An artificial membrane-cable: decay and delay of electrical potentials along a lipid bilayer with ion channels

A model membrane is described which exhibits the properties of a neurite with respect to passive propagation of electrical potentials. A groove in a glass plate is covered by a black lipid membrane of glycerol monooleate. Gramicidin is incorporated. The stationary and transient response of the assembly is tested by two experiments: (i) One end of the groove is clamped at a constant voltage. The voltage at the other end and the total electrical current are measured. (ii) A charge pulse is injected at one end of the groove. The time-dependent voltage at the other end is measured. The results with respect to the lateral decay and delay of voltage are in quantitative agreement with the stationary and transient solutions of Kelvin's equation for a homogeneous cable. If gramicidin is incorporated unevenly along the membrane, the lateral decay of voltage is found to be asymmetric with respect to both directions. The cable is a partial one-way transmission line.     

Stacking interaction of guanine with netropsin in the minor groove of d(CGTATATACG)2

We present the structure of the decanucleotide d(CGTATATACG) determined by single crystal X-ray diffraction at 1.58 A resolution. A netropsin drug is found in the minor groove with guanine stacked on a pyrrole ring of the drug, a feature described here for the first time. The stacked guanine is an extra-helical base coming from the end of a neighbour oligonucleotide. This observation may open the way to the development of minor groove binding drugs with a higher sequence selectivity. The oligonucleotide is in the B-conformation, but the terminal base-pairs are disrupted: the cytosine residues are disordered while the guanine residues penetrate into the minor groove of neighbouring duplexes. Four hydrated Ni ions with octahedral co-ordination are found associated with the N7 atoms of each guanine. The high affinity of these ions with guanine suggests that they may be used as probes for specific guanine residues.     

Variable diversity joining recombination: nonhairpin coding ends in thymocytes of SCID and wild-type mice

Initiation of V(D)J recombination results in broken DNA molecules with blunt recombination signal ends and covalently sealed (hairpin) coding ends. In SCID mice, coding joint formation is severely impaired and hairpin coding ends accumulate as a result of a deficiency in the catalytic subunit of DNA-dependent protein kinase, an enzyme involved in the repair of DNA double-strand breaks. In this study, we report that not all SCID coding ends are hairpinned. We have detected open Jdelta1 and Ddelta2 coding ends at the TCRdelta locus in SCID thymocytes. Approximately 25% of 5'Ddelta2 coding ends were found to be open. Large deletions and abnormally long P nucleotide additions typical of SCID Ddelta2-Jdelta1 coding joints were not observed. Most Jdelta1 and Ddelta2 coding ends exhibited 3' overhangs, but at least 20% had unique 5' overhangs not previously detected in vivo. We suggest that the SCID DNA-dependent protein kinase deficiency not only reduces the efficiency of hairpin opening, but also may affect the specificity of hairpin nicking, as well as the efficiency of joining open coding ends.     

Ca2+-activated K channel of the BK-type in the inner mitochondrial membrane of a human glioma cell line

A single channel current was recorded from mitoplasts (i.e., inner mitochondrial membrane) of the human glioma cell line LN229 using patch-clamp techniques in the mitoplast-attached mode. We frequently found a 295 +/- 18 pS channel that showed a straight i-E relation in the range +/-60 mV in 150 mM KCl solutions on either side of the mitoplast. If KCl in the bath was exchanged against NaCl, outward currents were undetectable, indicating potassium selectivity. Channel activity determined as open probability increased with increasing Ca2+ concentrations (EC50 = 0.9 microM at 60 mV). Open probability was voltage dependent. An e-fold increase of time spent in the open state was induced by a depolarization of 10.5 mV. Open probability was decreased by charybdotoxin concentration and voltage dependently (EC50 = 1.4 nM). In conclusion, we show for the first time that the inner mitochondrial membrane in human glioma cells contains a calcium-dependent K channel of the BK-type.     

Ultrastructure of Trimastix pyriformis (Klebs) Bernard et al.: similarities of Trimastix species with retortamonad and jakobid flagellates.

Trimastix pyriformis (Klebs 1893) Bernard et al. 1999, is a quadriflagellate, free-living, bacterivorous heterotrophic nanoflagellate from anoxic freshwaters that lacks mitochondria. Monoprotist cultures of this species contained naked trophic cells with anterior flagellar insertion and a conspicuous ventral groove. Bacteria were ingested at the posterior end of the ventral groove, but there was no persistent cytopharyngeal complex. The posterior flagellum resided in this groove, and bore two prominent vanes. A Golgi body (dictyosome) was present adjacent to the flagellar insertion. The kinetid consisted of four basal bodies, four microtubular roots, and associated fibers and bands. Duplicated kinetids, each with four basal bodies and microtubular root templates, appeared at the poles of the open mitotic spindle. Trimastix pyriformis is distinguishable from other Trimastix species on the basis of external morphology, kinetid architecture and the distribution of endomembranes. Trimastix species are most similar to jakobid flagellates, especially Malawimonas jakobiformis, and to species of the retortamonad genus Chilomastix. Retortamonads may have evolved from a Trimastix-like ancestor through loss of "canonical" (easily seen with electron microscopy) endomembrane systems and elaboration of cytoskeletal elements associated with the cytostome/cytopharynx complex.     

Nonselective cation channels in intact human T lymphocytes.

Nonselective cation channels were found in single channel recordings from cell-attached patches on human T lymphocytes. These channels were active under conditions that should lead to cell swelling (hypotonic bath solutions with NaCl or KCl); however, a definite dependence of activity on cell swelling has not been proven. Under these conditions similar channels were found in 20 of 23 patches from 11 different blood donors. The current-voltage relation was approximately linear for outward current (11-14 pS) and inwardly rectifying (to 23 pS) when the intact cells were depolarized with high KCl in the bath. The voltage dependence of channel activity is consistent with closing at hyperpolarized membrane potentials (Vm less than or equal to -50 mV) and block of open channels at strongly depolarized membrane potentials (Vm greater than 0 mV). Reversal potentials under all ionic gradients tested are consistent with a channel that is poorly selective between Na+ and K+ ions. Active channels in cell-attached patches were rapidly blocked by bath addition of the membrane-permeant inhibitor quinine. Channels that were active in cell-attached became quiescent after patch excision; however, two patches remained active long enough to obtain current-voltage relations. These were linear with a slope conductance for outward current of 8-11 pS. Because of the clustering of single-channel openings, detailed voltage dependence of kinetics and probability of opening were not studied.     

Cyclic GMP diffusion coefficient in rod photoreceptor outer segments.

Cyclic GMP (cGMP) is the intracellular messenger that mediates phototransduction in retinal rods. As photoisomerizations of rhodopsin molecules are local events, the longitudinal diffusion of cGMP in the rod outer segment should be a contributing factor to the response of the cell to light. We have employed the truncated rod outer segment preparation from bullfrog (Rana catesbeiana) and tiger salamander (Ambystoma tigrinum) to measure the cGMP diffusion coefficient. In this preparation, the distal portion of a rod outer segment was drawn into a suction pipette for measuring membrane current, and the rest of the cell was then sheared off with a glass probe, allowing bath cGMP to diffuse into the outer segment and activate the cGMP-gated channels on the surface membrane. Addition and removal of bath cGMP were fast enough to produce effectively step changes in cGMP concentration at the open end of the outer segment. When cGMP hydrolysis is inhibited by isobutylmethylxanthine (IBMX), the equation for the diffusion of cGMP inside the truncated rod outer segment has a simple analytical solution, which we have used to analyze the rise and decay kinetics of the cGMP-elicited currents. From these measurements we have obtained a cGMP diffusion coefficient of approximately 70 x 10(-8) cm2 s-1 for bullfrog rods and approximately 60 x 10(-8) cm2 s-1 for tiger salamander rods. These values are six to seven times lower than the expected value in aqueous solution. The estimated diffusion coefficient is the same at high (20-1000 microM) and low (5-10 microM) concentrations of cGMP, suggesting no significant effect from buffering over these concentration ranges.     

A Method of Orienting Paraffin Sections for Three-Dimensional Reconstructions

To make reconstructions from serial sections, reference points are needed to orient the sections. Such points can be provided after paraffin embedding by cutting the bottom face of the block to form a plane and adding a groove along the center of this plane. The plane and groove are coated with Mimeograph Correction Fluid and the block is built up by dipping in hot paraffin so that the marked plane lies inside the block. Each section will have a blue line with a notch in it representing the plane and groove. This line remains through staining and is used to orient each section with respect to an eyepiece reticle. The reticle, in effect, supplies X and Y coordinates for every point in the specimen while the number of each section counted from one end is a Z coordinate.     

Regulation of the hyperpolarization-activated K+ channel in the lateral membrane of the cortical collecting duct [published erratum appears in J Gen Physiol 1995 Sep;106(3):579]

An intermediate-conductance K+ channel (I.K.), the activity of which is increased by hyperpolarization, was previously identified in the lateral membrane of the cortical collecting duct (CCD) of the rat kidney (Wang, W. H., C. M. McNicholas, A. S. Segal, and G. Giebisch. 1994. American Journal of Physiology. 266:F813-F822). The biophysical properties and regulatory mechanisms of this K+ channel have been further investigated with patch clamp techniques in the present study. The slope conductance of the channel in inside-out patches was 50 pS with 140 mM KCl in the pipette and 5 mM KCl, 140 mM NaCl (NaCl Ringer''s solution) in the bath. Replacement of the bath solution with symmetrical 140 mM KCl solution changed the slope conductance of the channel to 85 pS and shifted the reversal potential by 55 mV, indicating that the selectivity ratio of K+/Na+ was at least 10:1. Channel open probability (Po) in inside-out patches was 0.12 at 0 mV and was increased by hyperpolarization. The voltage-dependent Po was fitted with the Boltzmann''s equation: Po = 1/[1 + exp(V-V1/2)zF/RT], with z = 1.2 and V1/2 = -40 mV. Addition of 2 mM tetraethylammonium or 500 mM quinidine to the bath blocked the activity of the K+ channel in inside-out patches. In addition, decrease in the bath pH from 7.40 to 6.70 reduced Po by 30%. Addition of the catalytic subunit of protein kinase A (PKAc; 20 U/ml) and 100 microM [corrected] MgATP to the bath increased Po from 0.12 to 0.49 at 0 mV and shifted the voltage dependence curve of channel activity toward more positive potentials by 40 mV. Two exponentials were required to fit both the open-time and the closed-time histograms. Addition of PKAc increased the long open-time constant and shortened the long closed-time constant. In conclusion, PKA-mediated phosphorylation plays an important role in the regulation of the voltage dependence of the hyperpolarization-activated K+ channel in the basolateral membrane of CCD.     

Voltage-Gated Lipid Ion Channels

Synthetic lipid membranes can display channel-like ion conduction events even in the absence of proteins. We show here that these events are voltage-gated with a quadratic voltage dependence as expected from electrostatic theory of capacitors. To this end, we recorded channel traces and current histograms in patch-experiments on lipid membranes. We derived a theoretical current-voltage relationship for pores in lipid membranes that describes the experimental data very well when assuming an asymmetric membrane. We determined the equilibrium constant between closed and open state and the open probability as a function of voltage. The voltage-dependence of the lipid pores is found comparable to that of protein channels. Lifetime distributions of open and closed events indicate that the channel open distribution does not follow exponential statistics but rather power law behavior for long open times.     

Nucleosome structure and conformational changes.

We have used a variety of chemical probes to measure the accessibility of DNA on the surface of the nucleosome. We review these results, and describe new experiments which show that T4 phage DNA can form complexes with the core histones, possessing the properties of normal nucleosomes. Since T4 DNA is largely occupied by glucose residues in the major groove, this suggests (as did earlier probe experiments) that the major groove is not filled with histone amino acid side chains. We also report results of recent measurements which appear to show that only a few strong charge interactions are involved in the attachment of the terminal 20 nucleotide pairs at each end of nucleosome core DNA. We speculate on the possible functional significance of the accessibility of DNA revealed by all of these experiments. We have also examined conformational changes induced in nucleosomes at high ionic strength (0.5-0.7M NaCl). The frictional coefficient is found to undergo a small increase in this region, not consistent with models in which the nucleosome is completely unfolded, but possibly reflecting the dissociation of terminal DNA from the nucleosome surface.     

Localization of the activation gate of a voltage-gated Ca2+ channel

Ion channels open and close in response to changes in transmembrane voltage or ligand concentration. Recent studies show that K+ channels possess two gates, one at the intracellular end of the pore and the other at the selectivity filter. In this study we determined the location of the activation gate in a voltage-gated Ca2+ channel (VGCC) by examining the open/closed state dependence of the rate of modification by intracellular methanethiosulfonate ethyltrimethylammonium (MTSET) of pore-lining cysteines engineered in the S6 segments of the alpha1 subunit of P/Q type Ca2+ channels. We found that positions above the putative membrane/cytoplasm interface, including two positions below the corresponding S6 bundle crossing in K+ channels, showed pronounced state-dependent accessibility to internal MTSET, reacting approximately 1,000-fold faster with MTSET in the open state than in the closed state. In contrast, a position at or below the putative membrane/cytoplasm interface was modified equally rapidly in both the open and closed states. Our results suggest that the S6 helices of the alpha1 subunit of VGCCs undergo conformation changes during gating and the activation gate is located at the intracellular end of the pore.     

Techniques for obtaining analytical solutions to the multicylinder somatic shunt cable model for passive neurones.

The somatic shunt cable model for neurones is extended to the case in which several equivalent cylinders, not necessarily of the same electrotonic length, emanate from the cell soma. The cable equation is assumed to hold in each cylinder and is solved with sealed end conditions and a lumped soma boundary condition at a common origin. A Green's function (G) is defined, corresponding to the voltage response to an instantaneous current pulse at an arbitrary point along one of the cylinders. An eigenfunction expansion for G is obtained where the coefficients are determined using the calculus of residues and compared with an alternative method of derivation using a modified orthogonality condition. This expansion converges quickly for large time, but, for small time, a more convenient alternative expansion is obtained by Laplace transforms. The voltage response to arbitrary currents injected at arbitrary sites in the dendritic tree (including the soma) may then be expressed as a convolution integral involving G. Illustrative examples are presented for a point charge input.     

Sequence-dependent recognition of DNA duplexes. Netropsin complexation to the AATT site of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

We have investigated intermolecular interactions and conformational features of the netropsin X d(G-G-A-A-T-T-C-C) complex by one- and two-dimensional NMR studies in aqueous solution. Netropsin removes the 2-fold symmetry of the d(G-G-A-A-T-T-C-C) duplex at the AATT binding site and to a lesser extent at adjacent dG X dC base pairs resulting in doubling of resonances for specific positions in the spectrum of the complex at 25 degrees C. We have assigned the amide, pyrrole, and CH2 protons of netropsin, and the base and sugar H1' protons of the nucleic acid from an analysis of the nuclear Overhauser effect (NOESY) and correlated (COSY) spectra of the complex at 25 degrees C. We observe intermolecular nuclear Overhauser effects (NOE) between all three amide and both pyrrole protons on the concave face of the antibiotic and the minor groove adenosine H2 proton of the two central A4 X T5 base pairs of the d(G1-G2-A3-A4-T5-T6-C7-C8) duplex. Weaker intermolecular NOEs are also observed between the pyrrole concave face protons and the sugar H1' protons of residues T5 and T6 in the AATT minor groove of the duplex. We also detect intermolecular NOEs between the guanidino CH2 protons at one end of netropsin and adenosine H2 proton of the two flanking A3 X T6 base pairs of the octanucleotide duplex. These studies establish a set of intermolecular contacts between the concave face of the antibiotic and the minor groove AATT segment of the d(G-G-A-A-T-T-C-C) duplex in solution. The magnitude of the NOEs require that there be no intervening water molecules sandwiched between the antibiotic and the DNA so that release of the minor groove spine of hydration is a prerequisite for netropsin complex formation.     

The localization of transport properties in the frog lens.

The selectivity of fiber-cell membranes and surface-cell membranes in the frog lens is examined using a combination of ion substitutions and impedance studies. We replace bath sodium and chloride, one at a time, with less permeant substitute ions and we increase bath potassium at the expense of sodium. We then record the time course and steady-state value of the intracellular potential. Once a new steady state has been reached, we perform a small signal-frequency-domain impedance study. The impedance study allows us to separately determine the values of inner fiber-cell membrane conductance and surface-cell membrane conductance. If a membrane is permeable to a particular ion, we presume that the conductance of that membrane will change with the concentration of the permeant ion. Thus, the impedance studies allow us to localize the site of permeability to inner or surface membranes. Similarly, the time course of the change in intracellular potential will be rapid if surface membranes are the site of permeation whereas it will be slow if the new solution has to diffuse into the intercellular space to cause voltage changes. Lastly, the value of steady-state voltage change provides an estimate of the lens' permeability, at least for chloride and potassium. The results for sodium are complex and not well understood. From the above studies we conclude: (a) surface membranes are dominated by potassium permeability; (b) inner fiber-cell membranes are permeable to sodium and chloride, in approximately equal amounts; and (c) inner fiber-cell membranes have a rather small permeability to potassium.     

Interactions of the human T-cell leukemia virus type-II integrase with the conserved CA in the retroviral long terminal repeat end

Retroviral integrases (INs) interact with termini of retroviral DNA in the conserved 5'-C(A/G)T. For most integrases, modifications of critical moieties in the major and minor grooves of these sequences decrease 3'-processing. However, for human immunodeficiency virus type-2 (HTLV-2) IN, the replacement of the guanine with 6-methylguanine or hypoxanthine not only reduced 3'-processing, but also promoted cleavage at a second site. This novel cleavage activity required an upstream ACA, unique to the HTLV-2 U5 end. 3'-Processing assays with additional isosteric modifications at Gua and filter binding experiments revealed that the mechanism of the second site cleavage differed among the major groove, minor groove, and mismatch modifications. Importantly, the decrease in 3'-processing activity noted with the minor groove and mismatch modifications were attributed to a decrease in binding. Major groove modifications, however, decreased the level of 3'-processing, but did not affect binding. This suggests that integrase binds the viral end through the minor groove, but relies on major groove contacts for 3'-processing. Several modifications were also examined in strand transfer and disintegration substrates. HTLV-2 IN showed reduced activity with strand transfer and disintegration substrates containing major groove, but not minor groove modifications. This suggests major groove interactions at guanine also provide an important role in these reactions.     

Regulation of connexons composed of human connexin26 (hCx26) by temperature

This report shows that temperature is a latent regulator of the voltage-dependent conductance of hemichannels composed of hCx26. The latter were expressed in Xenopus oocytes by injection of a mixture of hCx26 cRNA and antisense of endogenous Cx38 (anti-Cx38). At 24-25 degrees C, voltage clamp of oocytes at potentials above -40 mV evoked outward currents which were not observed in control oocytes. These currents were reversibly affected by change in temperature. Increasing temperature of the bath solution amplified gradually, whereas decreasing bath temperatures below 20 degrees C reduced the current. Furthermore analysis revealed that temperature-dependent increase of the conductance of the hemichannels did not correlate with a change of the apparent gating charge, whereas the half-activation voltage V(1/2) of the hemichannel was affected by a temperature change. It is proposed that this finding correlates with a temperature-dependent transition into an open state above 20 degrees C. In addition, a temperature-dependent release of Lucifer Yellow from loaded liposomes containing reconstituted purified and hCx26 hemichannels was observed, which indicate that a temperature-dependent regulation of the permeability of hCx26 hemichannels is not related to intracellular mediators. The involvement of temperature to modulate hemichannels as well as of the corresponding gap junction channel composed of hCx26 at physiological condition is discussed.     

Evaluation of some chemotherapeutics against Endamoeba histolytica in cultures with Trypanosoma cruzi

Ten antibiotics and amebacidal agents have been re-studied for their “amebacidal” end points. These determinations were obtained by use of E. histolytica plus T. cruzi and were compared with the end points previously obtained by others utilizing “open” and “sealed” culture technics of E. histolytica plus organism ‘t.’. The end point ranges corresponded closely with those obtained with the “sealed culture” technic. The cultures of E. histolytica growing in association with T. cruzi alone provide an opportunity for study of direct amebacidal action in vitro, without the complication that associated bacterial flora has introduced in previous methods.     

Na-K pump current in the Amphiuma collecting tubule

There is strong evidence supporting the hypothesis of an electrogenic Na-K pump in the basolateral membrane of several epithelia. Thermodynamic considerations and results in nonepithelial cells indicate that the current carried by the pump could be voltage dependent. In order to measure the pump current and to determine its voltage dependence in a tight epithelium, we have used the isolated perfused collecting tubule of Amphiuma and developed a technique for clamping the basolateral membrane potential (Vbl) through transepithelial current injection. The transcellular current was calculated by subtracting the paracellular current (calculated from the transepithelial conductance measured in the presence of luminal amiloride) from the total transepithelial current. Basolateral membrane current-voltage (I-V) curves were obtained in conditions where the ratio of the pump current to the total basolateral membrane current had been maximized by loading the cells with Na+ (exposure to low-K+ bath), and by blocking the basolateral K+ conductance with barium. The pump current was defined as the difference of the current across the basolateral membrane measured before and 10-15 s after the addition of strophanthidin (20 microM) to the bath solution. With a bath solution containing 3 mM K+, the pump current was nearly constant in the Vbl range of -20 to -80 mV (52 +/- 5 microA.cm-2 at -60 mV) but showed a marked voltage dependence at higher negative Vbl (pump current decreased to 5 +/- 9 microA.cm-2 at -180 mV). In a 1.0 mM K bath, the shape of the pump I-V curve was similar but the amplitude of the current was decreased (24 +/- 4 microA.cm-2 at -60 mV). In a 0.1 mM K bath, the pump current was not significantly different from 0. Our results indicate that the basolateral Na-K pump generates a current which depends on the extracellular potassium concentration. With physiological peritubular concentration of K+ and in the physiological range of potential, the pump activity, measured as the pump-generated current, was independent of the membrane potential.