The Proton Permeability of Liposomes Made from Mitochondrial Inner Membrane Phospholipids: Comparison with Isolated Mitochondria

Unilamellar liposomes with native phospholipid fatty acid composition were prepared from rat liver mitochondrial inner membrane phospholipids by extrusion in medium containing 50 mm potassium. They were diluted into low potassium medium to establish a transmembrane potassium gradient. A known membrane potential was imposed by addition of valinomycin, and proton flux into liposomes was measured. Valinomycin in the range 10 pm–1nm was sufficient to fully establish membrane potential. Valinomycin concentrations above 3 nm catalyzed additional proton flux and were avoided. At 300 pm valinomycin, proton flux depended nonlinearly on membrane potential. At 160 mV membrane potential the flux was 30 nmol H⁺/min/mg phospholipid—approximately 5% of the proton leak flux under comparable conditions in isolated mitochondria, indicating that leak pathways through bulk phospholipid bilayer account for only a small proportion of total mitochondrial proton leak. Received: 5 August 1996/Revised: 1 October 1996     

Characteristics of the Quenching of 9-Aminoacridine Fluorescence by Liposomes Made from Plant Lipids

Several laboratories have determined the surface charge density of membranes utilizing methods based on vesicle-induced quenching of the fluorescence of 9-aminoacridine and its relief by other cations. However, the computational methods by which surface charge density were calculated have not been verified in a model system. In this study, the quenching of 9-aminoacridine fluorescence by liposomes made from varying amounts of digalactosyldiacylglyceride and phosphatidic acid and relief of quenching by salts was examined. Quenching of 9-aminoacridine fluorescence increased with increasing amounts of phosphatidic acid added, independent of the composition of the added liposomes. In certain instances, the computational methods did not yield the surface charge density of the liposomes expected from their composition. However, when the effects of background ionic strength on surface potential were considered, there was a positive correlation between expected and calculated values. Therefore, the data support the contention that changes in the fluorescence of 9-aminoacridine can be used to calculate surface charge density of membranes. Received: 29 November 1999/Revised: 31 July 2000     

Molecular Mechanisms of Polymyxin B-Membrane Interactions: Direct Correlation Between Surface Charge Density and Self-Promoted Transport

We have studied the interaction of the polycationic peptide antibiotic polymyxin B (PMB) with asymmetric planar bilayer membranes via electrical measurements. The bilayers were of different compositions, including those of the lipid matrices of the outer membranes of various species of Gram-negative bacteria. One leaflet, representing the bacterial inner leaflet, consisted of a phospholipid mixture (PL; phosphatidylethanolamine, -glycerol, and diphosphatidylglycerol in a molar ratio of 81:17:2). The other (outer) leaflet consisted either of lipopolysaccharide (LPS) from deep rough mutants of PMB-sensitive (Escherichia coli F515) or -resistant strains (Proteus mirabilis R45), glycosphingolipid (GSL-1) from Sphingomonas paucimobilis IAM 12576, or phospholipids (phosphatidylglycerol, diphytanoylphosphatidylcholine). In all membrane systems, the addition of PMB to the outer leaflet led to the induction of current fluctuations due to transient membrane lesions. The minimal PMB concentration required for the induction of the lesions and their size correlated with the charge of the lipid molecules. In the membrane system resembling the lipid matrix of a PMB-sensitive strain (F515 LPS/PL), the diameters of the lesions were large enough (d= 2.4 nm ± 8%) to allow PMB molecules to permeate (self-promoted transport), but in all other systems they were too small. A comparison of these phenomena with membrane effects induced by detergents (dodecyltriphenylphosphonium bromide, dodecyltrimethylammonium bromide, sodiumdodecylsulfate) revealed a detergent-like mechanism of the PMB-membrane interaction. Received: 16 September 1997/Revised: 25 November 1997     

Phylogeny of Nematoda and Cephalorhyncha Derived from 18S rDNA

Phylogenetic relationships of nematodes, nematomorphs, kinorhynchs, priapulids, and some other major groups of invertebrates were studied by 18S rRNA gene sequencing. Kinorhynchs and priapulids form the monophyletic Cephalorhyncha clade that is the closest to the coelomate animals. When phylogenetic trees were generated by different methods, the position of nematomorphs appeared to be unstable. Inclusion of Enoplus brevis, a representative of a slowly evolving nematode lineage, in the set of analyzed species refutes the tree patterns, previously derived from molecular data, where the nematodes appear as a basal bilateral lineage. The nematodes seem to be closer to the coelomate animals than was speculated earlier. According to the results obtained, nematodes, nematomorphs, tardigrades, arthropods, and cephalorhynchs are a paraphyletic association of closely related taxa. Received: 1 December 1997 / Accepted: 9 April 1998     

Phylogenetic Relationships of Acanthocephala Based on Analysis of 18S Ribosomal RNA Gene Sequences

Acanthocephala (thorny-headed worms) is a phylum of endoparasites of vertebrates and arthropods, included among the most phylogenetically basal tripoblastic pseudocoelomates. The phylum is divided into three classes: Archiacanthocephala, Palaeacanthocephala, and Eoacanthocephala. These classes are distinguished by morphological characters such as location of lacunar canals, persistence of ligament sacs in females, number and type of cement glands in males, number and size of proboscis hooks, host taxonomy, and ecology. To understand better the phylogenetic relationships within Acanthocephala, and between Acanthocephala and Rotifera, we sequenced the nearly complete 18S rRNA genes of nine species from the three classes of Acanthocephala and four species of Rotifera from the classes Bdelloidea and Monogononta. Phylogenetic relationships were inferred by maximum-likelihood analyses of these new sequences and others previously determined. The analyses showed that Acanthocephala is the sister group to a clade including Eoacanthocephala and Palaeacanthocephala. Archiacanthocephala exhibited a slower rate of evolution at the nucleotide level, as evidenced by shorter branch lengths for the group. We found statistically significant support for the monophyly of Rotifera, represented in our analysis by species from the clade Eurotatoria, which includes the classes Bdelloidea and Monogononta. Eurotatoria also appears as the sister group to Acanthocephala. Received: 12 October 1999 / Accepted: 8 February 2000     

Formation of Folds and Vesicles by Dipalmitoylphosphatidylcholine Monolayers Spread in Excess

Lipid monolayers exist in several biological systems, including the stratum corneum of the skin, the fluid tear film of the eye, the Eustachian tube of the ear, and airway and alveolar pulmonary surfactants. In this paper, the monolayer-to-bilayer transition was studied using dipalmitoylphosphatidylcholine (DPPC) as the model. Depositing DPPC organic solvent solutions in excess at an air:buffer interface led to the formation of elongated structures which could be imaged on carbon grids by transmission electron microscopy. The structures appeared to be DPPC folds protruding into the sol. The structures were frequently ordered with respect to one another, suggesting that they arose during lateral compression due to excess DPPC and are characteristic of a type of monolayer collapse phase. In some cases, series of short folds in an extended line and series of vesicles in line or parallel to the folds were observed. This suggests the elongated folds are unstable and can resolve by forming vesicles. Fold formation occurred at defined lipid concentrations above which more vesicles were observed. Surfactant protein-A did not influence fold or vesicle formation but bound to the edges of these structures preferentially. It is concluded that DPPC monolayers can form bilayers spontaneously in the absence of surfactant apoproteins, other proteins or agents. Received: 18 May 2000/Revised: 20 November 2000     

A Glycoprotein from Rat Liver Endoplasmic Reticulum Promotes Both Aggregation and Fusion of Liposomes at Acidic pH

Low-pH-induced fusion of liposomes with rat liver endoplasmic reticulum was evidenced. Fusion was inactivated by treatment of microsomes with trypsin or EEDQ (N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline), indicating the involvement of a protein. The protein was purified 555-fold by chromatographic steps. The identification and purification to homogeneity was obtained by electroelution from a slab gel, which gave a still active protein of about 50 kDa. The protein promoted the fusion of liposomes; laser light scattering showed an increase of mean radius of vesicles from 60 up to about 340 nm. Fusion was studied as mass action kinetics, describing the overall fusion as a two-step sequence of a second order aggregation followed by a first order fusion of liposomes. For phosphatidylcholine containing liposomes aggregation was not rate-limiting at pH 5.0 and fusion followed first order kinetics with a rate constant of 13 · 10⁻³ sec⁻¹. For phosphatidylethanolamine/phosphatidic acid liposomes aggregation was rate-limiting; however, the overall fusion was first order process, suggesting that fusogenic protein influences both aggregation and fusion of liposomes. The protein binds to the lipid bilayer of liposomes, independently of pH, probably by a hydrophobic segment. Exposed carboxylic groups might be able to trigger pH-dependent aggregation and fusion. It is proposed that the protein inserted in the lipid bilayer bridges with an adjacent liposome forming a fused doublet. Since at endoplasmic reticulum level proton pumps are operating to generate a low-pH environment, the membrane bound fusogenic protein may be responsible for both aggregation and fusion of neighboring membranes and therefore could operate in the exchange of lipidic material between intracellular membranes. Received: 25 August 1997/Revised: 28 April 1998     

Effects of ATP-sensitive Potassium Channel Regulators on Chloride Channels in the Sarcoplasmic Reticulum Vesicles from Rabbit Skeletal Muscle

The lipid bilayer technique was used to examine the effects of the ATP-sensitive K⁺ channel inhibitor (glibenclamide) and openers (diazoxide, minoxidil and cromakalim) and Cl⁻ channel activators (GABA and diazepam) on two types of chloride channels in the sarcoplasmic reticulum (SR) from rabbit skeletal muscle. Neither diazepam at 100 μm nor GABA at 150 μm had any significant effect on the conductance and kinetics of the 75 pS small chloride (SCl) channel. Unlike the 150 pS channel, the SCl channel is sensitive to cytoplasmic glibenclamide with K _i ∼ 30 μm. Glibenclamide induced reversible decline in the values of current (maximal current amplitude, I _max and average mean current, I′) and kinetic parameters (frequency of opening F _o , probability of the channel being open P _o and mean open time, T _o , of the SCl channel. Glibenclamide increased mean closed time, T _c , and was a more potent blocker from the cytoplasmic side (cis) than from the luminal side (trans) of the channel. Diazoxide increased I′, P _o , and T _o in the absence of ATP and Mg²⁺ but it had no effect on I _max and also failed to activate or remove the glibenclamide- and ATP-induced inhibition of the SCl channel. Minoxidil induced a transient increase in I′ followed by an inhibition of I _max, whereas cromakalim reduced P _o and I′ by increasing channel transitions to the closed state and reducing T _o without affecting I _max. The presence of diazoxide, minoxidil or cromakalim on the cytoplasmic side of the channel did not prevent [ATP] _cis or [glibenclamide] _cis from blocking the channel. The data suggest that the action(s) of these drugs are not due to their effects on the phosphorylation of the channel protein. The glibenclamide- and cromakalim-induced effects on the SCl channel are mediated via a ``flicker' type block mechanism. Modulation of the SCl channel by [diazoxide] _cis and [glibenclamide] _cis highlights the therapeutic potential of these drugs in regulating the Ca²⁺-counter current through this channel. Received: 2 September 1997/Revised: 20 March 1998     

A Group I Intron in the 18S Ribosomal DNA from the Parasitic Fungus Isaria japonica

The nucleotide sequence of the 18S rDNA coding gene in the ascomycetes parasitic fungus Isaria japonica contains a group I intron with a length of 379 nucleotides. The identification of the DNA sequence as a group I intron is based on its sequence homology to other fungal group I introns. Its group I intron contained the highly conserved sequence elements P, Q, R, and S found in other group I introns. Surprisingly, the intron sequence of I. japonica is more similar to that of Ustilago maydis than to the one found in Sclerotinia sclerotiorum. This is in contrast to the sequence identity found on the neighboring rDNA. This is an interesting finding and suggests a horizontal transfer of group I intron sequences. Received: 19 September 1997 / Accepted: 10 September 1998     

Membrane Fusion and Rupture in Liposomes: Effect of Biodegradable pH-Sensitive Surfactants

Biodegradable pH-sensitive surfactants (BPS) are a unique family of easily metabolized compounds that demonstrate pH-dependent surface activity. These agents, in combination with other delivery systems, have demonstrated effects in enhancing transnucleic acid activity. The increased activity has been hypothesized to occur from a release of endosomal contents. Simply, the BPS delivery system containing nucleic acids enters the cell through an endocytotoic process. It encounters an acidic pH and becomes surface active leading to defects in the endosomal membrane. In the current study, an in vitro model membrane was used to better understand the liposome defect mechanisms that BPS elicit. Using this system, it is shown that BPS can induce both liposome fusion and rupture depending upon the pH and mole ratio of BPS to membrane lipids. Futhermore, liposome fusion induced by BPS was dependent on the total numbers of liposome particles while rupture was independent of interacting liposome particles. The generated data indicate that BPS agents act differently from other typical surface active agents and fuosgenic compounds. Instead of facilitating membrane fusion through the hexagonal II phase, BPS appeared to contribute and participate in the membrane fusion at different stages. Received: 18 February 1998/Revised: 14 July 1998     

Effect of Adenosine on Na+ and Cl− Currents in A6 Monolayers. Receptor Localization and Messenger Involvement

The effect of adenosine regulation on sodium and chloride transport was examined in cultured A₆ renal epithelial cells. Adenosine and its analogue N⁶-cyclopentyladenosine (CPA) had different effects on short-circuit current (I _sc) depending on the side of addition. Basolateral CPA addition induced an approximately threefold increase of the I _sc that reached a maximum effect 20 min after addition and was completely inhibited by preincubation with either an A₂ selective antagonist, CSC, or the sodium channel blocker, amiloride. Apical CPA addition induced a biphasic I _sc response characterized by a rapid fourfold transient increase over its baseline followed by a decline and a plateau phase that were amiloride insensitive. The A₁ adenosine antagonist, CPX, completely prevented this response. This I _sc response to apical CPA was also strongly reduced in Cl⁻-free media and was significantly inhibited either by basolateral bumetanide or apical DPC preincubation. Only basolateral CPA addition was able to induce an increase in cAMP level. CPA, added to cells in suspension, caused a rapid rise in [Ca²⁺] _i that was antagonized by CPX, not affected by CSC and prevented by thapsigargin preincubation. These data suggest that basolateral CPA regulates active sodium transport via A₂ adenosine receptors stimulating adenylate cyclase while apical CPA regulates Cl⁻ secretion via A₁ receptor-mediated changes in [Ca²⁺] _i .     

Phylogenetic Position of the Phacotaceae Within the Chlamydophyceae as Revealed by Analysis of 18S rDNA and rbcL Sequences

Four genera of the Phacotaceae (Phacotus, Pteromonas, Wislouchiella, Dysmorphococcus), a family of loricated green algal flagellates within the Volvocales, were investigated by means of transmission electron microscopy and analysis of the nuclear encoded small-subunit ribosomal RNA (18S rRNA) genes and the plastid-encoded rbcL genes. Additionally, the 18S rDNA of Haematococcus pluvialis and the rbcL sequences of Chlorogonium elongatum, C. euchlorum, Dunaliella parva, Chloromonas serbinowii, Chlamydomonas radiata, and C. tetragama were determined. Analysis of ultrastructural data justified the separation of the Phacotaceae into two groups. Phacotus, Pteromonas, and Wislouchiella generally shared the following characters: egg-shaped protoplasts, a single pyrenoid with planar thylakoid double-lamellae, three-layered lorica, flagellar channels as part of the central lorica layer, mitochondria located in the central cytoplasm, lorica development that occurs in mucilaginous zoosporangia that are to be lysed, and no acid-resistant cell walls. Dysmorphococcus was clearly different in each of the characters mentioned. Direct comparison of sequences of Phacotus lenticularis, Pteromonas sp., Pteromonas protracta, and Wislouchiella planctonica revealed DNA sequence homologies of ≥98.0% within the 18S gene and 93.9% within the rbcL gene. D. globosus was quite different from these species, with a maximum of 92.9% homology in the 18S rRNA and ≤86.6% in the rbcL gene. It showed major similarities to the 18S rDNA of Dunaliella salina, with 95.3%, and to the rbcL sequence of Chlamydomonas tetragama, with 90.3% sequence homology. Additionally, the Phacotaceae sensu stricto exclusively shared 10 (rbcL: 4) characters which were present neither in other Chlamydomonadales nor in Dysmorphococcus globosus. Different phylogenetic analysis methods confirmed the hypothesis that the Phacotaceae are polyphyletic. The Phacotaceae sensu stricto form a stable cluster with affinities to the Dunaliellaes and possibly Haematococcus pluvialis. Dysmorphococcus globosus represented an independent lineage that is possibly related to Chlamydomonas moewusii and C. tetragama. Received: 9 June 1997 / Accepted: 17 October 1997     

Performance of 18S rRNA in Littorinid Phylogeny (Gastropoda: Caenogastropoda)

In the past, 18S rRNA sequences have proved to be very useful for tracing ancient divergences but were rarely used for resolving more recent ones. Moreover, it was suggested that the molecule does not contain useful information to resolve divergences which took place during less than 40 Myr. The present paper takes littorinid phylogeny as a case study to reevaluate the utility of the molecule for resolving recent divergences. Two data sets for nine species of the snail family Littorinidae were analyzed, both separately and combined. One data set comprised 7 new complete 18S rRNA sequences aligned with 2 published littorinid sequences; the other comprised 12 morphological, 1 biochemical, and 2 18S rRNA secondary structure characters. On the basis of its ability to confirm generally accepted relationships and the congruence of results derived from the different data sets, it is concluded that 18S rRNA sequences do contain information to resolve ``rapid' cladogenetic events, provided that they occurred in the not too distant past. 18S rRNA sequences yielded support for (1) the branching order (L. littorea, (L. obtusata, (L. saxatilis, L. compressa))) and (2) the basal position of L. striata in the Littorina clade. Received: 6 February 1998 / Accepted: 20 March 1998     

Regulation of Ca2+-activated K+ Channel from Rabbit Distal Colon Epithelium by Phosphorylation and Dephosphorylation

The Ca²⁺-activated maxi K⁺ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na⁺ reabsorption from the intestinal lumen. Previous measurements of these basolateral K⁺ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca²⁺ with a K _0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca²⁺. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K⁺ channels lost their high sensitivity to Ca²⁺, yet they could still be activated by high concentrations of Ca²⁺ (10 μm). Furthermore, the high sensitivity to Ca²⁺ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca²⁺ of the maxi K⁺ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca²⁺ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca²⁺ sensitivities for maxi K⁺ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. Received: 28 February 1995/Revised: 22 December 1995     

Differential Transmembrane Diffusion of Triiodothyronine and Thyroxine in Liposomes: Regulation by Lipid Composition

How thyroid hormones move across biological or model membranes is a subject of controversy. The passage of the 3,5,3′triiodo l-thyronine and 3,5,3′,5′ tetraiodo l-thyronine across model membranes was evaluated by the addition of the hormones to liposomes containing 2,4,6-trinitrobenzene sulfonic acid. Results indicate that hormones can react with an amino-reactive compound pre-encapsulated into phosphatidylcholine liposomes. The transversal motions of thyroid hormones were characterized by using physiological concentration levels of (¹²⁵I) 3,5,3′triiodo l-thyronine and (¹²⁵I) 3,5,3′,5′ tetraiodo l-thyronine. The hormone distribution between the two monolayers was time-dependent and kinetic data were fitted to a single exponential. Results obtained show that 3,5,3′ triiodo l-thyronine can permeate phospholipid membranes and the diffusion time increases in the gel and liquid-ordered phase. On the contrary, 3,5,3′, 5′ tetraiodo l-thyronine could not diffuse the liposomal membrane from dimyristoyl and dipalmitoyl phosphatidylcholine in gel phase and egg yolk phosphatidylcholine:cholesterol in the liquid-ordered phase. Our results in the liquid-ordered phase suggest that diffusion movement of thyroid hormones across cell membranes depends on the amount of cholesterol in the bilayer. Received: 1 June 1998/Revised: 14 October 1998     

Protein Kinase Inhibitors and the Dynamics of Tight Junction Opening and Closing in A6 Cell Monolayers

This study focuses, in A6 cell monolayers, on the role of protein kinases in the dynamics of tight junction (TJ) opening and closing. The early events of TJ dynamics were evaluated by the fast Ca⁺⁺-switch assay (FCSA), which consisted of opening the TJs by removing basolateral Ca⁺⁺ (Ca⁺⁺ _bl), and closing them by returning Ca⁺⁺ _bl to normal values. Changes in TJ permeability can be reliably gauged through changes of transepithelial electrical conductance (G) determined in the absence of apical Na⁺. The FCSA allows the evaluation of the effects of drugs and procedures acting upon the mechanism controlling the TJs. The time courses of TJ opening and closing in response to the FCSA followed single-exponential time courses. A rise of apical Ca⁺⁺ (Ca⁺⁺ _ap) causes a reduction of TJ opening rate in an FCSA or even a partial recuperation of G, an effect that is interpreted as mediated by Ca⁺⁺ _ap entering the open TJs. Protein kinase C (PKC) inhibition by H7 at low concentrations caused a reduction of the rate of junction opening in response to Ca⁺⁺ _bl removal, without affecting junction closing, indicating that PKC in this preparation is a key element in the control of TJ opening dynamics. H7 at 100 μm completely inhibits TJ opening in response to Ca⁺⁺ _bl withdrawal. Subsequent H7 removal caused a prompt inhibition release characterized by a sharp G increase, a process that can be halted again by H7 reintroduction into the bathing solution. Differently from the condition in which Ca⁺⁺ is absent from the apical solution, in which H7 halts the process of G increase in response to a FCSA, when Ca⁺⁺ is present in the apical solution, addition of H7 during G increase in an FCSA not only induces a halt of the G increase but causes a marked recuperation of the TJ seal, indicated by a drop of G, suggesting a cooperative effect of Ca⁺⁺ and H7 on the TJ sealing process. Staurosporine, another PKC inhibitor, differently from H7, slowed both G increase and G decrease in an FCSA. Even at high concentrations (400 nm) staurosporine did not completely block the effect of Ca⁺⁺ withdrawal. These discrepancies between H7 and staurosporine might result from distinct PKC isoforms participating in different steps of TJ dynamics, which might be differently affected by these inhibitors. Immunolocalizations of TJ proteins, carried out in conditions similar to the electrophysiological experiments, show a very nice correlation between ZO-1 and claudin-1 localizations and G alterations induced by Ca⁺⁺ removal from the basolateral solution, both in the absence and presence of H7. Received: 18 April 2001/Revised: 16 July 2001     

The Role of Yeast VDAC Genes on the Permeability of the Mitochondrial Outer Membrane

In addition to the POR1 gene, which encodes the well-characterized voltage dependent anion-selective channel (YVDAC1) of the mitochondrial outer membrane, the yeast Saccharomyces cerevisiae contains a second gene (POR2) encoding a protein (YVDAC2) with 50% sequence identity to YVDAC1. Mitochondria isolated from yeast cells deleted for the POR1 gene (Δpor1) had a profoundly reduced outer membrane permeability as measured by the ability of an intermembrane space dehydrogenase to oxidize exogenously added NADH. Mitochondria missing either YVDAC1 or both YVDAC1 and YVDAC2 showed a 2-fold increase in the rate of NADH oxidation when the outer membrane was deliberately damaged. Mitochondria from parental cells showed only a 10% increase indicating that the outer membrane is highly permeable to NADH. In the absence of YVDAC1, we calculate that the outer membrane permeability to NADH is reduced 20-fold. The low NADH permeability in the presence of YVDAC2 was not due to the low levels of YVDAC2 expression as mitochondria from cells expressing levels of YVDAC2 comparable to those of YVDAC1 in parental cells showed no substantial increase in NADH permeability, indicating a minimal role of YVDAC2 in this permeability. The residual permeability may be due to other pathways because cells missing both genes can still grow on nonfermentable carbon sources. However, YVDAC1 is clearly the major pathway for NADH flux through the outer membrane in these mitochondria. Received: 23 May 1997/Revised: 3 October 1997     

Algae or Protozoa: Phylogenetic Position of Euglenophytes and Dinoflagellates as Inferred from Mitochondrial Sequences

The chloroplasts of euglenophytes and dinoflagellates have been suggested to be the vestiges of endosymbiotic algae acquired during the process of evolution. However, the evolutionary positions of these organisms are still inconclusive, and they have been tentatively classified as both algae and protozoa. A representative gene of the mitochondrial genome, cytochrome oxidase subunit I (coxI), was chosen and sequenced to clarify the phylogenetic positions of four dinoflagellates, two euglenophytes and one apicomplexan protist. This is the first report of mitochondrial DNA sequences for dinoflagellates and euglenophytes. Our COXI tree shows clearly that dinoflagellates are closely linked to apicomplexan parasites but not with algae. Euglenophytes and algae appear to be only remotely related, with euglenophytes sharing a possible evolutionary link with kinetoplastids. The COXI tree is in general agreement with the tree based on the nuclear encoded small subunit of ribosomal RNA (SSU rRNA) genes, but conflicts with that based on plastid genes. These results support the interpretation that chloroplasts present in euglenophytes and dinoflagellates were captured from algae through endosymbioses, while their mitochondria were inherited from the host cell. We suggest that dinoflagellates and euglenophytes were originally heterotrophic protists and that their chloroplasts are remnants of endosymbiotic algae. Received: 24 March 1997 / Accepted: 21 April 1997     

The Action of Blocking Agents Applied to the Inner Face of Ca2+-activated K+ Channels from Human Erythrocytes

The actions of clotrimazole and cetiedil, two drugs known to inhibit the Gardos channel, have been studied on single intermediate conductance calcium-activated potassium (IK_Ca) channels in inside out patches from human red blood cells, and compared with those of TEA and Ba²⁺ applied to the cytoplasmic face of the membrane. TEA produced a fast block which was observed as a reduction in the amplitude of the single channel current. This effect was weakly voltage dependent with the fraction of the membrane potential sensed by TEA at its binding site (δ) of 0.18 and a K _d at 0 mV of 20.5 mm. Ba²⁺ was a very potent blocker of the channel, breaking the single channel activity up into bursts, interspersed with silent periods lasting several seconds. The effect of Ba²⁺ was very voltage sensitive, δ= 0.44, and a K _d at 0 mV of 0.15 μm. Clotrimazole applied to the inner face of the membrane at a concentration ≤1 μm produced a slow block resulting in bursts of channel activity separated by quiescent periods lasting many seconds. The effect of clotrimazole was mimicked by a quaternary derivative UCL 1559, in keeping with an action at the cytoplasmic face of the channel. A high concentration of cetiedil (100 μm) produced only a weak block of the channel. The kinetics of this action were very slow, with burst and inter-burst intervals lasting several minutes. While inhibition of the Gardos channel by cetiedil is unlikely to involve an intracellular site of action, if clotrimazole is able to penetrate the membrane, part of its effect may result from binding to an intracellular site on the channel. Received; 18 February 1998/Received: 5 June 1998     

Lateral Microheterogeneity of Diphenylhexatriene-Labeled Choline Phospholipids in the Erythrocyte Ghost Membrane as Determined by Time-Resolved Fluorescence Spectroscopy

Choline phospholipids are the major constituents of the outer layer of the erythrocyte membrane. To investigate their lateral membrane organization we determined the fluorescence lifetime properties of diphenylhexatriene analogues of phosphatidylcholine, choline plasmalogen, (the respective enolether derivative), and sphingomyelin inserted into the outer layer of hemoglobin-free ghosts. Fluorescence lifetimes were recorded by time-resolved phase and modulation fluorometry and analyzed in terms of Continuous Lorentzian distributions. To assess the influence of membrane proteins on the fluorescence lifetime of the labeled lipids in the biomembrane, lipid vesicles were used as controls. In general, the lifetime distributions in the ghost membranes are broad compared to vesicles. Phosphatidylcholine and sphingomyelin exhibit very similar lifetime distributions in contrast to an increased plasmalogen lifetime heterogeneity in both systems. Orientational effects of side chain mobilities on the observed lifetimes can be excluded. Fluorescence anisotropies revealed identical values for all three labeled phospholipids in the biomembrane. Received: 22 July 1999/Revised: 6 January 2000