Photofrin II Sensitized Modifications of Ion Transport Across the Plasma Membrane of an Epithelial Cell Line: II. Analysis at the Level of Membrane Patches

In the first part of this study, photofrin II sensitized membrane modifications of OK-cells were investigated at the level of macroscopic membrane currents. In this second part, the inside-out configuration of the patch-clamp technique is applied to analyze the phenomena at the microscopic level. It is shown that the characteristic single channel fluctuations of the electric current disappear after the start of illumination of membrane patches in the presence of photofrin II. This holds for all three types of ion channels investigated: the large-conductance Ca²⁺-dependent K⁺ channel (maxi-K_Ca), a K⁺ channel of small conductance (sK), and a stretch-activated nonselective cation channel (SA-cat). Part of the experiments show a transient activation of the channels (indicated by an increase of the probability in the open-channel state) before the channels are converted into a closed nonconductive state. Inactivation of all three channel types proceeds by a continuous reduction of their open probability, while the single channel conductance values are not affected. The process of photodynamically induced channel inactivation is followed by a pronounced increase of the leak conductance of the plasma membrane. The latter process — after light-induced initiation — is found to continue in the dark. The ionic pathways underlying the leak conductance also allow permeation of Ca²⁺ ions. The resulting Ca²⁺-flux may contribute to the photodynamically induced increase of the intracellular Ca²⁺ concentration observed in various cell lines. Received: 26 May 1998/Revised: 8 September 1998     

Photomodification of the Electrical Properties of the Plasma Membrane: A Comparison Between 6 Different Membrane-Active Photosensitizers

The present study deals with photomodification of the electrical properties of the plasma membrane of an epithelial cell line (opossum kidney (OK) cells). The effect of photofrin II (previously investigated) is compared with that of 5 other membrane-active sensitizers: sulfonated Zn-phthalocyanine, merocyanine 540, rose bengal, methylene blue and protoporphyrin IX (an endogenous sensitizer induced by addition of its biosynthetic precursor 5-aminolaevulinic acid). The study was performed in order to investigate whether photomodification of the ion transport properties of the plasma membrane by membrane-active sensitizers is a general and early event in cellular photosensitization. The changes in the electrical properties were monitored by application of the whole-cell and the inside-out configuration of the patch-clamp technique. Illumination in the presence of the compounds (apart from merocyanine 540) gave rise to similar changes of the electrical properties of the membrane: depolarization of the membrane potential, inactivation of a large-conductance, Ca²⁺-dependent K⁺-channel (maxi-K_Ca), and a strong increase of the leak conductance of the membrane. This similarity indicates the general character of the functional photomodifications by membrane-active sensitizers previously reported for photofrin II. Received: 5 September 2000/Revised: 28 December 2000     

Magnesium Transport Across the Basolateral Plasma Membrane of the Fish Enterocyte

In tilapia (Oreochromis mossambicus) intestine, Mg²⁺ transport across the epithelium involves a transcellular, Na⁺- and Na⁺/K⁺-ATPase dependent pathway. In our search for the Mg²⁺ extrusion mechanism of the basolateral compartment of the enterocyte, we could exclude Na⁺/Mg²⁺ antiport or ATP-driven transport. Evidence is provided, however, that Mg²⁺ movement across the membrane is coupled to anion transport. In basolateral plasma membrane vesicles, an inwardly directed Cl⁻ gradient stimulated Mg²⁺ uptake (as followed with the radionuclide ²⁷Mg) twofold. As Cl⁻-stimulated uptake was inhibited by the detergent saponin and by the ionophore A23187, Mg²⁺ may be accumulated intravesicularly above chemical equilibrium. Valinomycin did not affect uptake, suggesting that electroneutral symport activity occurred. The involvement of anion coupled transport was further indicated by the inhibition of Mg²⁺ uptake by the stilbene derivative, 4,4′-diisothiocyanato-stilbene-2,2′-disulfonic acid. Kinetic analyses of the Cl⁻-stimulated Mg²⁺ uptake yielded a K _m (Mg²⁺) of 6.08 ± 1.29 mmol · l⁻¹ and a K _m (Cl⁻) of 26.5 ± 6.5 mmol · l⁻¹, compatible with transport activity at intracellular Mg²⁺- and Cl⁻-levels. We propose that Mg²⁺ absorption in the tilapia intestine involves an electrically neutral anion symport mechanism. Received: 19 January 1996/Revised: 1 August 1996     

Kinetics of Chloride-Bicarbonate Exchange Across the Human Red Blood Cell Membrane

We had previously shown that an influx of extracellular Ca²⁺ (Ca²⁺ _e ), though it occurs, is not strictly required for aminoethyldextran (AED)-triggered exocytotic membrane fusion in Paramecium. We now analyze, by quenched-flow/freeze-fracture, to what extent Ca²⁺ _e contributes to exocytotic and exocytosis-coupled endocytotic membrane fusion, as well as to detachment of ``ghosts' — a process difficult to analyze by any other method or in any other system. Maximal exocytotic membrane fusion (analyzed within 80 msec) occurs readily in the presence of [Ca<sup>2+</sup>] <sub>e</sub> ≥ 5 × 10<sup>−6</sup> m, while normally a [Ca<sup>2+</sup>] <sub>e</sub> = 0.5 mm is in the medium. A new finding is that exocytosis and endocytosis is significantly stimulated by increasing [Ca<sup>2+</sup>] <sub>e</sub> even beyond levels usually available to cells. Quenching of [Ca<sup>2+</sup>] <sub>e</sub> by EGTA application to levels of resting [Ca<sup>2+</sup>] <sub>i</sub> or slightly below does reduce (by ∼50%) but not block AED-triggered exocytosis (again tested with 80 msec AED application). This effect can be overridden either by increasing stimulation time or by readdition of an excess of Ca<sup>2+</sup> <sub>e</sub> . Our data are compatible with the assumption that normally exocytotic membrane fusion will include a step of rapid Ca<sup>2+</sup>-mobilization from subplasmalemmal pools (``alveolar sacs') and, as a superimposed step, a Ca²⁺-influx, since exocytotic membrane fusion can occur at [Ca²⁺] _e even slightly below resting [Ca²⁺] _i . The other important conclusion is that increasing [Ca²⁺] _e facilitates exocytotic and endocytotic membrane fusion, i.e., membrane resealing. In addition, we show for the first time that increasing [Ca²⁺] _e also drives detachment of ``ghosts' — a novel aspect not analyzed so far in any other system. According to our pilot calculations, a flush of Ca²⁺, orders of magnitude larger than stationary values assumed to drive membrane dynamics, from internal and external sources, drives the different steps of the exo-endocytosis cycle. Received: 27 September 1996/Revised: 11 February 1997     

The Calculation of Intracellular Ion Concentrations and Membrane Potential from Cell-attached and Excised Patch Measurements. Cytosolic K+ Concentration and Membrane Potential in Vicia faba Guard Cells

Ion channel activity in cell-attached patch recordings shows channel behavior under more physiological conditions than whole-cell and excised patch measurements. Yet the analysis of cell-attached patch measurements is complicated by the fact that the system is ill defined with respect to the intracellular ion activities and the electrical potential actually experienced by the membrane patch. Therefore, of the several patch-clamp configurations, the information that is obtained from cell-attached patch measurements is the most ambiguous. The present study aims to achieve a better understanding of cell-attached patch measurements. Here we describe a method to calculate the intracellular ion concentration and membrane potential prevailing during cell-attached patch recording. The first step is an analysis of the importance of the input resistance of the intact cell on the cell-attached patch measurement. The second step, and actual calculation, is based on comparison of the single channel conductance and reversal potential in the cell-attached patch and excised patch configurations. The method is demonstrated with measurements of membrane potential and cytosolic K⁺ concentrations in Vicia faba guard cells. The approach described here provides an attractive alternative to the measurement of cytosolic ion concentrations with fluorescent probes or microelectrodes. Received: 3 April 1998/Revised: 6 August 1998     

Modeling Plasmalemma Ion Transport of the Aquatic Plant Egeria densa

Fresh-water plants generate extraordinarily high electric potential differences at the plasma membrane. For a deeper understanding of the underlying transport processes a mathematical model of the electrogenic plasmalemma ion transport was developed based on experimental data mainly obtained from Egeria densa. The model uses a general nonlinear network approach and assumes coupling of the transporters via membrane potential. A proton pump, an outward-rectifying K⁺ channel, an inward-rectifying K⁺ channel, a Cl⁻ channel and a (2H-Cl)⁺ symporter are considered to be elements of the system. The model takes into consideration the effects of light, external pH and ionic content of the bath medium on ion transport. As a result it does not only satisfactorily describe the membrane potential as a function of these external physiological factors but also succeeds in simulating the effects of specific inhibitors as well as I-V-curves obtained with the patch-clamp technique in the whole cell mode. The quality of the model was checked by stability and sensitivity analyses. Received: 18 March 1996/Revised: 17 July 1996     

Electrogenic Properties of the Sodium Pump in a Dynamic Model of Membrane Transport

The general purpose of this theoretical work is to contribute to understand the physiological role of the electrogenic properties of the sodium pump, by studying a dynamic model that integrates diverse processes of ionic and water transport across the plasma membrane. For this purpose, we employ a mathematical model that describes the rate of change of the intracellular concentrations of Na⁺, K⁺ and Cl⁻, of the cell volume, and of the plasma membrane potential (V _m ). We consider the case of a nonexcitable, nonpolarized cell expressing the sodium pump; Na⁺, K⁺, Cl⁻ and water channels, and cotransporters of KCl and NaCl in its plasma membrane. We particularly analyze here the conditions under which the physiological V _m can be generated in a predominantly electrogenic fashion, as a result of the activity of the sodium pump. A major conclusion of this study is that, for the cell model considered, a low potassium permeability is not a sufficient condition for a predominantly electrogenic generation of the V _m by the sodium pump. The presence of an electroneutral exchange of Na⁺ and K⁺ represents a necessary additional requirement. Received: 8 September 1999/Revised: 21 March 2000     

Discontinuities in the Temperature Function of Transmembrane Water Transport in Chara: Relation to Ion Transport

The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P _d ) were observed delimiting a region of low activation energy (E _a around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region water transport was of a different type with high E _a (about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at 15–30°C and lipid matrix-mediated transport beyond this region. The K⁺ channel inhibitor, tetraethylammonium as well as the Cl⁻ channel inhibitor, ethacrynic acid, diminished P _d in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate) the water transport inhibitor in erythrocytes also known to affect K⁺ transport in Chara, only increased P _d below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K⁺ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence of the conductance of a single K⁺ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K⁺ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at 13 and 29°C. Received: 12 August 1998/Revised: 13 November 1998     

Protein Translocation Across the Membrane of the Endoplasmic Reticulum

Saccharomyces cerevisiae and mammals concerning the mechanisms of the translocation step and discuss the roles of the proteins implicated in this process. Received: 5 June 1996/Revised: 20 September 1996     

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     

Partners in Protection: Interdependence of Cytoskeleton and Plasma Membrane in Adaptations to Applied Forces

In mechanically active environments mammalian cells must cope with potentially injurious forces to survive, but the most proximal mechanosensors are largely unknown. How mechanoprotective responses to applied forces are generated and regulated is still a mystery. We consider recent evidence that suggests cellular mechanoprotective adaptations involve a coordinated remodeling of the cell membrane and the associated cytoskeleton. The plasma membrane ``protects' the cytoskeleton by maintenance of intracellular ionic balance and can modulate force-induced cytoskeletal rearrangements by stretch-activated (e.g., Ca²⁺) ion channels and mechanosensitive enzymes (e.g., Phospholipase A₂ and Phospholipase C). Conversely, the cytoskeleton protects the plasma membrane by providing structural support, reinforcement of the cortical framework at sites of force application, modulation of mechanosensitive ion channels and by potentially contributing to the membrane resealing process after mechanical rupture. We suggest that the plasma membrane and the cytoskeleton are partners in the cytoprotective response to physical forces. Received: 8 September 1999/Revised: 15 December 1999     

Influence of Membrane Physical State on the Lysosomal Proton Permeability

Influence of membrane physical state on the proton permeability of isolated lysosomes was assessed by measuring the membrane potential with 3,3′-dipropylthiadicarbocyanine iodide and monitoring their proton leakage with p-nitrophenol. Changes in the membrane order were examined by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene. Both the membrane potential and proton leakage increased with fluidizing the lysosomal membranes by benzyl alcohol and decreased with rigidifying the membranes by cholesteryl hemisuccinate. The proton permeability increased to the maximum of 42% by the benzyl alcohol treatment and decreased to the minimum of 38.1% by the cholesteryl hemisuccinate treatment. Treating the lysosomes with protonophore CCCP increased the proton permeability by 58%. The effects of the membrane fluidization and rigidification can be reversed by rigidifying the fluidized membranes and fluidizing the rigidified membranes, respectively. The results indicate that the proton permeability of lysosomes increased and decreased with increasing and decreasing their membrane fluidity, respectively. Moreover, the lysosomal proton permeability did not alter further if the changes, either an increase or a decrease, in the fluidity exceeded some amount. The results suggest that the proton permeability of lysosomes can be modulated finitely by the alterations in their membrane physical state. Received: 27 September 1999 / Revised: 27 December 1999     

Determination of the Individual Electrical and Transport Properties of the Plasmalemma and the Tonoplast of the Giant Marine Alga Ventricaria ventricosa by Means of the Integrated Perfusion/Charge-Pulse Technique: Evidence for a Multifolded Tonoplast

The charge-pulse relaxation spectrum of nonperfused and perfused (turgescent) cells of the giant marine alga Ventricaria ventricosa showed two main exponential decays with time constants of approximately 0.1 msec and 10 msec, respectively, when the cells were bathed in artificial sea water (pH 8). Variation of the external pH did not change the relaxation pattern (in contrast to other giant marine algae). Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the vacuolar perfusion solution resulted in the disappearance of the slow exponential, whereas external nystatin decreased dramatically the time constant of the fast one. This indicated (by analogy to corresponding experiments with Valonia utricularis, J. Wang, I. Spiess, C. Ryser, U. Zimmermann, J. Membrane Biol. 157: 311-321, 1997) that the fast relaxation must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. Consistent with this, external variation of [K+]o or of [Cl-]o as well as external addition of K+- or Cl--channel/carrier inhibitors (TEA, Ba2+, DIDS) affected only the fast relaxation, but not the slow one. In contrast, addition of these inhibitors to the vacuolar perfusion solution had no measurable effect on the charge-pulse relaxation spectrum. The analysis of the data in terms of the "two membrane model" showed that K+- and (to a smaller extent) Cl--conducting elements dominated the plasmalemma conductance. The analysis of the charge-pulse relaxation spectra also yielded the following area-specific data for the capacitance and the conductance for the plasmalemma and tonoplast (by assuming that both membranes have a planar surface): (plasmalemma) Cp = 0.82 * 10(-2) F m-2, Rp = 1.69 * 10(-2) Omega m2, Gp = 5.9 * 10(4) mS m-2, (tonoplast) Ct = 7. 1 * 10(-2) F m-2, Rt = 14.9 * 10(-2) Omega m2 and Gt = 0.67 * 10(4) mS m-2. The electrical data for the tonoplast show that (in contrast to the literature) the area-specific membrane resistance of the tonoplast of these marine giant algal cells is apparently very high as reported already for V. utricularis. The exceptionally high value of the area-specific capacitance could be explained - among other interpretations - by assuming a 9-fold enlargement of the tonoplast surface. The hypothesis of a multifolded tonoplast was supported by transmission electronmicroscopy of cells fixed under maintenance of turgor pressure and of the electrical parameters of the membranes. This finding indicates that the tonoplast of this species exhibited a sponge-like appearance. Taking this result into account, it can be easily shown that the tonoplast exhibits a high-resistance (1.1 Omega m2). Vacuolar membrane potential measurements (performed in parallel with charge-pulse relaxation studies) showed that the potential difference across the plasmalemma was mainly controlled by the external K+-concentration which suggested that the resting membrane potential of the plasmalemma is largely a K+-diffusion potential. After permeabilization of the tonoplast with nystatin the potential of the intact membrane barrier dropped from about slightly negative or positive (-5.1 to +18 mV, n = 13) to negative values (-15 up to -68 mV; n = 8). This indicated that the cytoplasm of V. ventricosa was apparently negatively charged relative to the external medium. Permeabilization of the plasmalemma by addition of external nystatin resulted generally in an increase in the potential to slightly more positive values (-0.8 to +4.3 mV; n = 5), indicating that the vacuole is positively charged relative to the cytoplasm. These findings apparently end the long-term debate about the electrical properties of V. ventricosa. The results presented here support the findings of Davis (Plant Physiol. 67: 825-831, 1981), but are contrary to the results of Lainson and Field (J. Membrane Biol. 29: 81-94, 1976).     

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     

Sugar Transport Heterogeneity in the Kidney: Two Independent Transporters or Different Transport Modes through an Oligomeric Protein? 1. Glucose Transport Studies

The kinetics of Na⁺/d-glucose cotransport (SGLT) were reevaluated in rabbit renal brush border membrane vesicles isolated from the whole kidney cortex using a fast-sampling, rapid-filtration apparatus (FSRFA, US patent #5,330,717) for uptake measurements. Our results confirm SGLT heterogeneity in this preparation, and both high (HAG) and low (LAG) affinity glucose transport pathways can be separated over the 15–30°C range of temperatures. It is further shown that: (i) Na⁺ is an essential activator of both HAG and LAG; (ii) similar energies of activation can be estimated from the linear Arrhenius plots constructed from the V _max data of HAG and LAG, thus suggesting that the lipid composition and/or the physical state of the membrane do not affect much the functioning of SGLT; (iii) similar V _max values are observed for glucose and galactose transport through HAG and LAG, thus demonstrating that the two substrates share the same carrier agencies; and (iv) phlorizin inhibits both HAG and LAG competitively and with equal potency (K _i = 15 μm). Individually, these data do not allow us to resolve conclusively whether the kinetic heterogeneity of SGLT results from the expression in the proximal tubule of either two independent transporters (rSGLT1 and rSGLT2) or from a unique transporter (rSGLT1) showing allosteric kinetics. Altogether and compared to the kinetic characteristics of the cloned SGLT1 and SGLT2 systems, they do point to a number of inconsistencies that lead us to conclude the latter possibility, although it is recognized that the two alternatives are not mutually exclusive. It is further suggested, from the differences in the K _m values of HAG transport in the kidney as compared to the small intestine and SGLT1 cRNA-injected oocytes, that renal SGLT1 activity is somehow modulated, maybe through heteroassociation with (a) regulatory subunit(s) that might also contribute quite significantly to sugar transport heterogeneity in the kidney proximal tubule. Received: 25 October 1995/Revised: 10 June 1996     

Structure-Function Relationship of the Ion Channel Formed by Diphtheria Toxin in Vero Cell Membranes

Diphtheria toxin (DT) forms cation selective channels at low pH in cell membranes and planar bilayers. The channels formed by wild-type full length toxin (DT-AB), wild-type fragment B (DT-B) and mutants of DT-B were studied in the plasma membrane of Vero cells using the patch-clamp technique. The mutations concerned certain negatively charged amino acids within the channel-forming transmembrane domain (T-domain). These residues might interact electrostatically with cations flowing through the channel, and were therefore exchanged for uncharged amino acids or lysine. The increase in whole-cell conductance induced by toxin, Δg _m , was initially determined. DT-AB induced a ∼10-fold lower Δg _m than DT-B. The mutations DT-B E327Q, DT-B D352N and DT-B E362K did not affect Δg _m , whereas DT-B D295K, DT-B D352K and DT-B D318K drastically reduced Δg _m . Single channel analysis of DT-B, DT-AB, DT-B D295K, DT-B D318K and DT-B E362K was then performed in outside-out patches. No differences were found for the single-channel conductances, but the mutants varied in their gating characteristics. DT-B D295K exhibited only a very transient channel activity. DT-AB as well as DT-B D318K displayed significantly lower open probability and mean dwell times than DT-B. Hence, the lower channel forming efficiency of DT-AB and DT-B D318K as compared to DT-B is reflected on the molecular level by their tendency to spend more time in the closed position and the fast flickering mode. Altogether, the present work shows that replacements of single amino acids distributed throughout a large part of the transmembrane domain (T-domain) strongly affect the overall channel activity expressed as Δg _m and the gating kinetics of single channels. This indicates clearly that the channel activity observed in DT-exposed Vero cells at low pH is inherent to DT itself and not due to DT-activation of an endogenous channel. Received: 20 June 1996/Revised: 8 November 1996     

Peptides and Membrane Fusion: Towards an Understanding of the Molecular Mechanism of Protein-Induced Fusion

Processes such as endo- or exocytosis, membrane recycling, fertilization and enveloped viruses infection require one or more critical membrane fusion reactions. A key feature in viral and cellular fusion phenomena is the involvement of specific fusion proteins. Among the few well-characterized fusion proteins are viral spike glycoproteins responsible for penetration of enveloped viruses into their host cells, and sperm proteins involved in sperm-egg fusion. In their sequences, these proteins possess a ``fusion peptide,' a short segment (up to 20 amino acids) of relatively hydrophobic residues, commonly found in a membrane-anchored polypeptide chain. To simulate protein-mediated fusion, many studies on peptide-induced membrane fusion have been conducted on model membranes such as liposomes and have employed synthetic peptides corresponding to the putative fusion sequences of viral proteins, or de novo synthesized peptides. Here, the application of peptides as a model system to understand the molecular details of membrane fusion will be discussed in detail. Data obtained from these studies will be correlated to biological studies, in particular those that involve viral and sperm-egg systems. Structure-function relationships will be revealed, particularly in the context of protein-induced membrane perturbations and bilayer-to-nonbilayer transition underlying the mechanism of fusion. We will also focus on the involvement of lipid composition of membranes as a potential regulating factor of the topological fusion site in biological systems. Received: 3 August 1998/Revised: 15 October 1998     

Angiotensin II receptors: Localization of type I and type II in rat epididymides of different developmental stages

Correspondence to: H.C. Chan--> Abstract. Previous studies from our laboratory have provided evidence for the existence of a local renin-angiotensin system in the rat epididymis. Evidence has also accumulated, indicating that locally formed angiotensin II from the rat epididymis may play a paracrine and/or autocrine role in regulating epididymal electrolyte and fluid transport. In the present study, specific anti-peptide antibodies against the second extracellular loops of angiotensin II type I (AT₁) and type II (AT₂) receptors were used to localize immunocytochemically these receptors in the rat cauda epididymides of three developmental stages, namely, immature (2-week), early mature (6-week) and fully mature (10-week). The immunostaining intensity for AT₁ receptors was found to be stronger than that for AT₂ receptors throughout rat epididymides of all stages. However, the immunostaining for both AT₁ and AT₂ receptors observed in the fully mature rat epididymis was much more intense than that observed in the epididymides of the two younger stages. While the immunostaining for both AT₁ and AT₂ receptors in the younger rat epididymides appeared to be distributed in both basal and apical regions, the immunostaining in the fully mature epididymis was predominantly localized in the basal region. The present finding of the differential patterns of angiotensin II receptor immunoreactivity in three different stages of the rat epididymis may reflect the fine tuning of rat epididymal function by angiotensin II, acting as a paracrine or autocrine agent, during the course of development. Received: 18 December 1995/Revised: 19 December 1996     

Hyperpolarization-activated Ca2+-permeable Channels in the Plasma Membrane of Tomato Cells

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca²⁺ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca²⁺ and Ba²⁺ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca²⁺ or Ba²⁺, respectively. These channels were 20–25 and 10–13 times more permeable to Ca²⁺ than to K⁺ and to Cl⁻, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca²⁺-permeable channels provide a mechanism for the influx of Ca²⁺ into tomato cells. Received: 13 February 1996/Revised: 12 August 1996