Cell Membrane Conductance Research Articles

Biological Compatibility of Electromanipulation Media

Electromanipulation of cells, which includes dielectrophoresis, electrorotation, and electrophoresis, as a label free cell manipulation and characterization tool has gained particular interest recently. However, the applicability of electromanipulation to biological cells is limited to cells suspended in special types of media containing lower amounts of salts in comparison to physiological buffers. Impedance measurements of cells are often performed in low conductive buffers, mainly due to parasitic capacitance that occurs at the electrode electrolyte interface. One might question the use of electromanipulation buffers as it results in lower transmembrane transport and possible increased cellular death. Therefore, in this study, effects of electromanipulation buffers with varying salt concentrations on cellular viability and transmembrane transport was studied for a human T-cell leukemia cell line (Jurkat). Cellular viability was measured in vitro using an MTT cell viability assay. Cellular death was also measured using the trypan blue dye exclusion method. Membrane capacitance and conductance was measured by impedance spectroscopy over a range of extracellular salt concentrations. We described the cell membrane conductance at different extracellular salt concentrations using a quantitative model. Even though membrane conductance measurements by impedance spectroscopy is indicative of number and activity of ion channels even performed at low extracellular salt levels; results indicate that cells in electromanipulation buffers have reduced viability. Strategies to increase metabolic activity by changing buffer composition while still retaining the performance of impedance spectroscopy and electromanipulation are also presented.

The Effects of Age on Lens Transport

Age-related nuclear cataracts involve denaturation and aggregation of intracellular proteins. We have documented age-dependent changes in membrane transport in the mouse lens to see what might initiate changes in the intracellular milieu. Microelectrode-based intracellular impedance studies of intact lenses were used to determine gap junction coupling conductance, fiber and surface cell membrane conductances, effective extracellular resistivity, and intracellular voltage. Fiber cell connexin expression was detected by Western blotting. Intracellular hydrostatic pressure was measured with a microelectrode/manometer system. Concentrations of intracellular sodium and calcium were measured by intracellular injection of sodium-binding benzofuran isophthalate and Fura2, respectively. In adult lenses, as age increased: fiber cell gap junction coupling conductance declined significantly, correlating with decreases in Cx46 and Cx50 labeling in Western blots; fiber and surface cell membrane conductances did not change systematically; effective extracellular resistivity increased monotonically; center to surface gradients for intracellular pressure, sodium, calcium, and voltage all increased, but in an interdependent manner that moderated changes. In newborn pup lenses, there were changes that did not simply fit with the above paradigm. In newborn pup lenses, the observed changes may relate to growth factors that are not related to age-dependent changes seen in adult lenses. The major change in adult lenses was an age-dependent decrease in gap junction coupling, probably due to oxidative damage leading to degradation of connexin proteins. These changes clearly lead to compromise of intracellular homeostasis and may be a causal factor in age-related nuclear cataracts.

Effects of oleic acid on ionic channels of plasma membranes of green alga Chara corallina

Extracellular application of 5–30 μM oleic acid (C18:1:9cis) produced a significant decrease in the amplitude of Ca2+ currents and Ca2+-activated chloride currents in plasma membrane of green alga Chara corallina. Introduction of 10 μM oleic acid intracellularly suppressed the development of the Ca2+ current. This inhibition of the Ca2+ current was reversible, and the Ca2+ conductivity of the cell membrane was restored after the removal of oleic acid. There were no changes in the non-specific leakage current, which indicates the intactness of the lipid component of the membrane. An increase of the oleic acid concentration up to 50–100 μM in the extracellular solution induced almost complete and irreversible suppression of inward currents and caused a substantial increase in the non-specific leakage current and conductivity of plasma membrane, suggesting its disruption. We suggest that the mechanism of cytotoxicity of oleic acid may be related to transport across plasma membrane of oleic acid and its complexes with α-lactalbumin (HAMLET and La-OA).

Downregulation of KCNQ4 by Janus Kinase 2

Janus kinase-2 (JAK2) participates in the signaling of several hormones, growth factors and cytokines. Further stimulators of JAK2 include osmotic cell shrinkage, and the kinase activates the cell volume regulatory Na(+)/H(+) exchanger. The kinase may thus participate in cell volume regulation. Cell shrinkage is known to inhibit K(+) channels. Volume-regulatory K(+) channels include the voltage-gated K(+) channel KCNQ4. The present study explored the effect of JAK2 on KCNQ4 channel activity. KCNQ4 was expressed in Xenopus oocytes with or without wild-type JAK2, constitutively active (V617F)JAK2 or inactive (K882E)JAK2; and cell membrane conductance was determined by dual-electrode voltage clamp. Expression of KCNQ4 was followed by the appearance of voltage-gated K(+) conductance. Coexpression of JAK2 or of (V617F)JAK2, but not of (K882E)JAK2, resulted in a significant decrease in conductance. Treatment of KCNQ4 and JAK2 coexpressing oocytes with the JAK2 inhibitor AG490 (40 μM) was followed by an increase in conductance. Treatment of KCNQ4 expressing oocytes with brefeldin A (5 μM) was followed by a decrease in conductance, which was similar in oocytes expressing KCNQ4 together with JAK2 as in oocytes expressing KCNQ4 alone. Thus, JAK2 apparently does not accelerate channel protein retrieval from the cell membrane. In conclusion, JAK2 downregulates KCNQ4 activity and thus counteracts K(+) exit, an effect which may contribute to cell volume regulation.

Specific sphingolipid content decrease in Cerkl knockdown mouse retinas

Sphingolipids (SPLs) are finely tuned structural compounds and bioactive molecules involved in membrane fluidity and cellular homeostasis. The core sphingolipid, ceramide (CER), and its derivatives, regulate several crucial processes in neuronal cells, among them cell differentiation, cell–cell interactions, membrane conductance, synaptic transmission, and apoptosis. Mutations in Ceramide Kinase-Like (CERKL) cause autosomal recessive Retinitis Pigmentosa and Cone Rod Dystrophy. The presence of a conserved lipid kinase domain and the overall similarity with CERK suggested that CERKL might play a role in the SPL metabolism as a CER kinase. Unfortunately, CERKL function and substrate(s), as well as its contribution to the retinal etiopathology, remain as yet unknown. In this work we aimed to characterize the mouse retinal sphingolipidome by UPLC-TOF to first, thoroughly investigate the SPL composition of the murine retina, compare it to our Cerkl −/− model, and finally assess new possible CERKL substrates by phosphorus quantification and protein-lipid overlay. Our results showed a consistent and notable decrease of the retinal SPL content (mainly ranging from 30% to 60%) in the Cerkl −/− compared to WT retinas, which was particularly evident in the glucosyl/galactosyl ceramide species (Glc/GalCer) whereas the phospholipids and neutral lipids remained unaltered. Moreover, evidence in favor of CERKL binding to GlcCer, GalCer and sphingomyelin has been gathered. Altogether, these results highlight the involvement of CERKL in the SPL metabolism, question its role as a kinase, and open new scenarios concerning its function.

The Effect of Size and Species on Lens Intracellular Hydrostatic Pressure

Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied. All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer-based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na(+) concentrations were measured by injecting the Na(+)-sensitive dye sodium-binding benzofuran isophthalate. Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na(+) concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from -45 mV in central fiber cells to -60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses. Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na(+)-leak channels in fiber cells of larger lenses.

Microfluidic Dielectric Spectroscopy of Costal Cartilage Chondrocytes

Dielectric spectroscopy allows non-invasive probing of biological cells. Information on the cell membrane, cytoplasm, and nucleus can be obtained by dielectric spectroscopy provided that appropriate experimental and numerical tools are used in specific frequency ranges. The focus of this study is to identify bioelectrical characteristics of human costal cartilage cells using a microfluidic device for dielectric spectroscopy. An in house computer program is developed to extract dielectric properties of biological cells from raw cell suspension impedance data. The algorithm utilizes Maxwell-Wagner mixture and double shell models to extract single cell dielectric spectrum and cellular dielectric parameters, respectively. The dielectric parameters of human costal chondrocytes are compared with melanoma, leukemia, and electrically excitable cardiomyoblast cells in order to comparatively assess the electrical nature of chondrocytes. The results suggest that electrical cell membrane characteristics of chondrocyte cells are closer to cardiomyoblast cells than melanoma or leukemia cells. In addition, blocking effects of Gadolinium is tested on chondrocyte, myoblast, and melanoma cells. The significant decreases in cell membrane conductance and capacitance values after treatment with Gadolinium are linked with ion channels activity on chondrocyte cell membranes.

Biochemical and Biophysical Response to Calcium Chloride Stress in Aspergillus niger and its Role in Malachite Green Degradation

Filamentous fungi show great promise in remediation of environmental contaminants such as industrial dyes. In the current study, Aspergillus niger (Genbank ID: JF437542) decolorized 82 % of the test dye malachite green (MG; 50 mg/l) during cultivation for 24 h. The organism decolorized only 6 % of the MG at higher concentration (250 mg MG/l) during the same time period and growth was inhibited at this higher MG concentration. Exposing A. niger to different types of stress resulted in variable impacts on ability to decolorize MG. CaCl2 had the largest positive impact on decolorization. A. niger cultures treated with CaCl2 (1 M) decolorized 46 % of the MG (250 mg/l) in 1 h compared to 6 % in untreated control cultures. CaCl2 also increased catalase production in A. niger which strongly supported a direct relationship between stress response and decolorizing ability. Spectrophotometric measurement confirmed MG decolorization while Fourier transform infrared spectroscopy suggested that biodegradation of MG occurred. Cultures treated with CaCl2 accumulated fewer toxic MG by-products than untreated cultures. CaCl2-induced stress increased the permeability and conductivity of the fungal cell membrane. An observed increase in medium [H(+)] also suggested a change in Ca(2+)/H(+) exchange capacity in the fungal cell. Calcium ions had a pronounced effect on membrane properties and this may have had an important impact on signal transduction. We conclude that A. niger decolorizes MG and that CaCl2 enhances this process; the CaCl2 effect appears to be associated with stress response.

Exploring Light and Life

Exploring Light and Life

Adenosine Release during Seizures Attenuates GABAAReceptor-Mediated Depolarization

Seizure-induced release of the neuromodulator adenosine is a potent endogenous anticonvulsant mechanism, which limits the extension of seizures and mediates seizure arrest. For this reason several adenosine-based therapies for epilepsy are currently under development. However, it is not known how adenosine modulates GABAergic transmission in the context of seizure activity. This may be particularly relevant as strong activation of GABAergic inputs during epileptiform activity can switch GABA(A) receptor (GABA(A)R) signaling from inhibitory to excitatory, which is a process that plays a significant role in intractable epilepsies. We used gramicidin-perforated patch-clamp recordings to investigate the role of seizure-induced adenosine release in the modulation of postsynaptic GABA(A)R signaling in pyramidal neurons of rat hippocampus. Consistent with previous reports, GABA(A)R responses during seizure activity transiently switched from hyperpolarizing to depolarizing and excitatory. We found that adenosine released during the seizure significantly attenuated the depolarizing GABA(A)R responses and also reduced the extent of the after-discharge phase of the seizure. These effects were mimicked by exogenous adenosine administration and could not be explained by a change in chloride homeostasis mechanisms that set the reversal potential for GABA(A)Rs, or by a change in the conductance of GABA(A)Rs. Rather, A(1)R-dependent activation of potassium channels increased the cell's membrane conductance and thus had a shunting effect on GABA(A)R currents. As depolarizing GABA(A)R signaling has been implicated in seizure initiation and progression, the adenosine-induced attenuation of depolarizing GABA(A)R signaling may represent an important mechanism by which adenosine can limit seizure activity.

Dielectrophoretic capture voltage spectrum for measurement of dielectric properties and separation of cancer cells

In this paper, a new dielectrophoresis (DEP) method based on capture voltage spectrum is proposed for measuring dielectric properties of biological cells. The capture voltage spectrum can be obtained from the balance of dielectrophoretic force and Stokes drag force acting on the cell in a microfluidic device with fluid flow and strip electrodes. The method was demonstrated with the measurement of dielectric properties of human colon cancer cells (HT-29 cells). From the capture voltage spectrum, the real part of Clausius-Mossotti factor of HT-29 cells for different frequencies of applied electric field was obtained. The dielectric properties of cell interior and plasma membrane were then estimated by using single-shell dielectric model. The cell interior permittivity and conductivity were found to be insensitive to changes in the conductivity of the medium in which the cells are suspended, but the measured permittivity and conductivity of cell membrane were found to increase with the increase of medium conductivity. In addition, the measurement of capture voltage spectrum was found to be useful in providing the optimum operating conditions for separating HT-29 cells from other cells (such as red blood cells) using dielectrophoresis.

Infrared Light Excites Cells via Transient Changes in Membrane Electrical Capacitance

Control of excitable cells using optical technologies such as optogenetics has enabled important advances in neuroscience and the development of clinical applications. Most existing methods of optical control require the use of genetic or chemical sensitizers that enable light to alter the ionic conductance of cell membranes. By contrast, infrared (IR) light of wavelengths > 1.5μm has been shown in vivo to excite neural and muscle tissue without any pre-treatment. Unfortunately, the mechanism of IR stimulation is unknown. Here, we describe how IR light excites cells by transiently altering their membrane electrical capacitance. Our data from voltage clamped Xenopus laevis oocytes, mammalian cells and artificial lipid bilayers shows that IR energy absorbed by water produces a rapid local increase in temperature at the cell membrane, transiently increasing its electrical capacitance, and generating depolarizing currents. Correspondingly, under current clamp conditions, IR pulses produce rapid changes in membrane potential. This unexpected mechanism is fully reversible and requires only the most basic properties of cell membranes. Changes in capacitance were verified by direct measurement in mammalian cells and artificial bilayers, and are consistent with a classical theoretical description of cell membranes as coupled double-layer capacitors. In shedding light on the mechanism of IR stimulation, our findings point to this technology's unique generality as a means to control excitable cells, and raise questions about other thermal phenomena that may meaningfully affect membrane electrostatics. Supported by the NIH: GM030376 and DC011481-01A1.

Downregulation of ClC-2 by JAK2

JAK2 (Janus kinase-2) is activated by cell shrinkage and may thus participate in cell volume regulation. Cell volume regulatory ion channels include the small conductance Cl^-channels ClC-2. The present study thus explored whether JAK2 influences ClC-2 activity. To this end, ClC-2 was expressed in Xenopus oocytes with or without wild type JAK2, active ^V617FJAK2 or inactive ^K882EJAK2 and the Cl channel activity determined by dual electrode voltage clamp. Expression of ClC-2 was followed by a marked increase of cell membrane conductance. The conductance was significantly decreased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Exposure of the oocytes expressing ClC-2 together with ^V617FJAK2 to the JAK2 inhibitor AG490 (40 µM) resulted in a gradual increase of the conductance. According to chemiluminescence JAK2 decreased the channel protein abundance in the cell membrane. The decline of conductance in ClC-2 and ^V617FJAK2 coexpressing oocytes following inhibition of channel protein insertion by brefeldin A (5 µM) was similar in oocytes expressing ClC-2 with ^V617FJAK2 and oocytes expressing ClC-2 alone, indicating that ^V617FJAK2 might slow channel protein insertion into rather than accelerating channel protein retrieval from the cell membrane. In conclusion, JAK2 down-regulates ClC-2 activity and thus counteracts Cl^-exit, an effect which may impact on cell volume regulation.

Epithelial Na⁺ sodium channels in magnocellular cells of the rat supraoptic and paraventricular nuclei.

The epithelial Na⁺ channels (ENaCs) are present in kidney and contribute to Na⁺ and water homeostasis. All three ENaC subunits (α, β, and γ) were demonstrated in the cardiovascular regulatory centers of the rat brain, including the magnocellular neurons (MNCs) in the supraoptic nucleus (SON) and the paraventricular nucleus (PVN). However, the functional significance of ENaCs in vasopressin (VP) and oxytocin (OT) synthesizing MNCs is completely unknown. In this study, we show with immunocytochemical double-labeling that the α-ENaC is colocalized with either VP or OT in MNCs in the SON and PVN. In addition, parvocellular neurons in the dorsal, ventrolateral, and posterior subregions of the PVN (not immunoreactive to VP or OT) are also immunoreactive for α-ENaC. In contrast, immunoreactivity to β- and γ-ENaC is colocalized with VP alone within the MNCs. Furthermore, immunoreactivity for a known target for ENaC expression, the mineralcorticoid receptor (MR), is colocalized with both VP and OT in MNCs. Using single-cell RT-PCR, we detected mRNA for all three ENaC subunits and MR in cDNA libraries derived from single MNCs. In whole cell voltage clamp recordings, application of the ENaC blocker benzamil reversibly reduced a steady-state inward current and decreased cell membrane conductance approximately twofold. Finally, benzamil caused membrane hyperpolarization in a majority of VP and about one-half of OT neurons in both spontaneously firing and quiet cells. These results strongly suggest the presence of functional ENaCs that may affect the firing patterns of MNCs, which ultimately control the secretion of VP and OT.

Cationic Contamination Effects on Polymer Electrolyte Membrane Fuel Cell Performance

There are significant impacts to fuel cell performance when contaminant cations replace protons in the ionomer. The purpose of this paper is to directly correlate extent of cationic contamination with fuel cell performance and also to elucidate main mechanisms responsible for this performance degradation. A series of standard membrane electrode assemblies (MEAs) were contaminated by varying the ratio of protons to cesium cations in the ionomer phase. The amount of contamination was determined using X-ray Fluorescence (XRF) Spectroscopy. These MEAs were then run in fuel cell mode to determine fuel cell performance as a function of cationic contamination level. A strip cell configuration was also utilized to distinguish between ohmic, thermodynamic, and kinetic effects. Additionally, the distribution of contaminants was measured using XRF. The decrease in fuel cell limiting current, power density, and membrane conductivity as a function of contamination level were quantified. Performance decreased monotonically with increased contamination. The cationic contaminant was preferentially located near the cathode of the strip cell under load. This distribution led to increases in thermodynamic and kinetic polarizations that were more important than changes in membrane conductivity.

Dopamine and Paraquat Enhance α-Synuclein-Induced Alterations in Membrane Conductance

We have previously demonstrated that α-synuclein overexpression increases the membrane conductance of dopaminergic-like cells. Although α-synuclein is thought to play a central role in the pathogenesis of several neurodegenerative diseases including Parkinson's disease, multiple system atrophy, and diffuse Lewy body disease, the mechanism of action is not completely understood. In this study, we sought to determine whether multiple factors act together with α-synuclein to engender cell vulnerability through an augmentation of membrane conductance. In this article, we employed a cell model that mimics dopaminergic neurons coupled with α-synuclein overexpression and oxidative stressors. We demonstrate an enhancement of α-synuclein-induced toxicity in the presence of combined treatment with dopamine and paraquat, two molecules known to incite oxidative stress. In addition, we show that combined dopamine and paraquat treatment increases the expression of heme oxygenase-1, an antioxidant response protein. Finally, we demonstrate for the first time that combined treatment of dopaminergic cells with paraquat and dopamine enhances α-synuclein-induced leak channel properties resulting in increased membrane conductance. Importantly, these increases are most robust when both paraquat and dopamine are present suggesting the need for multiple oxidative insults to augment α-synuclein-induced disruption of membrane integrity.

Mechanism of Cancer Growth Suppression of Alpha-Fetoprotein Derived Growth Inhibitory Peptides (GIP): Comparison of GIP-34 versus GIP-8 (AFPep). Updates and Prospects

The Alpha-fetoprotein (AFP) derived Growth Inhibitory Peptide (GIP) is a 34-amino acid segment of the full-length human AFP molecule that inhibits tumor growth and metastasis. The GIP-34 and its carboxy-terminal 8-mer segment, termed GIP-8, were found to be effective as anti-cancer therapeutic peptides against nine different human cancer types. Following the uptake of GIP-34 and GIP-8 into the cell cytoplasm, each follows slightly different signal transduction cascades en route to inhibitory pathways of tumor cell growth and proliferation. The parallel mechanisms of action of GIP-34 versus GIP-8 are demonstrated to involve interference of signaling transduction cascades that ultimately result in: (1) cell cycle S-phase/G2-phase arrest; (2) prevention of cyclin inhibitor degradation; (3) protection of p53 from inactivation by phosphorylation; and (4) blockage of K+ ion channels opened by estradiol and epidermal growth factor (EGF). The overall mechanisms of action of both peptides are discussed in light of their differing modes of cell attachment and uptake fortified by RNA microarray analysis and electrophysiologic measurements of cell membrane conductance and resistance. As a chemotherapeutic adjunct, the GIPs could potentially aid in alleviating the negative side effects of: (1) tamoxifen resistance, uterine hyperplasia/cancer, and blood clotting; (2) Herceptin antibody resistance and cardiac (arrest) arrhythmias; and (3) doxorubicin's bystander cell toxicity.

Assessment of microalgae viability employing insulator-based dielectrophoresis

Microscale bioparticle analysis has advanced significantly providing advantages over bench-scale studies such as the use of a reduced amount of sample and reagents, higher sensitivity, faster response, and portability. Insulator-based dielectrophoresis (iDEP) is a microscale technique where particles are driven by polarization effects under a non-uniform electrical field created by the inclusion of insulators between two electrodes. iDEP possesses attractive advantages over traditional electrode-based dielectrophoresis since there is no electrode degradation and manufacture of the device is simpler and economical. This novel and powerful technique has been applied successfully in the manipulation of macromolecules and cells. In this study, differences in dielectric properties (cell membrane conductivity) of viable and non-viable microalgae, Selenastrum capricornutum, were employed to concentrate and separate a mixture of live and dead cells. A microchannel, manufactured in glass and containing an array of cylindrical insulating posts, was employed to dielectrophoretically immobilize and concentrate the mixture of cells employing direct current electric fields. Experiments showed that live cells exhibited a stronger dielectrophoretic response than dead cells, which allowed cell differentiation. Separation and enrichment of viable and non-viable microalgae was achieved in 35 s with a concentration yield of 10.36 and 15.87 times the initial cell concentration, respectively. These results demonstrate the use of iDEP as a technique for rapid assessment of microalgae viability; unveiling the potential of this powerful technique for environmental applications on lab-on-a-chip platforms.

Self-incompatibility in Papaver rhoeas activates nonspecific cation conductance permeable to Ca2+ and K+.

Cellular responses rely on signaling. In plant cells, cytosolic free calcium is a major second messenger, and ion channels play a key role in mediating physiological responses. Self-incompatibility (SI) is an important genetically controlled mechanism to prevent self-fertilization. It uses interaction of matching S-determinants from the pistil and pollen to allow "self" recognition, which triggers rejection of incompatible pollen. In Papaver rhoeas, the S-determinants are PrsS and PrpS. PrsS is a small novel cysteine-rich protein; PrpS is a small novel transmembrane protein. Interaction of PrsS with incompatible pollen stimulates S-specific increases in cytosolic free calcium and alterations in the actin cytoskeleton, resulting in programmed cell death in incompatible but not compatible pollen. Here, we have used whole-cell patch clamping of pollen protoplasts to show that PrsS stimulates SI-specific activation of pollen grain plasma membrane conductance in incompatible but not compatible pollen grain protoplasts. The SI-activated conductance does not require voltage activation, but it is voltage sensitive. It is permeable to divalent cations (Ba(2+) ≥ Ca(2+) > Mg(2+)) and the monovalent ions K(+) and NH(4)(+) and is enhanced at voltages negative to -100 mV. The Ca(2+) conductance is blocked by La(3+) but not by verapamil; the K(+) currents are tetraethylammonium chloride insensitive and do not require Ca(2+). We propose that the SI-stimulated conductance may represent a nonspecific cation channel or possibly two conductances, permeable to monovalent and divalent cations. Our data provide insights into signal-response coupling involving a biologically important response. PrsS provides a rare example of a protein triggering alterations in ion channel activity.

Cyclosis-related asymmetry of chloroplast–plasma membrane interactions at the margins of illuminated area in Chara corallina cells

Cytoplasmic streaming in plant cells is an effective means of intracellular transport. The cycling of ions and metabolites between the cytosol and chloroplasts in illuminated cell regions may alter the cytoplasm composition, while directional flow of this modified cytoplasm may affect the plasma membrane and chloroplast activities in cell regions residing downstream of the illumination area. The impact of local illumination is predicted to be asymmetric because the cell regions located downstream and upstream in the cytoplasmic flow with respect to illumination area would be exposed to flowing cytoplasm whose solute composition was influenced by photosynthetic or dark metabolism. This hypothesis was checked by measuring H(+)-transporting activity of plasmalemma and chlorophyll fluorescence of chloroplasts in shaded regions of Chara corallina internodal cells near opposite borders of illuminated region (white light, beam width 2mm). Both the apoplastic pH and chlorophyll fluorescence, recorded in shade regions at equal distances from illuminated area, exhibited asymmetric light-on responses depending on orientation of cytoplasmic streaming at the light-shade boundary. In the region where the cytoplasm flowed from illuminated area to the measurement area, the alkaline zone (a zone with high plasma membrane conductance) was formed within 4-min illumination, whereas no alkaline zone was observed in the area where cytoplasm approached the boundary from darkened regions. The results emphasize significance of cyclosis in lateral distribution of a functionally active intermediate capable of affecting the membrane transport across the plasmalemma, the functional activity of chloroplasts, and pattern formation in the plant cell.