Cation Metabolism in Relation to Cell Size in Synchronously Grown Tissue Culture Cell

In randomly grown tissue culture cells (mouse leukemic lymphoblast, L5178Y) the number, volume, and Na⁺ and K⁺ content increase as an exponential function with a doubling time of 11.3 hr. In synchronously grown cells the volume increase of the population and of single cells follows the same exponential function as in randomly grown cells. In contrast, the cation content fluctuates during a single cell cycle. About 1½ hr after the cell division burst (at the beginning of the S period), a net loss of K⁺ occurs for a period of about 1 hr amounting to about 20% of the total K. Over the next 5 to 6 hr, the deficit in K⁺ is eliminated. The Na⁺ content shows a double fluctuation. It falls during the cell division burst, rises when the K⁺ content decreases, falls again when K⁺ content rises, and then increases again before the next cell division burst. The net fluxes of both Na⁺ and K⁺ are very small compared to the unidirectional fluxes (less than 5%), thus small changes in the balance of influx and efflux account for the changes in cation content during the growth cycle. Both unidirectional fluxes increase dramatically (by a factor of two) about 2 hr after the cell division burst, and then remain constant until after the next cell division. The pattern of electrolyte regulation during cell division does not follow a simple function such as cell number, cell surface, or cell volume, but must be related to specific internal events in the cell.     

K+ Channel Expression in Human Breast Cancer Cells: Involvement in Cell Cycle Regulation and Carcinogenesis

K⁺ channels are a most diverse class of ion channels in the plasma membrane and are distributed widely throughout a variety of cells including cancer cells. Evidence has been accumulating from fundamental studies indicating that tumour cells possess various types of K⁺ channels and that these K⁺ channels play important roles in regulating tumor cell proliferation, cell cycle progression and apoptosis. Moreover, a significant increase in K⁺ channel expression has been correlated with tumorigenesis, suggesting the possibility of using these proteins as transformation markers and perhaps reducing the tumor growth rate by selectively inhibiting their functional activity. Significant progress has been made in defining the properties of breast K⁺ channels, including their biophysical and pharmacological properties and distribution throughout different phases of the cell cycle in breast cell line MCF-7. This review aims to provide a comprehensive overview of the current state of research into K⁺ channels/currents in breast cancer cells. The possible mechanisms by which K⁺ channels affect tumor cell proliferation and cell cycle progression are discussed.     

Characterization of Erg K+ Channels in ��- and ��-Cells of Mouse and Human Islets

Voltage-gated eag-related gene (Erg) K⁺ channels regulate the electrical activity of many cell types. Data regarding Erg channel expression and function in electrically excitable glucagon and insulin producing cells of the pancreas is limited. In the present study Erg1 mRNA and protein were shown to be highly expressed in human and mouse islets and in α-TC6 and Min6 cells α- and β-cell lines, respectively. Whole cell patch clamp recordings demonstrated the functional expression of Erg1 in α- and β-cells, with rBeKm1, an Erg1 antagonist, blocking inward tail currents elicited by a double pulse protocol. Additionally, a small interference RNA approach targeting the kcnh2 gene (Erg1) induced a significant decrease of Erg1 inward tail current in Min6 cells. To investigate further the role of Erg channels in mouse and human islets, ratiometric Fura-2 AM Ca²⁺-imaging experiments were performed on isolated α- and β-cells. Blocking Erg channels with rBeKm1 induced a transient cytoplasmic Ca²⁺ increase in both α- and β-cells. This resulted in an increased glucose-dependent insulin secretion, but conversely impaired glucagon secretion under low glucose conditions. Together, these data present Erg1 channels as new mediators of α- and β-cell repolarization. However, antagonism of Erg1 has divergent effects in these cells; to augment glucose-dependent insulin secretion and inhibit low glucose stimulated glucagon secretion.Voltage-gated eag-related gene (Erg)² potassium (K⁺) channels are part of the larger family of voltage dependent K⁺ (Kv) channels (1). Three channel isoforms Erg1, Erg2, and Erg3 have been discovered (2, 3), and they differ by their activation and inactivation voltage dependence, gating properties, and pharmacological profile (4–7). Erg channels control cellular activity by controlling the repolarization of the action potential (AP). In atrial cells and ventricular myocytes, Erg regulates plateau formation and AP repolarization, as blocking Erg channels increases AP length (8, 9). These channels are also strongly involved in the pacemaking activity of cardiac cells (10, 11). Interestingly, a rare congenital heart condition, the inherited form of long QT syndrome is caused by mutations of Erg channel genes (9, 12). Erg channels also control the resting membrane potential in various cell types. For example, in neurons of the medial vestibular nucleus, blocking Erg channels produce an increase in AP discharge or in smooth muscle cells, blocking Erg channels mediates depolarization up to 20 mV (13–15). Hormone secretion studies also demonstrated the involvement of Erg channels in the secretion of prolactin from neurons of the anterior pituitary. Thyrotropin-releasing factor decreases Erg current, which depolarizes neurons and thereby stimulates prolactin secretion (16, 17).In the pancreas, Kv channels and more specifically Kv2.1, regulate insulin secretion by controlling the repolarization of β-cell membrane potential (18–20), although the contribution of this isoform in humans has recently been questioned (21). In α-cells, Kv2.1 and Kv1.4 channels repolarize the membrane potential (22, 23); however, the involvement of Kv channels in the secretion of glucagon is yet to be investigated. One study showed that Erg1, -2, and -3 are expressed in rat α- and β-cells and the rat insulinoma cell line, INS-1, and that they are involved in decreasing membrane potential. Blocking Erg channels with the channel antagonist E4031 increases insulin secretion from INS1 cells (24); however, definitive data regarding the role of Erg channels in insulin and glucagon secretion is limited.Therefore this study aimed to define the functions of Erg channels in α- and β-cells. We found that Erg1 channels are strongly expressed in pancreatic α- and β-cells. Pharmacological and genetic manipulation combined with whole cell recordings in pancreatic cell lines and primary islet cells determined that Erg1 produces a functional current in α- and β-cells. Blocking Erg1 increased intracellular calcium ([Ca²⁺]_i) in mouse β-cells, but only in a minority of mouse and human α-cells. Secretion studies using isolated mouse islets demonstrated that Erg1 are negative regulators of insulin secretion, but positive regulators of glucagon secretion, suggesting distinct roles for Erg1 in β- and α-cells.     

Cell Volume Changes Regulate Slick (Slo2.1), but Not Slack (Slo2.2) K+ Channels

Slick (Slo2.1) and Slack (Slo2.2) channels belong to the family of high-conductance K⁺ channels and have been found widely distributed in the CNS. Both channels are activated by Na⁺ and Cl⁻ and, in addition, Slick channels are regulated by ATP. Therefore, the roles of these channels in regulation of cell excitability as well as ion transport processes, like regulation of cell volume, have been hypothesized. It is the aim of this work to evaluate the sensitivity of Slick and Slack channels to small, fast changes in cell volume and to explore mechanisms, which may explain this type of regulation. For this purpose Slick and Slack channels were co-expressed with aquaporin 1 in Xenopus laevis oocytes and cell volume changes of around 5% were induced by exposure to hypotonic or hypertonic media. Whole-cell currents were measured by two electrode voltage clamp. Our results show that Slick channels are dramatically stimulated (196% of control) by cell swelling and inhibited (57% of control) by a decrease in cell volume. In contrast, Slack channels are totally insensitive to similar cell volume changes. The mechanism underlining the strong volume sensitivity of Slick channels needs to be further explored, however we were able to show that it does not depend on an intact actin cytoskeleton, ATP release or vesicle fusion. In conclusion, Slick channels, in contrast to the similar Slack channels, are the only high-conductance K⁺ channels strongly sensitive to small changes in cell volume.     

Ion Channels in Cell Proliferation and Apoptotic Cell Death

Cell proliferation and apoptosis are paralleled by altered regulation of ion channels that play an active part in the signaling of those fundamental cellular mechanisms. Cell proliferation must - at some time point - increase cell volume and apoptosis is typically paralleled by cell shrinkage. Cell volume changes require the participation of ion transport across the cell membrane, including appropriate activity of Cl⁻ and K⁺ channels. Besides regulating cytosolic Cl⁻ activity, osmolyte flux and, thus, cell volume, most Cl⁻ channels allow HCO₃⁻ exit and cytosolic acidification, which inhibits cell proliferation and favors apoptosis. K⁺ exit through K⁺ channels may decrease intracellular K⁺ concentration, which in turn favors apoptotic cell death. K⁺ channel activity further maintains the cell membrane potential, a critical determinant of Ca²⁺ entry through Ca²⁺ channels. Cytosolic Ca²⁺ may trigger mechanisms required for cell proliferation and stimulate enzymes executing apoptosis. The switch between cell proliferation and apoptosis apparently depends on the magnitude and temporal organization of Ca²⁺ entry and on the functional state of the cell. Due to complex interaction with other signaling pathways, a given ion channel may play a dual role in both cell proliferation and apoptosis. Thus, specific ion channel blockers may abrogate both fundamental cellular mechanisms, depending on cell type, regulatory environment and condition of the cell. Clearly, considerable further experimental effort is required to fully understand the complex interplay between ion channels, cell proliferation and apoptosis.     

Roles of heterotrimeric G proteins in guard cell ion channel regulation

Stomata are formed by pairs of surrounding guard cells and perform important roles in photosynthesis, transpiration and innate immunity of terrestrial plants. Ionic solutes in the cytosol of guard cells are important for cell turgor and volume change. Consequently, trans-membrane flux of ions such as K⁺, Cl⁻, and malate2⁻ through K⁺ channels and anion channels of guard cells are a direct driving force for turgor change, while the opening of calcium permeable channels can serve as a trigger of cytosolic free calcium concentration elevations or oscillations, which play second messenger roles. In plants, heterotrimeric G proteins have fewer members than in animals, but they are well investigated and found to regulate these channels and to play fundamental roles in guard cell function. This mini-review focuses on the recent understanding of G-protein regulation of ion channels on the plasma membrane of guard cells and their participation in stomatal movements.Key words: guard cell, heterotrimeric G protein, ion channel, arabidopsis thaliana, stomata, plasma membrane, patch clampHeterotrimeric G proteins, composed of Gα, Gβ and Gγ subunits, are key elements of cellular signal transduction networks. In plant species, fewer members of G proteins are present than in animals. For example, only one Gα subunit (GPA1), one Gβ subunit (AGB1) and two Gγ subunits (AGG1 and AGG2) are reported in Arabidopsis while 23 Gα, 5 Gβ and 12 Gγ subunits have been identified in human.¹ All three kinds of subunits are expressed in guard cells. Ubiquitous expression of GPA1 throughout plant was ascertained by northern and promoter::GUS analyses and RT-PCR results also indicate guard cell expression.^2–4 AGB1 is ubiquitously expressed throughout the plant and its promoter::GUS transgenic lines show strong expression in guard cells.^5–7 For Gγ subunits, RNA blots show AGG1 and AGG2 expression throughout the plant, however, reporter gene analysis shows guard cell expression of AGG2 but not AGG1.^7–9 The guard cell expression of G protein subunits implies the function of G protein in guard cell signaling and stomatal movement regulation.Stomata are microscopic pores in the epidermis of terrestrial plants, which serve as the mouths of plants for gas change since through them CO₂ enters leaves for photosynthesis and water vapor is lost as transpiration.^10–13 In addition, stomatal movements induced by pathogen and pathogen/microbe-associated molecular patterns (PAMPs or MAMPs) are a component of the plant innate immunity system.^14–16 Biotic and abiotic stresses (e.g. water deficiency, cold, pathogens) and their induced phytohormone changes (e.g. abscisic acid [ABA], ethylene) have been widely investigated in stomatal movement regulation, and stomatal apertures are directly regulated by volume change of the surrounding guard cell pairs. The accumulation/release of ionic solutes through ion channels on the guard-cell plasma membrane together with malate production/metabolism induces water influx/efflux driving increase/decrease of cell turgor and volume which co-operates with the radial reinforcement of the guard cell walls to widen/shrink stomatal aperture.^10,17 Given that mature guard cells lack plasmodesmata with neighboring cells, all ion uptake and efflux must pass through ion channels and ion transporters on the plasma membrane.In Arabidopsis guard cells, the model cell type for cell signaling of the model plant species, all three kinds of ion channels (K⁺ channels, anion channels and Ca²⁺-permeable channels) have been investigated and found to be regulated by heterotrimeric G proteins.^10,17 Their ion channel activities can be measured in intact guard cells, guard cell protoplasts, or cell membrane patches using the patch clamp technique.^15,18,19 Patch clamping can be used to measure ion fluxes in whole cells or even through a single ion channel.^20,21 The patch clamp technique under the whole-cell recording configuration can measure the currents through hyperpolarization-activated inward K⁺ channels which account for K⁺ accumulation during stomatal opening, and the depolarizationactivated outward K⁺ channels which, together with R-type and S-type anion channels, mediate solute removal during stomatal closure. Besides these ionic fluxes which directly elicit changes in turgor, Ca²⁺-permeable channels which participate in Ca²⁺ signaling are also regulated by G proteins. For better visualization of the currents through K⁺, anion and Ca²⁺permeable channels, real current traces and their idealized current/voltage relationships are indicated in Figure 1. The G-protein regulation of inward and outward K⁺ channels, S-type anion channels, and Ca²⁺-permeable channels and their significance for stomatal movements will be discussed below, and the genes encoding them which have been explored up to now also will be discussed.Open in a separate windowFigure 1Current traces and idealized current/voltage relationships of wild type guard cell plasma membrane ion channels involved in G-protein regulation (A–C), ABA inhibition of whole-cell inward K⁺ currents. (A) indicates inward K⁺ currents of wild type guard cell protoplasts in response to hyperpolarizing voltages under control conditions [Scale bar is shown in (B)]; (B) indicates inward K+ currents of wild type guard cell protoplasts with ABA treatment; (C) indicates the idealized current/voltage relationship of inward K⁺ currents for control (gray) and ABA treatments (black). (D–F), ABA activation of slow anion currents. (D) indicates anion currents of wild type under control condition and (E) shows current after ABA treatment; (F) indicates the idealized current/voltage relationship of anion currents for control (gray) and ABA treatments (black). (G–I), ABA activation of currents through Ca²⁺-permeable channels. (G) indicates currents through Ca²⁺-permeable channels of wild type under control condition and (H) shows current after ABA treatments; (I) indicates the idealized current/voltage relationship of currents through Ca²⁺-permeable channels for control (gray) and ABA treatments (black).     

Cl? and K+ channels and their role in primary brain tumour biology

Profound cell volume changes occur in primary brain tumours as they proliferate, invade surrounding tissue or undergo apoptosis. These volume changes are regulated by the flux of Cl⁻ and K⁺ ions and concomitant movement of water across the membrane, making ion channels pivotal to tumour biology. We discuss which specific Cl⁻ and K⁺ channels are involved in defined aspects of glioma biology and how these channels are regulated. Cl⁻ is accumulated to unusually high concentrations in gliomas by the activity of the NKCC1 transporter and serves as an osmolyte and energetic driving force for volume changes. Cell volume condensation is required as cells enter M phase of the cell cycle and this pre-mitotic condensation is caused by channel-mediated ion efflux. Similarly, Cl⁻ and K⁺ channels dynamically regulate volume in invading glioma cells allowing them to adjust to small extracellular brain spaces. Finally, cell condensation is a hallmark of apoptosis and requires the concerted activation of Cl⁻ and Ca²⁺-activated K⁺ channels. Given the frequency of mutation and high importance of ion channels in tumour biology, the opportunity exists to target them for treatment.     

Role of voltage‐gated potassium channels in the fate determination of embryonic stem cells

Embryonic stem cells (ESCs) possess two unique characteristics: self‐renewal and pluripotency. In this study, roles of voltage‐gated potassium channels (K_v) in maintaining mouse (m) ESC characteristics were investigated. Tetraethylammonium (TEA⁺), a K_v blocker, attenuated cell proliferation in a concentration‐dependent manner. Possible reasons for this attenuation, including cytotoxicity, cell cycle arrest and differentiation, were examined. Blocking K_v did not change the viability of mESCs. Interestingly, K_v inhibition increased the proportion of cells in G₀/G₁ phase and decreased that in S phase. This change in cell cycle distribution can be attributed to cell cycle arrest or differentiation. Loss of pluripotency as determined at both molecular and functional levels was detected in mESCs with K_v blockade, indicating that K_v inhibition in undifferentiated mESCs directs cells to differentiate instead of to self‐renew and progress through the cell cycle. Membrane potential measurement revealed that K_v blockade led to depolarization, consistent with the role of K_v as the key determinant of membrane potential. The present results suggest that membrane potential changes may act as a “switch” for ESCs to decide whether to proliferate or to differentiate: hyperpolarization at G₁ phase would favor ESCs to enter S phase while depolarization would favor ESCs to differentiate. Consistent with this notion, S‐phase‐synchronized mESCs were found to be more hyperpolarized than G₀/G₁‐phase‐synchronized mESCs. Moreover, when mESCs differentiated, the differentiation derivatives depolarized at the initial stage of differentiation. This investigation is the first study to provide evidence that K_v and membrane potential affect the fate determination of ESCs. J. Cell. Physiol. 224:165–177, 2010 © 2010 Wiley‐Liss, Inc.     

Differential expression of the Kv1 voltage-gated potassium channel family in the rat nephron

Several potassium (K⁺) channels contribute to maintaining the resting membrane potential of renal epithelial cells. Apart from buffering the cell membrane potential and cell volume, K⁺ channels allow sodium reabsorption in the proximal tubule (PT), K⁺ recycling and K⁺ reabsorption in the thick ascending limb (TAL) and K⁺ secretion and K⁺ reabsorption in the distal convoluted tubule (DCT), connecting tubule (CNT) and collecting duct. Previously, we identified Kv.1.1, Kv1.3 and Kv1.6 channels in collecting ducts of the rat inner medulla. We also detected intracellular Kv1.3 channel in the acid secretory intercalated cells, which is trafficked to the apical membrane in response to dietary K⁺ to function as a secretory K⁺ channel. In this work we sought to characterize the expression of all members of the Kv1 family in the rat nephron. mRNA and protein expression were detected for all Kv1 channels. Immunoblots identified differential expression of each Kv1 in the cortex, outer and inner medulla. Immunofluorescence labeling detected Kv1.5 in Bowman´s capsule and endothelial cells and Kv1.7 in podocytes, endothelial cells and macula densa in glomeruli; Kv1.4, Kv1.5 and Kv1.7 in PT; Kv1.2, Kv1.4 and Kv1.6 in TAL; Kv1.1, Kv1.4 and Kv1.6 in DCT and CNT and Kv1.3 in DCT, and all the Kv1 family in the cortical and medullary collecting ducts. Recently, some hereditary renal syndromes have been attributed to mutations in K⁺ channels. Our results expand the repertoire of K⁺ channels that contribute to K⁺ homeostasis to include the Kv1 family.     

Cell proliferation,potassium channels,polyamines and their interactions: a mini review

Polyamines, which are obligatory molecules involved in cell cycling and proliferation, are subject to a change in their free intracellular concentrations during the cell cycle. Potassium (K⁺) channels are also considered, but less well recognized, to be necessary for cell proliferation by either hyperpolarizing or depolarizing cells during the cell cycle. A block of polyamine synthesis as well as block or knockout of K⁺ channels can halt cell proliferation. K⁺ channels like BK (maxi calcium (Ca²⁺)-activated K⁺), Kir (inward rectifier), M-type K⁺-and TASK (two-pore domain K⁺) channels or the delayed rectifier K⁺ channels are modulated in their electrical properties by polyamines. Polyamines are most effective in blocking these channels when applied to the intracellular face of these channels except for TASK channels where they act only from the extracellular side. Quinidine, a general K⁺ channel blocker, was found to reduce putrescine concentrations, to block the ornithine decarboxylase and halt cell proliferation. From these results, the question arises if there is an interaction between polyamines, K⁺ channels and proliferation. It might be speculated that a decrease of intracellular polyamines allows more K⁺ channels to be active, thus inducing hyperpolarization, while an increase of the polyamine concentration may block K⁺ channel activity leading to depolarization of the membrane potential. On the other hand, a block or a deletion of K⁺ channels may cause a decrease of the polyamine concentration in cells. More research is needed to test these hypotheses.     

Cytoskeleton and ion movements during volume regulation in cultured PC12 cells

Summary The present study investigates the role of cytoskeletal elements, microtubules and microfilaments, on ion transport systems activated during volume regulatory processes in PC12 pheochromocytoma cells. Disruption of microtubule network by colchicine (0.1 mm) or vinblastine sulfate (10 m) has no significant effect on PC12 cell hydration or on changes of the intracellular K⁺, Cl^– and Na⁺ content observed in hypo-osmotic conditions. Disruption of microfilament network by cytochalasin B strongly affects volume regulation in a dose-dependent manner. Cytochalasin B leads to a potentiation of the initial cell swelling and the regulatory volume decrease is suppressed. Although, the internal K⁺ and Cl^– level decreases significantly, as demonstrated by measurements of intracellular ion content and ⁸⁶Rb fluxes. Using the patch-clamp technique, we could demonstrate in PC12 cell membranes an ion channel whose gating is affected by application of a negative hydrostatic pressure (mechanical stress) to the membrane patch, by exposure of the cell to hypoosmotic medium (osmotic stress), or by disruption of the microfilament network with cytochalasin B.Water and ion content measurements, as well as ⁸⁶Rb fluxes have been carried out in the Laboratory of Animal Physiology from Professor R. Gilles, University of Liège, Belgium. M. Cornet was supported by the F.N.R.S., Belgium.     

Potassium-dependent,bipolar gating of K+ channels in guard cells

Summary Guard cells of higher plants control transpirational water loss and gas exchange for photosynthesis by opening and closing pores in the epidermis of the leaf. To power these turgordriven movements, guard cells accumulate (and lose) 200 to 400mm (1 to 3 pmol/cell) K⁺, fluxes thought to pass through K⁺ channels in the guard cells plasma membrane. Steady-state current-voltage (I–V) relations of intactVicia guard cells frequently show large, outward-going currents at potentials approaching 0 mV. Since this current could be carried by K⁺ channels, its pharmacology and dependence on external K⁺ (K _v ⁺ ) has been examined under voltage clamp over an extended potential range. Measurements were carried out on cells which showed little evidence of primary electrogenic transport, thus simplifying analyses. Clamping these cells away from the free-running membrane potential (V _m ) revealed an outward-rectifying current with instantaneous and time-dependent components, and sensitive to the K⁺ channel blocker tetraethylammonium chloride. The current declined also under metabolic blockade with NaCN and in the presence of diethylstilbesterol, responses which were attributed to secondary effects of these inhibitors. The putative K⁺ current rose with voltage positive toV _m but it decayed over two voltage ranges, one negative toV _m and one near +100 mV, to give steady-stateI–V relations with two regions of negative (slope) conductance. Voltage-dependent and kinetic characteristics of the current were affected by K _v ⁺ and followed the K⁺ equilibrium potential. Against a (presumably) low background of primary ion transport, the K⁺ current contributed appreciably to charge balance atV _m in 0.1mm as well as in 1 to 10mm K _v ⁺ . Thus, gating of these K⁺ channels compensates for the prevailing K⁺ conditions to ensure net K⁺ movement out of the cell.     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium     

Calculations of K+, Na+, and Cl? fluxes across cell membrane with Na+/K+ pump, NKCC, NC cotransport and ionic channels with non-Goldman rectification in K+-channels: Normal and apoptotic cells

The balance of K⁺, Na⁺, and Cl⁻ fluxes across the cell membrane with the Na⁺/K⁺ pump, ion channels, and Na⁺K⁺2Cl⁻ (NKCC) and Na⁺-Cl⁻ (NC) cotransport was calculated to determine the mechanism of cell shrinkage in apoptosis. It is shown that all unidirectional K⁺, Na⁺, and Cl⁻ fluxes; the ion channel permeability; and the membrane potential can be found using the principle of the flux balance if the following experimental data are known: K⁺, Na⁺, and Cl⁻ concentrations in cell water; total Cl⁻ flux; total K⁺ influx; and the ouabain-inhibited pump component of the Rb⁺(K⁺) influx. The change in different ionic pathways during apoptosis was estimated by calculations based on the data reported in the preceded paper (Yurinskaya et al., 2010). It is found that cell shrinkage and the shift in ion balance in U937 cells induced to apoptosis with 1 μM staurosporine occur due to the coupling of reduced pump activity with a decrease in the integral permeability of Na⁺ channels, whereas K⁺ and Cl⁻ channel permeability remains almost unchanged. Calculations show that only a small part of the total fluxes of K⁺, Na⁺, and Cl⁻ account for the fluxes mediated by NKCC and NC cotransporters. Despite the importance of cotransport fluxes for maintaining the nonequilibrium steady-state distribution of Cl⁻, they cannot play a significant role in apoptotic cell shrinkage because of their minority and cannot be revealed by inhibitors.     

Ion channels in volume regulation of clonal kidney cells

Objectives: Clonal kidney cells (Vero cells) are extensively utilized in the manufacture of biological preparations for disease diagnostics and therapeutics and also in preparation of vaccines. In all cells, regulation of volume is an essential function coupled to a variety of physiological processes and is a topic of interest. The objective here was to investigate involvement of ion channels in the process of volume regulation of Vero cells. Methods: Involvement of ion channels in cell volume regulation was studied using video‐microscopy and flow cytometry. Pharmacologically unaltered cells of different sizes, which are presumably at different phases of the cell cycle, were used. Results: Ion transport inhibitors altered all phases of regulatory volume decrease (RVD) of Vero cells, rate of initial cell swelling, V_max and volume recovery. Effects were dependent on type of inhibitor and on cell size (cell cycle phase). Participation of aquaporins in RVD was suggested. Inhibitors decelerated growth, arresting Vero cells at the G₀/G₁ phase boundary. Electrophysiological study confirmed presence of volume‐activated Cl^? channels and K⁺ channels in plasmatic membranes of the cells. Conclusion: Vero cells of all sizes maintained the ability to recover from osmotic swelling. Activity of ion channels was one of the key factors that controlled volume regulation and proliferation of the cells.     

Cell Volume Regulation in Cultured Human Retinal Müller Cells Is Associated with Changes in Transmembrane Potential

Müller cells are mainly involved in controlling extracellular homeostasis in the retina, where intense neural activity alters ion concentrations and osmotic gradients, thus favoring cell swelling. This increase in cell volume is followed by a regulatory volume decrease response (RVD), which is known to be partially mediated by the activation of K⁺ and anion channels. However, the precise mechanisms underlying osmotic swelling and subsequent cell volume regulation in Müller cells have been evaluated by only a few studies. Although the activation of ion channels during the RVD response may alter transmembrane potential (V_m), no studies have actually addressed this issue in Müller cells. The aim of the present work is to evaluate RVD using a retinal Müller cell line (MIO-M1) under different extracellular ionic conditions, and to study a possible association between RVD and changes in V_m. Cell volume and V_m changes were evaluated using fluorescent probe techniques and a mathematical model. Results show that cell swelling and subsequent RVD were accompanied by V_m depolarization followed by repolarization. This response depended on the composition of extracellular media. Cells exposed to a hypoosmotic solution with reduced ionic strength underwent maximum RVD and had a larger repolarization. Both of these responses were reduced by K⁺ or Cl⁻ channel blockers. In contrast, cells facing a hypoosmotic solution with the same ionic strength as the isoosmotic solution showed a lower RVD and a smaller repolarization and were not affected by blockers. Together, experimental and simulated data led us to propose that the efficiency of the RVD process in Müller glia depends not only on the activation of ion channels, but is also strongly modulated by concurrent changes in the membrane potential. The relationship between ionic fluxes, changes in ion permeabilities and ion concentrations –all leading to changes in V_m– define the success of RVD.     

Ion channels in the membrane ofChara inflata

Summary Voltage-clamped steps in the electric potential difference (PD) across the membrane in cells of the green alga,Chara inflata, cause voltage- and time-dependent current flows, interpreted to arise from opening and closing of various types of ion channel in the membrane. With cells in the light, these channels are normally closed, and the resting PD is probably determined by the operation of an H⁺ efflux pump. Positive steps in PD from the resting level often caused the opening of K⁺ channels with sigmoid kinetics. The channels began to show opening when the PD–120 mV for an external concentration of K⁺ of 1.0mm. Return of the PD to the resting level caused closing of the channels with complex kinetics. Various treatments of the cell could cause these K⁺ channels to open, and remain open continuously, with the PD then lying closer to the Nernst PD for K⁺. The K⁺ channels have been identified by the blocking effects of TEA⁺. Another group of channels, probably Cl^– and Ca²⁺ associated with the action potential open when the PD is stepped to values less negative than –50 mV. Negative steps from the resting PD cause the slow opening, with a time course of seconds, of yet another type of channel, probably Cl^–.