Chemoreception inParamecium tetraurelia: acetate and folate-induced membrane hyperpolarization

Summary Acetic and folic acids hyperpolarize the membrane potential ofParamecium tetraurelia in a concentration-dependent manner. The membrane responses are accompanied by small changes in cell resistance, and are significantly reduced by increasing extracellular cation concentrations, suggesting that the attractants bring about the membrane potential change by increasing cell permeability to cations. The inability to show a reversal potential for the hyperpolarization to attractants suggests that the effects of cations on the response are non-specific, however. The possible roles of Ca⁺⁺, K⁺, and Na⁺ in the attractant-induced responses were further investigated by applying acetate and folate to cells with genetic defects in specific ion conductances, by collapsing the driving forces for these ions, and by testing the effects of ion channel blockers on the responses. These studies suggest that the membrane responses to attractants are not due to the direct effects of increased or decreased membrane permeability to cations.Attempts to block the acetate and folate-induced hyperpolarization by collapsing surface potential or using a mutant with reduced surface charge were inconclusive, as were studies on the possible role of attractant transport in the membrane responses.We hypothesize that the membrane hyperpolarization may be due to either the indirect effects of increased calcium permeability, to extrusion of calcium through activation of a calcium pump, or to a proton efflux.     

Effects of cytochalasin B and local anesthetics on electrical and morphological properties in L cells

Spontaneous oscillations of membrane potential observed in L cells were inhibited rapidly and reversibly in the presence of cytochalasin B (CB). Sustained hyperpolarization induced by high external Ca²⁺ was also depressed by the drug. However, Ca²⁺ injection into the cytoplasm elicited a sustained hyperpolarization, even in the presence of CB. These observations strongly suggest that CB inhibits calcium transport system in cell membrane. Morphological alterations associated with the CB treatment were decreased adhesiveness and rounding of the cells, with concomitant changes in surface architecture. Similar changes in electrophysiological and morphological properties were observed in cells treated with local anesthetics. Since such morphological changes induced by CB and local anesthetics were always preceded by electrical changes, it was suggested that the morphological changes are secondary phenomena resulting from inhibition of the Ca²⁺ transport.     

The electrical potential difference through the foot epithelium of the snail Achatina achatina, Lameere during mechanical and chemical stimulation

An important electrophysiological variable--the transepithelial potential difference reflects the electrogenic transepithelial ion currents, which are produced and modified by ion transport processes in polarized cells of epithelium. These processes result from coordinated function of transporters in apical and basolateral cell membranes and have been observed in all epithelial tissues studied so far. The experiments were performed on isolated specimens of snail foot. In the experiments, the baseline transepithelial electrical potential difference--PD, changes of transepithelial difference during mechanical stimulation--dPD and the transepithelial resistance were measured with an Ussing apparatus. A total of 60 samples of foot ventral surface of 28 snails were studied. The transepithelial electrical potential difference of isolated foot ranged from -6.0 to 10.0 mV under different experimental conditions. Mechanical stimulation of foot ventral surface caused changes of electrogenic ion transport, observed as transient hyperpolarization (electrical potential difference became more positive). When the transepithelial electrical potential difference decreased during stimulation, the reaction was described as depolarization. When amiloride and bumetanide were added to the stimulating fluid so that the sodium and chloride ion transport pathways were inhibited, prolonged depolarization occurred. Under the influence of different stimuli: mechanical (gentle rinsing), chemical (changes of ion concentrations) and pharmacological (application of ion inhibitors), transient changes of potential difference (dPD) were evoked, ranging from about -0.7 to almost 2.0 mV. Changes in transepithelial potential difference of the pedal surface of the snail's foot related to these physiological stimuli are probably involved in the locomotion of the animal and are under control of the part of the nervous system in which tachykinin related peptides (TRP) act as transmitters.     

Inhibition of chloride secretion in human bronchial epithelial cells by cigarette smoke extract

Chronic bronchitis, a disease mainly of cigarette smokers, shares many clinical features with cystic fibrosis, a disease of altered ion transport, suggesting that the negative effects of cigarette smoke on mucociliary clearance may be mediated through alterations in ion transport. We tested the hypothesis that cigarette smoke extract would inhibit chloride secretion in human bronchial epithelial cells. In agreement with studies in canine trachea, cigarette smoke extract inhibited net chloride secretion without affecting sodium transport. We performed microelectrode impalements and impedance analysis studies to investigate the physiological mechanisms of this inhibition. These data demonstrated that cigarette smoke extract caused an acute increase in membrane resistances in conjunction with apical membrane hyperpolarization, an effect consistent with inhibition of an apical membrane anion conductance. After this acute phase, both membrane resistances decreased while membrane potentials continued to hyperpolarize, indicating that cigarette smoke extract also inhibited the basolateral entry of chloride into the cell. Furthermore, cigarette smoke extract caused an increase in mucin secretion. Therefore, the ion transport phenotype of human bronchial epithelial cells exposed to cigarette smoke extract is similar to that of cystic fibrosis epithelia in which there is sodium absorption out of proportion to chloride secretion in the setting of increased mucus secretion.     

Calcium-dependent sodium currents inParamecium: Mutational manipulations and effects of hyper- and depolarization

Summary The membrane ofParamecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation,fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that theParamecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca²⁺ concentration. The function of the Ca-dependent Na conductance is discussed.     

Regulation of transient receptor potential melastatin 7 (TRPM7) currents by mitochondria

Mitochondria play a central role in energy-generating processes and may be involved in the regulation of channels and receptors. Here we investigated TRPM7, an ion channel and functional kinase, and its regulation by mitochondria. Proton ionophores such as CCCP elicited a rapid decrease in outward TRPM7 whole-cell currents but a slight increase in inward currents with pipette solutions containing no MgATP. With pipette solutions containing 3 mM MgATP, however, CCCP increased both outward and inward TRPM7 currents. This effect was reproducible and fully reversible, and repeated application of CCCP yielded similar decreases in current amplitude. Oligomycin, an inhibitor of F1/FO-ATP synthase, inhibited outward whole-cell currents but did not affect inward currents. The respiratory chain complex I inhibitor, rotenone, and complex III inhibitor, antimycin A, were without effect as were kaempferol, an activator of the mitochondrial Ca2+ uniporter, and ruthenium red, an inhibitor of the mitochondrial Ca2+ uniporter. These results suggest that the inner membrane potential (as regulated by proton ionophores) and the F1/FO-ATP synthase of mitochondria are important in regulating TRPM7 channels.     

Alkali cation binding and permeation in the rat organic cation transporter rOCT2

Organic cation transporters of the OCT family mediate downhill transport of organic cations, compatible with carrier, pore, or gate-lumen-gate mechanisms. We studied rat OCT2 expressed in Xenopus oocytes by the two-electrode voltage-clamp technique, including membrane capacitance (C(m)) monitoring. Choline, a transported cationic substrate, elicited the expected inward currents but also elicited decreases of C(m). Similar C(m) decreases were caused by the non-transported inhibitors tetrabutylammonium (a cation) and corticosterone (uncharged). Effects on C(m) were voltage-dependent, with a maximum at -140 mV. These findings suggest that the empty rOCT2 protein can undergo an electrogenic conformation change, with one conformation highly favored at physiological voltage. Moreover, alkali cations elicited considerable inward currents and inhibited uptake of [(14)C]tetraethylammonium with a sequence Cs(+) > Rb(+) > K(+) > Na(+) approximately Li(+). Cs(+) affected current and capacitance with similar affinity (K(0.5) approximately 50 mm). Tetraethylammonium inhibited Cs(+) currents in a concentration-dependent manner. Conversely, Cs(+) inhibited tetraethylammonium uptake by a competitive mechanism. Activation energy of the currents estimated from measurements between 12 degrees C and 32 degrees C was approximately 81 kJ/mol for Cs(+) and 39 kJ/mol for tetramethylammonium, compatible with permeation of Cs(+) through rOCT2 along the same path as organic substrates and by a mechanism different from simple electrodiffusion. Rationalization of Cs(+) selectivity in terms of a pore pointed to a pore diameter of approximately 4 A. Intriguingly, that value matches the known selectivity of rOCT2 for organic compounds. Our data show that selective permeability of rOCT2 is not determined by ligand affinity but might rather be understood in terms of the ion channel concept of a distinct "selectivity filter."     

Identification and regulation of whole-cell chloride currents in airway epithelium

We used the whole-cell patch-clamp technique to study membrane currents in human airway epithelial cells. The conductive properties, as described by the instantaneous current-voltage relationship, rectified in the outward direction when bathed in symmetrical CsCl solutions. In the presence of Cl concentration gradients, currents reversed near ECl and were not altered significantly by cations. Agents that inhibit the apical membrane Cl conductance inhibited Cl currents. These conductive properties are similar to the conductive properties of the apical membrane Cl channel studied with the single-channel patch-clamp technique. The results suggest that the outwardly rectifying Cl channel is the predominant Cl-conductive pathway in the cell membrane. The steady-state and non-steady-state kinetics indicate that current flows through ion channels that are open at hyperpolarizing voltages and close with depolarization. These Cl currents were regulated by the cAMP-dependent protein kinase: when the catalytic subunit of cAMP-dependent protein kinase was included in the pipette solution, Cl channel current more than doubled. We also found that reducing extracellular osmolarity by 30% increased Cl current, suggesting that cell-swelling stimulated Cl current. Studies of transepithelial Cl transport in cell monolayers suggest that a reduction in solution osmolarity activates the apical Cl channel: reducing extracellular osmolarity stimulated a short-circuit current that was inhibited by Cl-free solution, by mucosal addition of a Cl channel antagonist, and by submucosal addition of a loop diuretic. These results suggest that apical membrane Cl channels may be regulated by cell volume and by the cAMP-dependent protein kinase.     

Calcium-dependent inactivation of the calcium current activated upon hyperpolarization of Paramecium tetraurelia

The Ca2+ current activated upon hyperpolarization of Paramecium tetraurelia decays over a period of 150-200 ms during sustained steps under voltage clamp. At membrane potentials between -70 and approximately -100 mV, the time course of this inactivation is described by a single exponential function. Steps negative to approximately -100 mV elicit currents that decay biexponentially, however. Three lines of evidence suggest that this current's inactivation is a function of intracellular Ca2+ concentration rather than membrane potential: (a) Comparing currents with similar amplitudes but elicited at widely differing membrane potentials suggests that their time course of decay is a sole function of inward current magnitude. (b) The extent of current inactivation is correlated with the amount of Ca2+ entering the cell during hyperpolarization. (c) The onset and time course of recovery from inactivation can be hastened significantly by injecting cells with EGTA. We suggest that the decay of this current during hyperpolarization involves a Ca(2+)-dependent pathway.     

Stable expression of gastric proton pump activity at the cell surface

Stable cell lines expressing the gastric proton pump alpha- and/or beta-subunits were constructed. The cell line co-expressing the alpha- and beta-subunits showed inward Rb(+) transport, which was activated by Rb(+) in a concentration-dependent manner. In the alpha+beta-expressing cell line, rapid recovery of intracellular pH was also observed after acid load, indicating that this cell line transported protons outward. These ion transport activities were inhibited by a proton pump inhibitor, 2-methyl-8-(phenylmethoxy)imidazo[1,2-a]pyridine-3-acetonitrile (SCH 28080). In a membrane fraction of the alpha+beta-expressing cell line, K(+)-stimulated ATPase (K(+)-ATPase) activity and the acylphosphorylation of the alpha-subunit were observed, both of which were also inhibited by SCH 28080. The specific activity and properties of the K(+)-ATPase were comparable to those found in the native gastric proton pump. In the stable cell lines, the alpha-subunit was retained in the intracellular compartment and was unstable in the absence of the beta-subunit, but it was stabilized and reached the cell surface in the presence of the beta-subunit. On the other hand, the beta-subunit was stable and able to travel to the cell surface in the absence of the alpha-subunit. These cell lines are ideal for the structure-function study of ion transport by the gastric proton pump as well as for characterization of the cellular regulation of surface expression of the functional proton pump.     

A novel Cl- inward-rectifying current in the plasma membrane of the calcifying marine phytoplankton Coccolithus pelagicus

We investigated the membrane properties and dominant ionic conductances in the plasma membrane of the calcifying marine phytoplankton Coccolithus pelagicus using the patch-clamp technique. Whole-cell recordings obtained from decalcified cells revealed a dominant anion conductance in response to membrane hyperpolarization. Ion substitution showed that the anion channels were selective for Cl(-) and Br(-) over other anions, and the sensitivity to the stilbene derivative 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, ethacrynic acid, and Zn(2+) revealed a pharmacological profile typical of many plant and animal anion channels. Voltage activation and kinetic characteristics of the C. pelagicus Cl(-) channel are consistent with a novel function in plants as the inward rectifier that tightly regulates membrane potential. Membrane depolarization gave rise to nonselective cation currents and in some cases evoked action potential currents. We propose that these major ion conductances play an essential role in membrane voltage regulation that relates to the unique transport physiology of these calcifying phytoplankton.     

Energy conservation by Rhodothermus marinus respiratory complex I

A sodium ion efflux, together with a proton influx and an inside-positive ΔΨ, was observed during NADH-respiration by Rhodothermus marinus membrane vesicles. Proton translocation was monitored by fluorescence spectroscopy and sodium ion transport by ²³Na-NMR spectroscopy. Specific inhibitors of complex I (rotenone) and of the dioxygen reductase (KCN) inhibited the proton and the sodium ion transport, but the KCN effect was totally reverted by the addition of menaquinone analogues, indicating that both transports were catalyzed by complex I. We concluded that the coupling ion of the system is the proton and that neither the catalytic reaction nor the establishment of the delta-pH are dependent on sodium, but the presence of sodium increases proton transport. Moreover, studies of NADH oxidation at different sodium concentrations and of proton and sodium transport activities allowed us to propose a model for the mechanism of complex I in which the presence of two different energy coupling sites is suggested.     

T-Type Ca2+ Current Activity during Oocyte Growth and Maturation in the Ascidian Styela plicata

Voltage-dependent calcium currents play a fundamental role during oocyte maturation, mostly L-type calcium currents, whereas T-type calcium currents are involved in sperm physiology and cell growth. In this paper, using an electrophysiological and pharmacological approach, we demonstrated, for the first time in oocytes, that T-type calcium currents are present with functional consequences on the plasma membrane of growing immature oocytes of the ascidian Styela plicata. We classified three subtypes of immature oocytes at the germinal vesicle stage on the basis of their size, morphology and accessory cellular structures. These stages were clearly associated with an increased activity of T-type calcium currents and hyperpolarization of the plasma membrane. We also observed that T-type calcium currents oscillate in the post-fertilization embryonic stages, with minimal amplitude of the currents in the zygote and maximal at 8-cell stage. In addition, chemical inhibition of T-type calcium currents, obtained by applying specific antagonists, induced a significant reduction in the rate of cleavage and absence of larval formation. We suggest that calcium entry via T-type calcium channels may act as a potential pacemaker in regulating cytosolic calcium involved in fertilization and early developmental events.     

Multiple acute effects on the membrane potential of PC12 cells produced by nerve growth factor (NGF)

We studied whether nerve growth factor (NGF) can affect the membrane potential and conductance of PC12 cells. We demonstrate that NGF depolarizes the membrane of PC12 cells within a minute and by using transfected NIH 3T3-Trk and -p75 cells we show that both the high affinity NGF receptor p140(trk) and the low affinity NGF receptor or p75(NGF) may be involved in the depolarization. Tyrosine kinase inhibitor, K252a, partially inhibited the depolarization, but two agents affecting intracellular calcium movements, Xestospongin C (XeC) and thapsigargin, did not. The early depolarization was eliminated in Na+ free solutions and under this condition, a 'prolonged' (> 2 min) hyperpolarization was observed in PC12 cells in response to NGF. This hyperpolarization was also induced in PC12 cells by epidermal growth factor (EGF). Voltage clamp experiments showed that NGF produced a late (> 2 min) increase in membrane conductance. The Ca2+-dependent BK-type channel blocker, iberiotoxin, and the general Ca2+-dependent K+ channel blocker, TEA, attenuated or eliminated the hyperpolarization produced by NGF in sodium free media. Under pretreatment with the non-selective cation channel blockers La3+ and Gd3+, NGF hyperpolarized the membrane of PC12 cells. These results suggest that three different currents are implicated in rapid NGF-induced membrane voltage changes, namely an acutely activated Na+ current, Ca2+-dependent potassium currents and non-selective cation currents.     

Glucose transport by mixed ruminal bacteria from a cow.

The glucose transport of mixed ruminal bacteria harvested from a holstein cow fed 5.0 kg of Italian ryegrass and 1.5 kg of flaked corn a day was investigated. The Eadie-Hofstee plot characterized two transport systems: a high-affinity, low-velocity system and a low-affinity, high-velocity system. The former system (K(m) = 16 microM; Vmax = 2.2 nmol/min/mg of protein) is considered dominant under this feeding condition based on the glucose concentration in the rumen (< 1 mM). In light of the facts that the protonophore SF6847 and the lipophilic triphenylmethyl phosphonium ion had no effect on the high-affinity system and an artificially generated proton gradient and electrical potential across the cell membrane did not increase glucose transport, a proton motive force is not be involved in the system. On the other hand, from the facts that chlorhexidine inhibited about 90% of the high-affinity system while iodoacetate showed no significant effect, and a high phosphoenolpyruvate-dependent phosphorylation of glucose was actually shown, the phosphoenolpyruvate-dependent phosphotransferase system is considered the main system in the high-affinity system. Moreover, as shown by the facts that harmaline inhibited about 30% of the high-affinity system and the artificially generated sodium gradient across the cell membrane significantly stimulated glucose transport, this system also includes sodium symport to some degree. The high-affinity system was sensitive to a decrease in pH (< 6.5) and was inhibited by the presence of sucrose, mannose, and fructose.     

Anticancer and antiproliferative activity of natural brassinosteroids

Brassinosteroids (BRs) are steroid plant hormones that are essential for many plant growth and developmental processes, including cell expansion, vascular differentiation and stress responses. Up to now the inhibitory effects of BRs on cell division of mammalian cells are unknown. To determine basic anticancer structure-activity relationships of natural BRs on human cells, several normal and cancer cell lines have been used. Several of the tested BRs were found to have high cytotoxic activity. Therefore, in our next series of experiments, we tested the effects of the most promising and readily available BR analogues with interesting anticancer properties, 28-homocastasterone (1) and 24-epibrassinolide (2), on the viability, proliferation, and cycling of hormone-sensitive/insensitive (MCF-7/MDA-MB-468) breast and (LNCaP/DU-145) prostate cancer cell lines to determine whether the discovered cytotoxic activity of BRs could be, at least partially, related to brassinosteroid-nuclear receptor interactions. Both BRs inhibited cell growth in a dose-dependent manner in the cancer cell lines. Flow cytometry analysis showed that BR treatment arrested MCF-7, MDA-MB-468 and LNCaP cells in G(1) phase of the cell cycle and induced apoptosis in MDA-MB-468, LNCaP, and slightly in the DU-145 cells. Our results provide the first evidence that natural BRs can inhibit the growth, at micromolar concentrations, of several human cancer cell lines without affecting the growth of normal cells. Therefore, these plant hormones are promising leads for potential anticancer drugs.     

Induction of divalent cation permeability by heterologous expression of a voltage sensor domain

The voltage sensor domain (VSD) is a protein domain that confers sensitivity to membrane potential in voltage-gated ion channels as well as the voltage-sensing phosphatase. Although VSDs have long been considered to function as regulatory units acting on adjacent effectors, recent studies have revealed the existence of direct ion permeation paths in some mutated VSDs and in the voltage-gated proton channel. In this study, we show that calcium currents are evoked upon membrane hyperpolarization in cells expressing a VSD derived from an ascidian voltage-gated ion channel superfamily. Unlike the previously reported omega-pore in the Shaker K⁺ channel and rNav1.4, mutations are not required. From electrophysiological experiments in heterologous expression systems, we found that the conductance is directly mediated by the VSD itself and is carried by both monovalent and divalent cations. This is the first report of divalent cation permeation through a VSD-like structure.     

Heme histidine ligands within gp91(phox) modulate proton conduction by the phagocyte NADPH oxidase

The membrane subunit of the phagocyte NADPH oxidase, gp91(phox), possesses a H(+) channel motif formed by membrane-spanning histidines postulated to coordinate the two heme groups forming the redox center of the flavocytochrome. To study the role of heme-binding histidines on proton conduction, we stably expressed the gp91(phox) cytochrome in human embryonic kidney 293 cells and measured proton currents with the patch clamp technique. Similar to its shorter homologue, NADPH oxidase homologue 1, which is predicted not to bind heme, gp91(phox) generated voltage-activated, pH-dependent, H(+)-selective currents that were reversibly blocked by Zn(2+). The gp91(phox) currents, however, activated faster, deactivated more slowly, and were markedly affected by the inhibition of heme synthesis. Upon heme removal, the currents had larger amplitude, activated faster and at lower voltages, and became sensitive to the histidine reagent diethylpyrocarbonate. Mutation of the His-115 residue to leucine abolished both the gp91(phox) characteristic 558-nm absorbance peak and voltage-activated currents, indicating that His-115 is involved in both heme ligation and proton conduction. These results indicate that the gp91(phox) proton channel is activated upon release of heme from its His-115 ligand. During activation of the oxidase complex, changes in heme coordination within the cytochrome might increase the mobility of histidine ligands, thereby coupling electron and proton transport.     

The MgtC virulence factor of Salmonella enterica serovar Typhimurium activates Na(+),K(+)-ATPase

The mgtC gene of Salmonella enterica serovar Typhimurium encodes a membrane protein of unknown function that is important for full virulence in the mouse. Since mgtC is part of an operon with mgtB which encodes a Mg(2+)-transporting P-type ATPase, MgtC was hypothesized to function in ion transport, possibly in Mg(2+) transport. Consequently, MgtC was expressed in Xenopus laevis oocytes, and its effect on ion transport was evaluated using ion selective electrodes. Oocytes expressing MgtC did not exhibit altered currents or membrane potentials in response to changes in extracellular H(+), Mg(2+), or Ca(2+), thus ruling out a previously postulated function as a Mg(2+)/H(+) antiporter. However, addition of extracellular K(+) markedly hyperpolarized membrane potential instead of the expected depolarization. Addition of ouabain to block the oocyte Na(+),K(+)-ATPase completely prevented hyperpolarization and restored the normal K(+)-induced depolarization response. These results suggested that the Na(+),K(+)-ATPase was constitutively activated in the presence of MgtC resulting in a membrane potential largely dependent on Na(+),K(+)-ATPase. Consistent with the involvement of Na(+),K(+)-ATPase, oocytes expressing MgtC exhibited an increased rate of (86)Rb(+) uptake and had increased intracellular free [K(+)] and decreased free [Na(+)] and ATP. The free concentrations of Mg(2+) and Ca(2+) and cytosolic pH were unchanged, although the total intracellular Ca(2+) content was slightly elevated. These results suggest that the serovar Typhimurium MgtC protein may be involved in regulating membrane potential but does not directly transport Mg(2+) or another ion.