Membrane potential induced by external electric field pulses can be followed with a potentiometric dye.

A potential-sensitive dye was recently used to measure the spatial variation in the membrane potential induced by an externally applied electric field. In this work, we demonstrate that the time course of these induced potentials can also be followed. Two experimental systems were explored. Dye fluorescence from HeLa cells could be modulated by a train of field pulses; the relative fluorescence change measured with a lock-in amplifier was linear with the field and similar to the fluorescence responses obtained in the static measurements. A model membrane system consisting of a hemispherical bilayer allowed convenient measurement of the dye absorbance change as a function of the bathing solution conductivity. The charging time of the membrane was inversely related to the aqueous conductance as predicted by the theoretical solution to Laplace's equation.     

Analysis of the effect of medium and membrane conductance on the amplitude and kinetics of membrane potentials induced by externally applied electric fields.

The kinetics and amplitudes of membrane potential induced by externally applied electric field pulses are determined for a spherical lipid bilayer using a voltage-sensitive dye. Several experimental parameters were systematically varied. These included the incorporation of gramicidin into the membrane to alter its conductivity and the variation of the external electrolyte conductivity via changes in salt concentration. The ability of the solution to Laplace's equation for a spherical dielectric shell to quantitatively describe the membrane potential induced on a lipid bilayer could thus be critically evaluated. Both the amplitude and the kinetics of the induced potential were consistent with the predictions of this simple model, even at the extremes of membrane conductance or electrolyte concentration. The success of the experimental approach for this system encourages its application to more complex problems such as electroporation and the influences of external electric fields in growth and development.     

Theoretical study of the product specificity in the hydroxylation of camphor, norcamphor, 5,5-difluorocamphor, and pericyclocamphanone by cytochrome P-450cam

The hydroxylations of d-camphor, norcamphor, pericyclocamphanone, and 5,5-difluorocamphor by cytochrome P-450cam have been examined using theoretical methods to identify and characterize properties which determine product specificity. Experimental results indicate that each molecule is hydroxylated with quite different regio-specificity when metabolized by P-450cam. This result is surprising in view of their overall structural similiarity. Herein we report the results of calculations on d-camphor and three of its analogues which suggest that all of these molecules should, when metabolized by P-450cam, form hydroxylation products and predict the product distribution for each. Our conclusions are based on two fundamental criteria which are consistent with a generally accepted radical mechanism in determining product specificity in these molecules: 1) relative heats of formation of the radicals formed by abstracting a hydrogen, and 2) orientation of the substrate molecule with respect to the putative active oxygen species bound to iron. Our results explain the experimental observations for camphor and 5,5-difluorocamphor but disagree with original published results for norcamphor and pericyclocamphanone. In light of our results, new experiments have been performed for norcamphor and the original data reexamined for pericyclocamphanone. Our predictions have recently been experimentally confirmed for norcamphor, and unpublished data (Dr. S. Sligar) suggest that the same is true for pericyclocamphanone.     

Localized membrane depolarizations and localized calcium influx during electric field-guided neurite growth.

Our study explores the mechanisms behind neurite galvanotropism. Using phase, differential interference contrast and ratiometric fluorescence microscopy, we reveal four responses of N1E-115 mouse neuroblastoma cells to 0.1-1.0 mV/microns uniform DC electric fields: cathode-directed neurite initiation and elongation, cathode-biased growth cone filopodial protrusions, transient cathode-localized calcium increases, and persistent cathode-localized membrane depolarizations. These newly demonstrated events are temporally and spatially correlated, suggesting that they are causally related. The calcium increases are prevented by calcium channel blockers and by the removal of extracellular calcium. We therefore propose that the observed field-induced membrane depolarizations activate voltage-dependent calcium channels, resulting in cathode-localized calcium influx. This, in turn, may initiate the observed cathode-biased growth cone filopodial protrusions, followed by the cathode-directed neurite elongation.     

Membrane potential can be determined in individual cells from the nernstian distribution of cationic dyes.

The distribution of a selection of cationic fluorescent dyes can be used to measure the membrane potential of individual cells with a microfluorometer. The essential attributes of these dyes include membrane permeability, low membrane binding, spectral properties which are insensitive to environment, and, of course, strong fluorescence. A series of dyes were screened on HeLa cells for their ability to meet these criteria and several commercially available dyes were found to be satisfactory. In addition, two new dyes were synthesized for this work by esterification of tetramethyl rhodamine. The analysis of the measured fluorescent intensities requires correction for fluorescence collected from outside the plane of focus of the cell and for nonpotentiometric binding of the dye. The measurements and analysis were performed on three different cell types for which there exists a body of literature on membrane potential; the potentials determined in this work were always within the range of literature values. The rhodamine esters are nontoxic, highly fluorescent dyes which do not form aggregates or display binding-dependent changes in fluorescence efficiency. Thus, their reversible accumulation is quantitatively related to the contrast between intracellular and extracellular fluorescence and allows membrane potentials in individual cells to be continuously monitored.     

Optical imaging of cell membrane potential changes induced by applied electric fields.

We report the first imaging of the spatial distributions of transmembrane potential changes induced in nonexcitable cells by applied external electric fields. These changes are indicated by the fluorescence intensity of a charge-shift potentiometric dye incorporated in the cell plasma membrane and measured by digital intensified video microscopy.     

Purification and pore-forming activity of two hydrophobic polypeptides from the secretion of the Red Sea Moses sole (Pardachirus marmoratus)

A new column chromatography procedure, based on ion exchange, chromatofocusing, and reverse phase high pressure liquid chromatography was employed to isolate the two main proteinaceous, toxic, cytolytic, pore-forming factors from the secretion of the Red Sea Moses sole Pardachirus marmoratus. Pardaxin I, comprising 10% of the gland secretion proteins, was shown to be 5-10 times more toxic, cytolytic, and active in membrane pore formation than pardaxin II (8% of gland secretion proteins). Gel electrophoresis, amino acid analysis, and NH2-terminal amino acid sequence reveals a high degree of homogeneity and resemblance between the two toxins. They are rich in aspartic acid, serine, glycine, and alanine and devoid of arginine, tyrosine, and tryptophan. Their NH2-terminal residue sequence was found to be NH2-Gly-Phe-Phe. Their hydrophobicity is evident from chromatographic behavior on a hydrophobic matrix, presence of 9 successive hydrophobic residues at the NH2 terminus, and a decrease in drop size during elution of active fractions during chromatographic purification. The minimal molecular weight of pardaxin I is about 3500 as determined by sodium dodecyl sulfate gel electrophoresis and amino acid analyses. It is composed of 35 amino acids and is free of carbohydrate and sialic acid residues. Mass spectrometry of the ethyl acetate extract of the gland secretion and purified toxin reveals the presence of sterols in the secretion but their absence in the purified toxins. Pardaxin I was iodinated without affecting its chemical and pore-forming properties. It binds to liposomes of different phospholipid compositions. In hyperpolarized unilamellar liposomes, pardaxin I produced a fast, nonspecific permeabilization and in multilamellar liposomes, a slow, cation-specific pore. It is suggested that pardaxins exert their effects due to their hydrophobic and pore-formation properties.     

Fluorometric analysis of transferable membrane pores

When pore-forming factors insert into the hyperpolarized membranes of lipid vesicles, ion gradients are rapidly equilibrated, effecting complete depolarization. This process can be conveniently followed with a potentiometric cyanine dye. The generality of the method is demonstrated by applications to three diverse materials. The well-studied gramicidin channel is used to demonstrate that the method is sensitive down to concentrations of 10(-12)M. An extract from the shark repellent skin secretion of the Red Sea flatfish displays activity in the assay and is used to demonstrate the potential of the method to elucidate some of the characteristics of the pore, including its molecularity. That membrane-active factors can be detected and assayed in crude preparations is demonstrated with an impure extract of "amoebapore" from Entamoeba histolytica. In addition, variation of the buffer composition surrounding the vesicles can provide information about the ion selectivity of the pore under investigation.