: in apomyoglobin most of the helices are arranged and folded around a helix core to form a compact structure as a whole, while in apo-alpha subunit some helices are not folded around the helix core. Helix D, which is absent in the alpha subunit, may play an important role in folding of the helices.     

Simultaneous measurement of electric birefringence and dichroism. A study on photosystem 1 particles.

We have developed a straightforward method to separate linear-dichroism and birefringence contributions to electric-field induced signals in a conventional birefringence setup. The method requires the measurement of electric birefringence for three different angular positions of the analyzer. It is demonstrated that the presence of linear dichroism can significantly influence the measured signals and lead to completely erroneous calculations of the birefringence signal and field-free decay times if its contribution is not taken into account. The new method is used to determine electric birefringence and linear dichroism of trimeric Photosystem 1 complexes from the cyanobacterium Synechocystis PCC 6803 in the detergents n-dodecyl-beta-D-maltoside and n-octyl-beta-D-glucoside. It is concluded that the orientation of the particles in the field is predominantly caused by a permanent electric dipole moment that is directed parallel to the symmetry axis of the particles. Comparison of the decay times obtained with dodecylmaltoside and octylglucoside supports a model in which the thickness of the disc-like complexes remains similar (7-8 nm) upon replacing dodecylmaltoside by octylglucoside, whereas the diameter increases from 14.4 +/- 0.2 to 16.6 +/- 0.2 nm because of an increased thickness of the detergent layer. This change in diameter is in good agreement with electron-microscopy results on Photosystem 2 complexes in dodecylmaltoside and octylglucoside (Dekker, J. P., E. J. Boekema, H. T. Witt, and M. Rögner. 1988. Biochim. Biophys. Acta 936:307-318). The value of approximately 16.6 nm for the diameter of Photosystem 1 trimers in dodecylmaltoside is in good agreement with recent results obtained from electron microscopy in combination with extensive image analysis (Kruip, J., E. J. Boekema, D. Bald, A. F. Boonstra, and M. Rögner. 1993. J. Biol. Chem. 268:23353-23360).     

Osmometrically determined characteristics of the cell membrane of squid and lobster giant axons

Lobster and squid giant nerve fibers respond differently when subjected to osmotic challenges. The axons proper, as distinct from the total (fiber) complex formed by the axon and connective sheath, both behave as "fast" osmometers for changes in the concentration of NaCl, but the maximum degree of swelling in hyposmotic media is by about 60% in lobster and only by 20% in squid. The relative volume intercepts of the van't Hoff relation are about 0.2 for lobster and 0.4 for squid. The sheaths of both axons undergo only small, apparently passive changes in volume. Lobster axons are permeable to Cl, but squid axons are impermeable to this anion. Lobster axons are also permeable to glycerol. The implications of the data as to the nature of volume regulation of cells are discussed.     

A proposed membrane model for generation of sodium currents in squid giant axons

An experimental review to show that axonal undercoat and cytoskeletal structures underneath the axolemma of squid giant axons play an important role in generating sodium currents is presented. Correspondingly, two alternative membrane models are proposed; one is that the undercoat and cytoskeletal structures support the functioning of sodium channels and the other is that they are directly incorporated with the molecular mechanism of generating sodium currents. This latter model is probable in squid giant axons. The model of direct participation of the underlying cytoskeleton in the sodium activation mechanism modifies the sodium activation gating kinetics in the Hodgkin-Huxley scheme; that is, the transition velocities between the open and closed states of the activation gate depend not only on membrane potentials but also on the time after the onset of application of a potential step.     

Interaction of filipin with dimyristoylphosphatidylcholine membranes studied by 2H-NMR,circular dichroism,electronic absorption and fluorescence

Interaction of the pentene antibiotic filipin with dimyristoylphosphatidylcholine (DMPC) membranes has been monitored by ²H-NMR, circular dichroism (CD), electronic absorption and fluorescence in the temperature range 10° to 60°C. Interaction appears to depend on whether filipin is added before or after membrane formation and also upon the temperature of the system.When filipin is added to preformed DMPC large unilamellar vesicles (LUV), the association constants, as determined by electronic absorption are 39×10³ M ^-1, 15×10³ M ^-1 and 0.6×10³ M ^-1 at 15°, 30° and 50°C, respectively. Under identical conditions, CD spectra of bound filipin exhibit features characteristic of an aggregation over the whole temperature range.When filipin is incorporated in membranes during their preparation, the ²H-NMR spectra of deuterated DMPC indicate that the drug has a slight disordering effect on the lipid matrix below the temperature, T _c ,of the gel-to-fluid phase transition and above T _c +11°C. Between these two temperature boundaries the system consists of two lipid regions of very different dynamic properties. One of the regions, which is attributed to a filipin-lipid complex, has the properties of gel-like lipids whereas the other has those of fluid-like lipids. The latter domain is however more ordered than the pure lipid at corresponding temperatures. CD spectra under the same conditions are found to be identical to spectra when the drug is added to preformed membranes, only in the region T _c to T _c +11°C.Filipin induced carboxyfluorescein release from DMPC-LUV is found to be complete when the filipin-to-lipid ratio is near 1, for temperatures below T _c +11°C.Results are compared to previous data on amphotericin B and provide evidence that the gel-like structure of phospholipid and membrane permeation may be induced by filipin even in the absence of cholesterol.Abbreviations NMR nuclear magnetic resonance - CD circular dichroism - DMPC dimyristoylphosphatidylcholine - EPA egg phosphatidic acid - LUV large unilamelar vesicles - SPC soybean phosphatidylcholine - DMSO dimethylsulfoxide - CF carboxyfluorescein