F-actin at Newly Invaginated Membrane in Neurons: Implications for Surface Area Regulation

Neuronal shape and volume changes require accompanying cell surface adjustments. In response to osmotic perturbations, neurons show evidence of surface area regulation; shrinking neurons invaginate membrane at the substratum, pinch off vacuoles, and lower their membrane capacitance. F-actin is implicated in reprocessing newly invaginated membrane because cytochalasin causes the transient shrinking-induced invaginations, vacuole-like dilations (VLDs), to persist indefinitely instead of undergoing recovery. To help determine if cortical F-actin indeed contributes to cell surface area regulation, we test, here, the following hypothesis: invaginating VLD membrane rapidly establishes an association with F-actin and this association contributes to VLD recovery. Cultured molluscan (Lymnaea) neurons, whose large size facilitates three-dimensional imaging, were used. In fixed neurons, fluorescent F-actin stains were imaged. In live neurons, VLD membrane was monitored by brightfield microscopies and actin was monitored via a fluorescent tag. VLD formation (unlike VLD recovery) is cytochalasin insensitive and consistent with this, VLDs formed readily in cytochalasin-treated neurons but showed no association with F-actin. Normally, however (i.e., no cytochalasin), VLDs were foci for rapid reorganization of F-actin. At earliest detection (1–2 min), nascent VLDs were entirely coated with F-actin and by 5 min, VLD mouths (i.e., at the substratum) had become annuli of F-actin-rich motile leading edge. Time lapse images from live neurons showed these rings to be motile filopodia and lamellipodia. The retrieval of VLD membrane (vacuolization) occurred via actin-associated constriction of VLD mouths. The interplay of surface membrane and cortical cytoskeleton in osmotically perturbed neurons suggests that cell surface area and volume adjustments are coordinated in part via mechanosensitive F-actin dynamics. Received: 25 March 1999/Revised: 15 June 1999     

Discrete and reversible vacuole-like dilations induced by osmomechanical perturbation of neurons

In cultured Lymnaea stagnalis neurons, osmolarity increases (upshocks) rapidly elicited large membranous dilations that could be dislodged and pushed around inside the cell with a microprobe. Subsequent osmolarity decreases (downshocks) caused these vacuole-like dilations (VLDs) to disappear. Additional upshock/downshock perturbations resulted in repeated appearance/disappearance (formation/reversal) of VLDs at discrete sites. Confocal microscopy indicated that VLDs formed as invaginations of the substrate-adherent surface of the neuron: extracellular rhodamine-dextran entered VLDs as they formed and was expelled during reversal. Our standard VLD-inducing perturbation was: 2–4 min downshock to distilled water, upshock to normal saline. However, a wide range of other osmotic perturbations (involving osmolarities up to 3.5x normal, perturbations with or without Ca²⁺, replacement of ions by sucrose) were also used. We concluded that mechanical, not chemical, aspects of the osmo-mechanical shocks drove the VLD formation and reversal dynamics and that extracellular Ca²⁺ was not required.Following a standard perturbation, VLDs grew from invisible to their full diameter (>10 m) in just over a minute. Over the next 0.5–3 hr in normal saline, neurons recovered. Recovery eliminated any visible VLDs and was accompanied by cytoplasmic turmoil around the VLDs. Recovery was prevented by cytochalasin B, brefeldin A and N-ethylmaleimide but not by nocodazole. In striking contrast, these drugs did not prevent repeated VLD formation and reversal in response to standard osmo-mechanical perturbations; VLD disappearance during reversal and during recovery are different.The osmo-mechanical changes that elicited VLDs may, in an exaggerated fashion, mimic tension changes in extending and retracting neuntes. In this context we postulate: (a) the trafficking or disposition of membrane between internal stores and plasma membrane is mechano sensitive, (b) normally, this mechanosensitivity provides an on demand system by which neurons can accommodate stretch/release perturbations and control cell shape but, (c) given sudden extreme mechanical stimuli, it yields VLDs. Present address: Biology Department, Washington University, St Louis, MO 63130-4899This work was supported by NSERC of Canada research grants to CEM and to LRM. CR was the recipient of a postdoctoral fellowship from the Ministere francais de la Recherche et de l'Espace. We thank J-M. Trifaro for the use of his image processing equipment.     

Neuronal swelling and surface area regulation: elevated intracellular calcium is not a requirement

Neurons aremechanically robust. During prolonged swelling, molluscan neurons cantriple their apparent membrane area. They gain surface area andcapacitance independent of extracellular Ca concentration([Ca]_e), but it isunknown if an increase in intracellular Ca concentration([Ca]_i) isnecessary. If Ca for stimulating exocytosis is unnecessary, it ispossible that swelling-induced membrane tension changes directlytrigger surface area readjustments. If, however, Ca-mediated but nottension-mediated membrane recruitment is responsible for surface areaincreases, swelling neurons should sustain elevated levels of[Ca]_i. The purpose ofthis investigation is to determine if the[Ca]_i in swellingneurons attains levels high enough to promote exocytosis and if anysuch increase is required. Lymnaeaneurons were loaded with the Ca concentration indicator fura 2. Calibration was performed in situ using 4-bromo-A-23187 and Ca-ethyleneglycol-bis(-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA), with free Ca concentration ranging from 0 to 5 µM. Swelling perturbations (medium osmolarity reduced to 25% for 5 min)were done at either a standard[Ca]_e or very low[Ca]_e level (0.9 mM or0.13 µM, respectively). In neither case did the[Ca]_i increase tolevels that drive exocytosis. We also monitored osmomechanically drivenmembrane dynamics [swelling, then formation and reversal ofvacuole-like dilations (VLDs)] with the[Ca]_i clamped below 40 nM via1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). [Ca]_idid not change with swelling, and VLD behavior was unaffected,consistent with tension-driven,[Ca]_i-independent surface area adjustments. In addition, neurons with[Ca]_i clamped at 0.1 µM via an ionophore could produce VLDs. We conclude that, undermechanical stress, neuronal membranes are compliant by virtue ofsurface area regulatory adjustments that operate independent of[Ca]_i. The findingssupport the hypothesis that plasma membrane area is regulated in partby membrane tension.

     

Cell Surface Area Regulation and Membrane Tension

The beautifully orchestrated regulation of cell shape and volume are central themes in cell biology and physiology. Though it is less well recognized, cell surface area regulation also constitutes a distinct task for cells. Maintaining an appropriate surface area is no automatic side effect of volume regulation or shape change. The issue of surface area regulation (SAR) would be moot if all cells resembled mammalian erythrocytes in being constrained to change shape and volume using existing surface membrane. But these enucleate cells are anomalies, possessing no endomembrane. Most cells use endomembrane to continually rework their plasma membrane, even while maintaining a given size or shape. This membrane traffic is intensively studied, generally with the emphasis on targeting and turnover of proteins and delivery of vesicle contents. But surface area (SA) homeostasis, including the controlled increase or decrease of SA, is another of the outcomes of trafficking. Our principal aims, then, are to highlight SAR as a discrete cellular task and to survey evidence for the idea that membrane tension is central to the task. Cells cannot directly ``measure' their volume or SA, yet must regulate both. We posit that a homeostatic relationship exists between plasma membrane tension and plasma membrane area, which implies that cells detect and respond to deviations around a membrane tension set point. Maintenance of membrane strength during membrane turnover, a seldom-addressed aspect of SA dynamics, we examine in the context of SAR. SAR occurs in both animal and plant cells. The review shows the latter to be a continuing source of groundbreaking work on tension-sensitive SAR, but is principally slanted to animal cells. Received: 1 May 2000/Revised: 14 August 2000     

Violet laser diodes in flow cytometry: an update.

INTRODUCTION: In previous studies we and others have demonstrated the usefulness of violet laser diodes (VLDs) as replacement laser sources for krypton-ion lasers on stream-in-air cytometers. Previously available VLDs had a maximum available power of less than 25 mW; this was sufficient for excitation of densely labeled cell surface antigens using fluorochromes such as Cascade Blue or Pacific Blue, but may have been insufficient for applications requiring higher levels of photon saturation, such as low-level expression of Cyan Fluorescent Protein (ECFP) in CFP-YFP FRET applications. In this follow-up study, we have tested more powerful VLDs emitting at 55 mW, and a beam-merged dual module VLD with 100 mW combined output, for their ability to excite a variety of violet-excited fluorochromes, including CFP. METHODS: A dual module VLD (two linear polarized VLDs with their beams merged by a polarized beam combiner) emitting at 404 nm was mounted on a BD FACSVantage DiVa stream-in-air cytometer. The individual polarized 55 mW beams or the 100 mW combined beams were used to analyze PBMCs labeled with the violet-excited probes Cascade Blue, Alexa Fluor 405, Cascade Yellow and Pacific Orange dyes. Violet-excited fluorescent microsphere mixtures with decreasing fluorescence levels were also used to detect the minimum sensitivity threshold and precision of these lasers. VLD excitation on a gel-coupled cuvette flow cytometer was used as a sensitivity baseline. RESULTS: The dual module 100 mW VLD gave both sensitivity and precision levels approaching that observed for lower-power sources on a cuvette cytometer. Single polarized VLD modules at 55 mW gave slightly decreased sensitivity for the microspheres standards and all the tested fluorochromes compared to the 100 mW source. CONCLUSIONS: While 55 mW laser sources performed adequately in the stream-in-air format, increasing the power to 100 mW did give a small but detectable increase in instrument sensitivity. This sensitivity level approached that of cuvette systems.     

Characterization of Inverted Membrane Vesicles from the Halophilic Archaeon Haloferax volcanii

Membrane-related processes in archaea, the third and most-recently described domain of life, are in general only poorly understood. One obstacle to a functional understanding of archaeal membrane-associated activities corresponds to a lack of archaeal model membrane systems. In the following, characterization of inverted archaeal membrane vesicles, prepared from the halophilic archaeon Haloferax volcanii, is presented. The inverted topology of the vesicles was revealed by defining the orientation of membrane-bound enzymes that in intact cells normally face the cytoplasm or of other protein markers, known to face the exterior medium in intact cells. Electron microscopy, protease protection assays and lectin-binding experiments confirmed the sealed nature of the vesicles. Upon alkalinization of the external medium, the vesicles were able to generate ATP, reflecting the functional nature of the membrane preparation. The availability of preparative scale amounts of inverted archaeal membrane vesicles provides a platform for the study of various membrane-related phenomena in archaea. Received: 27 March 2001/Revised: 13 June 2001     

Voltage-Induced Activation of Mechanosensitive Cation Channels of Leech Neurons

The voltage dependence of stretch-activated cation channels in leech central neurons was studied in cell-free configurations of the patch-clamp technique. We established that stretch-activated channels excised from identified cell bodies of desheathed ganglia, as well as from neurons in culture, were slowly and reversibly activated by depolarizing membrane potentials. Negative pressure stimuli, applied to the patch pipette during a slow periodical modulation of membrane potential, enhanced channel activity, whereas positive pressures depressed it. Voltage-induced channel activation was observed, with soft glass pipettes, both in inside-out and outside-out membrane patches, at negative and positive reference potentials, respectively. The results presented in this study demonstrate that membrane depolarization induces slow activation of stretch-activated channels of leech central neurons. This phenomenon is similar to that found in Xenopus oocytes, however, some peculiar features of the voltage dependence in leech stretch-activated channels indicate that specific membrane-glass interactions might not necessarily be involved. Moreover, following depolarization, stretch-activated channels in membrane patches from neurons in culture exhibited significantly shorter delay to activation (sec) than their counterparts from neurons of freshly isolated ganglia (hundreds of sec).     

Thermodynamics and Energetics of the Tonoplast Membrane Operating as a Hysteresis Switch in an Oscillatory Model of Crassulacean Acid Metabolism

The observed endogenous circadian rhythm in plants performing Crassulacean acid metabolism is effected by malate transport at the tonoplast membrane. Experimental and theoretical work asks for a hysteresis switch, regulating this transport via the ordering state of the membrane. We apply a schematic molecular model to calculate the thermally averaged order parameter of the membrane lipid structure in its dependence on external parameters temperature and area per molecule. The model shows a first order structural phase transition in a biologically relevant temperature range. Osmotic consequences of malate accumulation can trigger a transition between the two phases by changing the surface area of the cell vacuole. Estimation of the energy needed to expand the vacuole under turgor pressure because of osmotic changes while acidifying shows that energy needed as latent heat for the calculated change between phases can easily be afforded by the cell. Thus, malate content and the coexisting two phases of lipid order, showing hysteretic behavior, can serve as a feedback system in an oscillatory model of Crassulacean acid metabolism, establishing the circadian clock needed for endogenous rhythmicity. Received: 2 September 1997/Revised: 24 April 1998     

Transient and Permanent Fusion of Vesicles in Zea mays Coleoptile Protoplasts Measured in the Cell-attached Configuration

Exocytosis in protoplasts from Zea mays L. coleoptiles was studied using patch-clamp techniques. Fusion of individual vesicles with the plasma membrane was monitored as a step increase of the membrane capacitance (C _m ). Vesicle fusion was observed as (i) An irreversible step increase in C _m . (ii) Occasionally, irreversible C _m steps were preceded by transient changes in C _m , suggesting that the electrical connection between the vesicle with the plasma membrane opens and closes reversibly before full connection is achieved. (iii) Most frequently, however, stepwise transient changes in C _m did not lead to an irreversible C _m step. Within one patch of membrane capacitance steps due to transient and irreversible fusions were of similar amplitude. This suggests that the exocytosis events do not result from the fusion of vesicles with different sizes but are due to kinetically different states in a fusion process of the same vesicle type. The dwell time histogram of the transient fusion events peaked at about 100 msec. Fusion can be described with a circular three-state model for the fusion process of two fused states and one nonfused state. It predicts that energy input is required to drive the system into a prevailing direction. Received: 27 August 1999/Revised: 28 October 1999     

Transient Activity of Excitatory Cl− Channels in Chara: Evidence for Quantal Release of a Gating Factor

In attached patches on the plasma membrane of nonexcited Chara corallina cells, randomly activating, transient Cl⁻ currents with variable amplitudes were recorded. The peak amplitudes of these currents could be grouped into distinct populations with approximately equidistant mean peak currents. Generally, the mean current of the smallest population measured about half of the distance between the means of subsequent populations. Currents of the smallest population occurred most frequently at all voltages; the frequency of observations decreased with increasing amplitudes of the currents. At all voltages transient currents from different populations were similar in duration with the exception of the smallest currents, which lasted only 0.6 times as long as larger currents. Furthermore, transient currents were most frequent at positive voltages, but once initiated at a positive conditioning pulse they were also observed during subsequent pulses to negative voltages. The results are consistent with the idea that Chara contains Ca²⁺ stores in the vicinity of the plasma membrane, which are indirectly filled from the external medium. Upon quantal Ca²⁺ discharge from adjacent stores, a process independent of membrane voltage, the concentration of Ca²⁺ in the cytoplasm increases transiently. Depending on the number of discharging stores, distinct numbers of Ca²⁺-stimulated Cl⁻ channels activate, giving rise to the macroscopic excitatory Cl⁻ current in these cells. Received: 27 October 1997/Revised: 26 February 1998     

Role of Actin Cytoskeleton in Regulation of Ion Transport: Examples from Epithelial Cells

The identification of molecular water transporters and the generation of transgenic mice lacking water transporting proteins has created a need for accurate methods to measure water permeability. This review is focused on methodology to characterize water permeability in living cells and complex multicellular tissues. The utility of various parameters defining water transport is critically evaluated, including osmotic water permeability (P _f ), diffusional water permeability (P _d ), Arrhenius activation energies (E _a ), and solute reflection coefficients (σ _p ). Measurements in cellular and complex tissues can be particularly challenging because of uncertainties in barrier geometry and surface area, heterogeneity in membrane transporting properties, and unstirred layer effects. Strategies to measure plasma membrane P _f in cell layers are described involving light scattering, total internal reflection fluorescence microscopy, confocal microscopy, interferometry, spatial filtering microscopy, and volume-sensitive fluorescent indicators. Dye dilution and fluorescent indicator methods are reviewed for measurement of P _f across cell and tissue barriers. Novel fluorescence and gravimetric methods are described to quantify microvascular and epithelial water permeabilities in intact organs, using as an example lungs from aquaporin knockout mice. Finally, new measurement strategies and applications are proposed, including high-throughput screening for identification of aquaporin inhibitors. Received: 3 August 1999/Revised: 22 September 1999     

Permeability of Boric Acid Across Lipid Bilayers and Factors Affecting It

Boron enters plant roots as undissociated boric acid (H₃BO₃). Significant differences in B uptake are frequently observed even when plants are grown under identical conditions. It has been theorized that these differences reflect species differences in permeability coefficient of H₃BO₃ across plasma membrane. The permeability coefficient of boric acid however, has not been experimentally determined across any artificial or plant membrane. In the experiments described here the permeability coefficient of boric acid in liposomes made of phosphatidylcholine was 4.9 × 10⁻⁶ cm sec⁻¹, which is in good agreement with the theoretical value. The permeability coefficient varied from 7 × 10⁻⁶ to 9.5 × 10⁻⁹ cm sec⁻¹ with changes in sterols (cholesterol), the type of phospholipid head group, the length of the fatty acyl chain, and the pH of the medium. In this study we also used Arabidopsis thaliana mutants which differ in lipid composition to study the effect of lipid composition on B uptake. The chs1-1 mutant which has lower proportion of sterols shows 30% higher B uptake compared with the wild type, while the act1-1 mutant which has an increased percentage of longer fatty acids, exhibited 35% lower uptake than the wild type. Lipid composition changes in each of the remaining mutants influenced B uptake to various extents. These data suggest that lipid composition of the plasma membrane can affect total B uptake. Received: 15 October 1999/Revised: 11 February 2000     

Probing Transmembrane Topology of the High-Affinity Sodium/Glucose Cotransporter (SGLT1) with Histidine-Tagged Mutants

To reexamine the existing predictions about the general membrane topology of the high-affinity Na⁺/glucose cotransporter (SGLT1) and in particular of the large loop at the C-terminal region, a small 6 × Histidine-tag was introduced at different positions of the SGLT1 sequence by site-directed mutagenesis. Eleven His-SGLT1 mutants were constructed and were transiently transfected into COS-7 cells. As demonstrated by immunofluorescent labeling with antipeptide antibodies against SGLT1, all mutants were expressed and inserted into the plasma membrane. Only mutants with the tag in the N-terminal region and the C-terminal region retained Na⁺/glucose cotransport activity at 0.1 mm d-glucose. The arrangement of the His-tag in the membrane was analyzed by indirect immunofluorescence, using a monoclonal antihistidine antibody. In nonpermeabilized cells the His-tag could be detected at the N-terminal end (insertion at aa 5) and at the C-terminal end (replacement between aa 584-589 and between aa 622-627), suggesting that these portions of the polypeptide are accessible from the extracellular space. Furthermore, an epitope-specific antibody directed against aa 606-630 reacted strongly with the cell surface. To support this topology intact stably transfected SGLT1 competent CHO cells were partially digested with an immobilized trypsin and subsequently subjected to electrophoresis and Western blot analysis. The size of the digestion product suggests that extravesicular trypsin removed the extracellular loop that contains the amino acid residues 549-664. Thus our results indicate that the last large loop (about aa 541–aa 639) towards the C-terminal end faces the cell exterior where it might be involved in substrate recognition. Received: 29 January 1999/Revised: 26 February 1999     

Complement Activation by Bacterial Surface Glycolipids: A Study with Planar Bilayer Membranes

Planar asymmetric glycolipid/phospholipid bilayer membranes were used as a reconstitution model of the lipid matrix of the outer membrane of Gram-negative bacteria to study complement (C) activation by various bacterial surface glycolipids with the aim of defining the C activation pathway. As glycolipids the lipopolysaccharides of Salmonella enterica serovar Minnesota R mutant strains R595 (Re LPS) and R4 (Rd₂ LPS), pentaacyl lipid A from the LPS of the Escherichia coli Re mutant F515, and glycosphingolipid GSL-1 of Sphingomonas paucimobilis IAM 12576 were used. Methylester and carboxyl-reduced derivatives of GSL-1 were used to elucidate the role of the carboxyl group as common functional group of LPS and GSL-1 for C activation. The formation of lytic pores was monitored via the measurement of changes in membrane current. For all glycolipids we observed a considerable increase in membrane current soon after addition of whole human serum due to the formation of lytic pores in the membranes. Pore formation was dependent on the presence of C9, indicating that the observed current changes were due to C activation. We found that in our reconstitution system of the outer membrane lipid A, Re LPS, and Rd₂ LPS activated the classical pathway, the activation being independent of specific anti-LPS antibodies. In contrast, GSL-1 and the methylester derivative of GSL-1 activated the alternative pathway even at the low serum concentrations used in this study (about 0.2% v/v). Interestingly, the carboxyl reduced GSL-1 activated the classical pathway. Received: 16 July 1998/Revised: 28 October 1998     

Effect of Two Tyrosine Mutations on the Activity and Regulation of the Renal Type II Na/P i -Cotransporter Expressed in Oocytes

The rat renal type II Na/Pi-cotransporter (NaPi2), which is regulated by mechanisms involving endocytosis and lysosomal degradation, contains two sequences that show high homology with two tyrosine (Y)-based consensus motifs previously reported to be involved in such intracellular trafficking: GY₄₀₂FAM matching the consensus sequence GYXXZ, and Y₅₀₉RWF matching the motif YXXO. Mutations of any of these two Y nearly abolished the NaPi2 mediated ³²P _i -uptake after cRNA-injection into oocytes. The mechanisms underlying these defects are however different. Mutation of the Y402 results in a lack of glycosylation and reduced surface expression of the cotransporter, that are specific for the Y402 mutation since substitution of the neighboring F403 did not have any effect. The inhibitory effect of the Y509 mutation is related to a functional inactivation of the protein expressed in the plasma membrane; mutation of the neighboring R510 also led to a decrease in the cotransporter activity. Pharmacological activation of the protein kinase C cascade by DOG induced the retrieval of both wild-type (WT) as well as Y509 cotransporters from the oocyte plasma membrane. These data suggest that the Y402 is important for the surface expression whereas Y509 for the function of the type II Na/P _i -cotransporter expressed in oocytes. Y509 seems not to be involved in the membrane retrieval of the cotransporter. Received: 3 November 1998/Revised: 20 January 1999     

Directly Observed Membrane Fusion Between Oppositely Charged Phospholipid Bilayers

A novel method was developed for the direct examination of pairwise encounters between positively and negatively charged phospholipid bilayer vesicles. Giant bilayer vesicles (unilamellar, 4–20 μm in diameter) prepared from 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine, a new cationic phospholipid derivative, were electrophoretically maneuvered into contact with individual anionic phospholipid vesicles. Fluorescence video microscopy revealed that such vesicles commonly underwent fusion within milliseconds (1 video field) after contact, without leakage. Fusion occurred at constant volume and, since flaccid vesicles were rare, the excess membrane was not available after fusion. Hemifusion (the outer monolayers of each vesicle fused while the inner monolayers remained intact) was inferred from membrane-bound dye transfer and a change in the contact area. Hemifusion was observed as a final stable state and as an intermediate to fusion of vesicles composed of charged phospholipids plus zwitterionic phospholipids. Hemifusion occurred in one of three ways following adhesion: either delayed with an abrupt increase in area of contact, immediately with a gradual increase in area of contact, or with retraction during which adherent vesicles dissociated from a flat contact to a point contact. Phosphatidylethanolamine strongly promoted immediate hemifusion; the resultant hemifused state was stable and seldom underwent complete fusion. Although sometimes single contacts between vesicles led to rupture of both, in other cases, a single vesicle underwent multiple fusion events. Direct observation has unequivocally demonstrated the fusion of two, isolated bilayer-bounded bodies to yield a stable, non-leaky product, as occurs in cells, in the absence of proteins. Received: 25 November 1998/Revised: 23 March 1999     

Identification of ATP-sensitive Potassium Channel in Frog Ventricular Myocytes

Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80 μm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with little yolk. Neither shortening τ_e (spin echo time) to 9 msec (from 18 msec) nor lengthening τ_r (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T₁ (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular medium (T₁= 3.0 sec), cytoplasm (T₁= 0.8 sec) and nucleoplasm (T₁= 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass) of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference between the nuclear and cytoplasmic compartments. Received: 18 January 1996/Revised: 29 April 1996     

pH-Induced Proton Permeability Changes of Plasma Membrane Vesicles

In vivo studies with leaf cells of aquatic plant species such as Elodea nuttallii revealed the proton permeability and conductance of the plasma membrane to be strongly pH dependent. The question was posed if similar pH dependent permeability changes also occur in isolated plasma membrane vesicles. Here we report the use of acridine orange to quantify passive proton fluxes. Right-side out vesicles were exposed to pH jumps. From the decay of the applied ΔpH the proton fluxes and proton permeability coefficients (P_H+) were calculated. As in the intact Elodea plasma membrane, the proton permeability of the vesicle membrane is pH sensitive, an effect of internal pH as well as external pH on P_H+ was observed. Under near symmetric conditions, i.e., zero electrical potential and zero ΔpH, P_H+ increased from 65 × 10⁻⁸ at pH 8.5 to 10⁻¹ m/sec at pH 11 and the conductance from 13 × 10⁻⁶ to 30 × 10⁻⁴ S/m². At a constant pH _i of 8 and a pH _o going from 8.5 to 11, P_H+ increased more than tenfold from 2 to 26 × 10⁻⁶ m/sec. The calculated values of P_H+ were several orders of magnitude lower than those obtained from studies on intact leaves. Apparently, in plasma membrane purified vesicles the transport system responsible for the observed high proton permeability in vivo is either (partly) inactive or lost during the procedure of vesicle preparation. The residue proton permeability is in agreement with values found for liposome or planar lipid bilayer membranes, suggesting that it reflects an intrinsic permeability of the phospholipid bilayer to protons. Possible implications of these findings for transport studies on similar vesicle systems are discussed. Received: 5 April 1995/Revised: 28 March 1996     

Modulation of Ca2+ Influx in Leech Retzius Neurons. I. Effect of Extracellular pH

We investigated the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca²⁺]_i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K⁺ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca²⁺]_i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca²⁺]_i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca²⁺]_i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca²⁺]_i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane. Received: 23 January 2001/Revised: 15 June 2001     

Thermal Stability of the Plasma Membrane Calcium Pump. Quantitative Analysis of Its Dependence on Lipid-Protein Interactions

Thermal stability of plasma membrane Ca²⁺ pump was systematically studied in three micellar systems of different composition, and related with the interactions amphiphile-protein measured by fluorescence resonance energy transfer. Thermal denaturation was characterized as an irreversible process that is well described by a first order kinetic with an activation energy of 222 ± 12 kJ/mol in the range 33–45°C. Upon increasing the mole fraction of phospholipid in the mixed micelles where the Ca²⁺ pump was reconstituted, the kinetic coefficient for the inactivation process diminished until it reached a constant value, different for each phospholipid species. We propose a model in which thermal stability of the pump depends on the composition of the amphiphile monolayer directly in contact with the transmembrane protein surface. Application of this model shows that the maximal pump stability is attained when 80% of this surface is covered by phospholipids. This analysis provides an indirect measure of the relative affinity phospholipid/detergent for the hydrophobic transmembrane surface of the protein (K _LD ) showing that those phospholipids with higher affinity provide greater stability to the Ca²⁺ pump. We developed a method for directly measure K _LD by using fluorescence resonance energy transfer from the membrane protein tryptophan residues to a pyrene-labeled phospholipid. K _LD values obtained by this procedure agree with those obtained from the model, providing a strong evidence to support its validity. Received: 5 August 1999/Revised: 20 October 1999