Model of the outer membrane potential generation by the inner membrane of mitochondria.

Voltage-dependent anion channels in the outer mitochondrial membrane are strongly regulated by electrical potential. In this work, one of the possible mechanisms of the outer membrane potential generation is proposed. We suggest that the inner membrane potential may be divided on two resistances in series, the resistance of the contact sites between the inner and outer membranes and the resistance of the voltage-dependent anion channels localized beyond the contacts in the outer membrane. The main principle of the proposed mechanism is illustrated by simplified electric and kinetic models. Computational behavior of the kinetic model shows a restriction of the steady-state metabolite flux through the mitochondrial membranes at relatively high concentration of the external ADP. The flux restriction was caused by a decrease of the voltage across the contact sites and by an increase in the outer membrane potential (up to +60 mV) leading to the closure of the voltage-dependent anion channels localized beyond the contact sites. This mechanism suggests that the outer membrane potential may arrest ATP release through the outer membrane beyond the contact sites, thus tightly coordinating mitochondrial metabolism and aerobic glycolysis in tumor and normal proliferating cells.     

Intramolecular and intermolecular enzymatic modulation of ion channels in excised membrane patches.

A calcium-activated potassium channel in posterior pituitary nerve terminals was modulated by phosphorylation and dephosphorylation. Nearly every patch of membrane containing this channel also contained both membrane bound protein phosphatase and membrane-bound protein kinase. By examining the statistical and kinetic nature of phosphorylation and dephosphorylation in excised patches, it was possible to evaluate two contrasting models for these enzymatic reactions. One of these models treated catalysis as an intermolecular process in which the enzyme and substrate are separate molecular species that diffuse and encounter one another during collisions. The second model treated catalysis as an intramolecular process in which the enzyme and substrate reside within a stable macromolecular complex. The study began with a Poisson analysis of the distribution of channel number in patches, and of the number of protein phosphatase-free and protein kinase-free patches. Subsequent kinetic analysis of dephosphorylation yielded an estimate of the mean number of protein phosphatase molecules per patch that was similar to the value obtained from Poisson analysis. Because these two estimates were independent predictions based on the intermolecular model, their agreement supported this model. Analysis of channel number in protein phosphatase-free patches and of the rarity of patches showing partial but incomplete rundown provided additional support for the intermolecular model over the intramolecular model. Furthermore, dephosphorylation exhibited monotonic kinetics with a rate well below the diffusion limit. Thus, several different lines of analysis support the intermolecular model for dephosphorylation, in which the protein phosphatase must encounter its substrate to effect catalysis. In contrast to the monotonic kinetics of dephosphorylation, the phosphorylation reaction exhibited sigmoidal kinetics, with a rate that depended on membrane potential. Voltage dependence is an unlikely property for a kinetic step involving encounters resulting from diffusion. Furthermore, the velocity of the phosphorylation reaction exceeded the diffusion limit, and this observation is inconsistent with the intermolecular model. Thus, both intermolecular and intramolecular enzymatic mechanisms operate in the modulation of the calcium-activated potassium channel of the posterior pituitary. These studies provide a functional characterization of the interactions between enzyme and substrate in intact patches of cell membrane.     

Patch voltage clamp of squid axon membrane.

A small area (patch) of the external surface of a squid axon can be "isolated" electrically from the surrounding bath by means of a pair of concentric glass pipettes. The seawater-filled inner pipette makes contact with the axon and constitutes the external access to the patch. The outer pipette is used to direct flowing sucrose solution over the area surrounding the patch of membrane underlying the inner pipette. Typically, sucrose isolated patches remain in good condition (spike amplitude greater than 90 mV) for periods of approximately one half hour. Patches of axon membrane which had previously been exposed to sucrose solution were often excitable. Membrane survival of sucrose treatment apparently arises from an outflow of ions from the axon and perhaps satellite cells into the interstitial cell space surrounding the exolemma. Estimate of the total access resistance (electrode plus series resistance) to the patch is about 100 komega (7 omega cm2). Patch capacitance ranges from 10-100 pF, which suggests areas of 10(-4) to 10(-5) cm2 and resting patch resistances of 10-100 Momega. Shunt resistance through the interstitial space exposed to sucrose solution, which isolates the patch, is typically 1-2 Momega. These parameters indicate that good potential control and response times can be achieved on a patch. Furthermore, spatial uniformity is demonstrated by measurement of an exoplasmic isopotential during voltage clamp of an axon patch. The method may be useful for other preparations in which limited membrane area is available or in special instances such as in the measurement of membrane conduction noise.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Intrinsic coherence resonance in excitable membrane patches

The influence of intrinsic channel noise on the spiking activity of excitable membrane patches is studied by use of a stochastic generalization of the Hodgkin-Huxley model. Internal noise stemming from the stochastic dynamics of individual ion channels does affect the electric properties of the cell-membrane patches. There exists an optimal size of the membrane patch for which the internal noise alone can cause a nearly regular spontaneous generation of action potentials. We consider the influence of intrinsic channel noise in presence of a constant and an oscillatory current driving for both, the mean interspike interval and the phenomenon of coherence resonance for neuronal spiking. Given small membrane patches, implying that channel noise dominates the excitable dynamics, we find the phenomenon of intrinsic coherence resonance. In this case, the relatively regular spiking behavior becomes essentially independent of an applied stimulus. We observed, however, the occurrence of a skipping of supra-threshold input events due to channel noise for intermediate patch sizes. This effect consequently reduces the overall coherence of the spiking.     

Rat liver glutaminase. Regulation by reversible interaction with the mitochondrial membrane

Partially purified rat liver mitochondrial glutaminase shows a sigmoidal dependence on glutamine concentration, and an absolute requirement for inorganic phosphate as activator. Reconstitution with a mitochondrial membrane fraction changes the kinetic properties of the enzyme making the glutamine dependence more hyperbolic and reducing the concentration of phosphate required for half-maximum activation. Glutaminase activity in isolated mitochondria is known to be increased as a result of mitochondrial swelling. In mitochondria suspended in isotonic medium, the properties of glutaminase resemble of the isolated enzyme while in swollen mitochondria the kinetic properties revert to those exhibited by the enzyme in association with the mitochondrial membrane. It is postulated that mitochondrial glutaminase is regulated in situ by reversible association with the inner mitochondrial membrane which is mediated by mitochondrial swelling. This mechanism may explain the short-term hormonally induced activation of the enzyme observed in isolated hepatocytes.     

Further analysis of spontaneous membrane potential activity and the hyperpolarizing response to parathyroid hormone in osteoblastlike cells

Whole cell voltage clamp measurements using the patch technique on well-attached and well-spread cells of an osteoblastlike line (ROS 17/2.8) show the same spontaneous membrane potential activity as measurements with inserted microelectrodes. Furthermore, membrane potential measurements during the first 80 milliseconds (ms) following microelectrode penetration of the cell membrane usually show no decay. There is also good agreement between values of cell membrane resistance obtained by the microelectrode technique, the whole cell patch clamp technique, and the single channel patch clamp technique. These results indicate that our microelectrode measurements are not dominated by leak-induced artifacts, and that the spontaneous membrane potential activity is not induced by Ca2+ leakage around the microelectrode. The spontaneous membrane potential activity is eliminated in the presence of the Ca2+ ionophore A23187, also in serum-free medium, and by K+ and Ca2+ channel blockers, but it is not affected by the hyperpolarizing responses to parathyroid hormone (PTH) and dibutyryl cAMP, which persist under all of these conditions. These results support the hypothesis that the spontaneous membrane potential activity is related to repeated fluctuations of internal [Ca2+] and that such fluctuations result from a feedback loop involving Ca2+ channels or Ca2+ pumps in the cell membrane.     

Hindered diffusion in excised membrane patches from retinal rod outer segments.

Excised inside-out membrane patches are useful for studying the cGMP-activated ion channels that generate the electrical response to light in retinal rod cells. We show that strong ionic current across a patch changes the driving force on the current by altering the ionic concentration near the surface membrane, an effect somewhat like that first described by Frankenhaeuser and Hodgkin (1956) in squid axons. The dominant concentration change occurs in the solution adjacent to the cytoplasmic (inner) surface of the membrane, where diffusion is impaired by intracellular material that adheres to the patch during excision. The magnitude and time course of the ionic changes are consistent with the expected volume of this material and with an effective diffusion coefficient about an order of magnitude less than that in free solution. Methods are described for correcting current transients observed in voltage clamp experiments, so that channel gating kinetics can be obtained without contamination by changes in driving force. We suggest that restricted diffusion may occur in patches excised from other types of cells and influence rapid kinetic measurements.     

The uptake of phosphatidylcholine by small intestinal brush border membrane is protein-mediated

Brush border membrane vesicles prepared from rabbit small intestine are essentially free of basolateral membranes and nuclear, mitochondrial, microsomal and cytosolic contaminants. The resulting brush border membrane is unstable due to intrinsic lipases and proteinases. The PC transfer between small unilamellar lipid vesicles or mixed lipid micelles as the donor and the brush border membrane vesicles as the acceptor is protein-mediated. After proteolytic treatment of brush border membrane with papain or proteinase K the PC transfer activity is lost and the kinetics of PC uptake are similar to those measured with erythrocytes under comparable conditions. Evidence is presented to show that the PC transfer activity resides in the apical membrane of the enterocyte and not in the basolateral part of the plasma membrane. Furthermore, the activity is localized on the external surface of the brush border membrane exposed to the aqueous medium with its active centre probably not in direct contact with the lipid bilayer of the membrane. Proteins released from brush border membrane by proteolytic treatment catalyze PC exchange between different populations of small unilamellar vesicles. Furthermore, these protein(s) bind(s) PC forming a PC-protein complex.     

A look at membrane patches with a scanning force microscope.

We combined scanning force microscopy with patch-clamp techniques in the same experimental setup and obtained images of excised membrane patches spanning the tip of a glass pipette. These images indicate that cytoskeleton structures are still present in such membrane patches and form a strong connection between the membrane and the glass wall. This gives the membrane patch the appearance of a tent, stabilized by a scaffold of ropes. The lateral resolution of the images depends strongly on the observed structures and can reach values as low as 10 nm on the cytoskeleton elements of a (inside-out) patch. The observations suggest that measurements of membrane elasticity can be made, opening the way for further studies on mechanical properties of cell membranes.     

Exocytosis of single chromaffin granules in cell-free inside-out membrane patches

In chromaffin cells, exocytosis of single granules and properties of the fusion pore--the first connection between vesicular lumen and extracellular space --can be studied by cell-attached patch amperometry, which couples patch-clamp capacitance measurements with simultaneous amperometric recordings of transmitter release. Here we have studied exocytosis of single chromaffin granules and endocytosis of single vesicles in cell-free inside-out membrane patches by patch capacitance measurements and patch amperometry. We excised patches from chromaffin cells by using methods developed for studying properties of single ion channels. With low calcium concentrations in the pipette and bath, the patches showed no spontaneous exocytosis, but exocytosis could be induced in some patches by applying calcium to the cytoplasmic side of the patch. Exocytosis was also stimulated by calcium entry through the patch membrane. Initial conductances of the fusion pore were undistinguishable in cell-attached and excised patch recordings, but the subsequent pore expansion was slower in excised patches. The properties of exocytotic fusion pores in chromaffin cells are very similar to those observed in mast cells and granulocytes. Excised patches provide a tool with which to study the mechanisms of fusion pore formation and endocytosis in vitro.     

The kinetic behavior of an artificial bienzyme membrane.

Artificial membranes bearing immobilized enzymes can be used to study some effects of membrane structure on enzyme kinetic behavior. The bienzyme system described is a mixture of beta-glucosidase and glucose oxidase. Gluconolactone, the product of thesecond enzyme, is an inhibitor of the first one. The resulting feedback effect has been compared using a mixed two-enzyme membrane, two separated one-enzyme membranes, and astirred bienzyme solution. The feedback effect is quicker and more efficient in the two-enzyme membrane than in solution; it is slower and less efficient in the case of the separated one-enzyme membranes. Effects of enzyme proximity in the structure are discussed. Conclusions are drawn concerning the efficiency of feedback mechanisms when enzymes are embedded within a single structure.     

A method for the rapid exchange of solutions bathing excised membrane patches.

In this communication we describe a technique for rapidly exchanging solutions bathing excised membrane patches, and present examples of its implementation using both outside-out and inside-out patches. The ability to make step changes in the concentration of channel-activating ligands (e.g., acetylcholine, calcium) offers a novel and direct means of measuring kinetic processes in the 10-100-ms range. The responses to step ligand concentration changes are well suited to ensemble variance analysis, yielding estimates of the number of channels in a patch, and testing assumptions of channel independence and homogeneity. Kinetic analysis of the pseudomacroscopic currents obtained by averaging large numbers of responses can be compared and correlated with analysis of the microscopic behavior of single channels, using the same membrane patch for both approaches. Practical and theoretical limitations associated with the method are briefly discussed.     

Cell membrane patches are supported by proteoglycans

A cell membrane patch isolated on a patch clamp pipette incorporates in addition to the phospholipid bilayer, an extracellular matrix and cytoskeletal components. The significance of the extracellular matrix for the patch formation was studied in aortic smooth muscle and cerebellar granule cells grow in the presence of an inhibitor of proteoglycan synthesis, -d -xyloside. The xyloside improved the seal success rate, and after patch excision membrane vesicles were formed instead of inside-out patches. When amphotericin B was included in the pipette solution, perforated outside-out vesicles were formed in 96% of cells. The findings suggest, that membrane patches are supported by the extracellular matrix or by structures that relate to this matrix.     

Dual localization of the mitochondrial phospholipase A2: outer membrane contact sites and inner membrane.

Mitochondria were fractionated according to a procedure which allowed to get free outer and inner membrane plus two distinct contact sites between the two membranes. The data indicate that phospholipase A2 is localized in outer membrane contact sites and in inner membrane. The enzyme activity is twice higher in the contact site fraction than in the free membrane. The major fatty acids released are linoleic and docosahexanoic acids.     

Proteolytic cleavage of the disease-related lysosomal membrane glycoprotein CLN7

CLN7 is a polytopic lysosomal membrane glycoprotein of unknown function and is deficient in variant late infantile neuronal ceroid lipofuscinosis. Here we show that full-length CLN7 is proteolytically cleaved twice, once proximal to the used N-glycosylation sites in lumenal loop L9 and once distal to these sites. Cleavage occurs by cysteine proteases in acidic compartments and disruption of lysosomal targeting of CLN7 results in inhibition of proteolytic cleavage. The apparent molecular masses of the CLN7 fragments suggest that both cleavage sites are located within lumenal loop L9. The known disease-causing mutations, p.T294K and p.P412L, localized in lumenal loops L7 and L9, respectively, did not interfere with correct lysosomal targeting of CLN7 but enhanced its proteolytic cleavage in lysosomes. Incubation of cells with selective cysteine protease inhibitors and expression of CLN7 in gene-targeted mouse embryonic fibroblasts revealed that cathepsin L is required for one of the two proteolytic cleavage events. Our findings suggest that CLN7 is inactivated by proteolytic cleavage and that enhanced CLN7 proteolysis caused by missense mutations in selected luminal loops is associated with disease.     

Homology modeling of the three membrane proteins of the dhurrin metabolon: catalytic sites, membrane surface association and protein-protein interactions

Formation of metabolons (macromolecular enzyme complexes) facilitates the channelling of substrates in biosynthetic pathways. Metabolon formation is a dynamic process in which transient structures mediated by weak protein-protein interactions are formed. In Sorghum, the cyanogenic glucoside dhurrin is derived from l-tyrosine in a pathway involving the two cytochromes P450 (CYPs) CYP79A1 and CYP71E1, a glucosyltransferase (UGT85B1), and the redox partner NADPH-dependent cytochrome P450 reductase (CPR). Experimental evidence suggests that the enzymes of this pathway form a metabolon. Homology modeling of the three membrane bound proteins was carried out using the Sybyl software and available relevant crystal structures. Residues involved in tight positioning of the substrates and intermediates in the active sites of CYP79A1 and CYP71E1 were identified. In both CYPs, hydrophobic surface domains close to the N-terminal trans-membrane anchor and between the F′ and G helices were identified as involved in membrane anchoring. The proximal surface of both CYPs showed positively charged patches complementary to a negatively charged bulge on CPR carrying the FMN domain. A patch of surface exposed, positively charged amino acid residues positioned on the opposite face of the membrane anchor was identified in CYP71E1 and might be involved in binding UGT85B1 via a hypervariable negatively charged loop in this protein.     

A dynamic loop provides dual control over the catalytic and membrane binding activity of a bacterial serine hydrolase

The bacterial acyl protein thioesterase (APT) homologue FTT258 from the gram-negative pathogen Francisella tularensis exists in equilibrium between a closed and open state. Interconversion between these two states is dependent on structural rearrangement of a dynamic loop overlapping its active site. The dynamics and structural properties of this loop provide a simple model for how the catalytic activity of FTT258 could be spatiotemporally regulated within the cell. Herein, we characterized the dual roles of this dynamic loop in controlling its catalytic and membrane binding activity. Using a comprehensive library of loop variants, we determined the relative importance of each residue in the loop to these two biological functions. For the catalytic activity, a centrally located tryptophan residue (Trp66) was essential, with the resulting alanine variant showing complete ablation of enzyme activity. Detailed analysis of Trp66 showed that its hydrophobicity in combination with spatial arrangement defined its essential role in catalysis. Substitution of other loop residues congregated along the N-terminal side of the loop also significantly impacted catalytic activity, indicating a critical role for this loop in controlling catalytic activity. For membrane binding, the centrally located hydrophobic residues played a surprising minor role in membrane binding. Instead general electrostatic interactions regulated membrane binding with positively charged residues bracketing the dynamic loop controlling membrane binding. Overall for FTT258, this dynamic loop dually controlled its biological activities through distinct residues within the loop and this regulation provides a new model for the spatiotemporal control over FTT258 and potentially homologous APT function.     

Dynamic Properties of Recruitment,Expansion and Mortality of Macrophyte Patches in Streams

Streams are physically perturbed habitats with high demands on the dispersal and recruitment to maintain plant populations. Yet, little is known about these important demographic processes for stream plants. Therefore, we studied the monospecific vegetation of Callitriche cophocarpa in a small Danish lowland stream to determine: 1) the importance of drifting shoots and seeds for recruitment of plants, and 2) the influence of water flow, light availability and patch size on recruitment, growth and mortality processes. We found that the majority (about 90%) of new colonising patches of plant stands derived from drifting shoots being caught around protruding stones, while few developed from seeds. Many new patches were lost in the flowing water before roots became well established. Flow exposure of the patches resulted in the main growth taking place in the downstream direction. Combined areal cover of Callitriche patches on the stream bottom reached an upper limit of about 70%, probably because areal expansion above this threshold was constrained by strong shear forces and coarse substrata developing in the flow channels between the patches. We discuss why efficient shoot dispersal and vegetative growth documented here for Callitriche is an optimal plant strategy in flow-perturbed streams in contrast to the production of numerous small seeds but limited vegetative spread among ruderal plants in perturbed habitats on land.     

S-adenosylmethionine synthetase in cultured normal and oncogenically-transformed human and rat cells

We have investigated the enzymatic formation of S-adenosylmethionine in extracts of a variety of normal and oncogenically-transformed human and rat cell lines which differ in their ability to grow in medium in which methionine is replaced by its immediate precursor homocysteine. We have localized the bulk of the S-adenosylmethionine synthetase activity to the post-mitochondrial supernatant. We show that in all cell lines a single kinetic species exists in a dialyzed extract with a Km for methionine of about 3-12 microM. In selected lines we have demonstrated a requirement for Mg2+ in addition to that needed to form the Mg X ATP complex for enzyme activity and have shown that the enzyme can be regulated by product feedback inhibition. Because we detect no differences in the enzymatic ability of these cell extracts to utilize methionine for S-adenosylmethionine formation in vitro, we suggest that the failure of oncogenically-transformed cell lines to grow in homocysteine medium may result from the decreased methionine pools in these cells or from the loss of ability of these cells to properly metabolize homocysteine, adenosine, or their cellular product S-adenosylhomocysteine.