Multiple potassium and chloride channels in the human colon carcinoma cell line SW1116

SW1116 cells have a profound capacity for secreting mucin molecules bearing the Lewisa epitope. Mucin molecules with the same epitope have been found to be elevated in the serum of patients with cystic fibrosis, a disease with defective ion channels. We therefore decided to study ion channels in this cell line. In the present work, we report the presence of two K(+)-channels and two Cl(-)-channels in the apical membrane of SW1116 cells. One of the K(+)-channels has a large conductance (approximately 278 pS), anomalous rectifying properties, and is inactivated rapidly. The second type exhibited a linear I/V curve (19 pS), was voltage insensitive and inactivation was not observed. In cell-attached patches, spontaneous openings of chloride channels were seen with higher frequency than previously reported in other colon carcinoma cell lines or airway epithelial cells. Inside-out experiments allowed identification of two different Cl(-)-channels (Cl(-)-1 and Cl(-)-2). Both exhibited rectification, but in opposite directions, and both were insensitive to NIPAB.     

Ultrastructural observation on sterilization of melittin

The effects of melittin on growth and bacteriostasis of four pathogens were extensively investigated using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results indicated that the melittin had a marked bacteriostatic effect on the four pathogenic bacteria. Among these, E. cacotowora was influenced most powerfully and quickly, the yeast and F. fulva were the second, and the S. aureus was inhibited by a low concentration but was killed by a high concentration. It was observed in the experiments that melittin killed pathogenic bacteria in three ways. One was by pore formation. After integrating melittin into the plasma membrane, a vacuole was formed then penetrated, resulting in bacterial content leakage. The vacuole also experienced plasmolysis and the growing cavity destroyed the membrane. A third effect was the formation of vacuoles in the cells which induced the pycnosis of the cytoplasm resulting in a cell death. The mechanism of melittin bacteriostasis was the result of integrating melittin with phospholipod double layers of the plasmalemma and the endomembranes.     

Effect of Salt Stress on Ion Channel Selective Permeability of Plasmalemma in Sorghum Roots

100 mmol/L, 200 mmol/L and 300 mmol/L NaC1 was used in proper order to treat three-day old seedlings of Sorghum vulgare Pets. The plasma membrane of roots was isolated and purified by aqueous biphasic partition device. The plasmalemma was incorporated into planar bilayer lipid membrane and ion channels were measured by electrical methods. Ion selective permeabilities (PK: PNa) were assayed in asymmetrical solutions containing 100 mmol/L NaC1 in Cis chamber and 100 mmol/L KC1 in Tran with chamber and were calculated from Goldman-Hodgkin-Kaltz equation. PK: PNa was 3.5 in plasmalemma of control roots and 1.5 in plasmalemma of salt stressed roots. It showed that the changes of ion selective permeability was very significant under salt stress. The importance of the change of ion selective permeability is discussed.     

盐胁迫对高粱根质膜离子通道通透性的影响

用１００ ｍ ｍ ｏｌ／Ｌ、２００ ｍ ｍ ｏｌ／Ｌ、３００ ｍ ｍ ｏｌ／ＬＮａＣｌ依次处理萌发后３ ｄ 的高粱（Ｓｏｒｇｈｕｍｖｕｌｇａｒｅ Ｐｅｒｓ．）幼苗，分离纯化根质膜。将质膜嵌入预先涂制好的平面脂双层，然后用电学方法测定该脂双层的离子选择通透性。膜的两测分别加入ＮａＣｌ和ＫＣｌ，在电压钳位下测定不同膜电位时的膜电流，通过ＧＨＫ 电压等式计算膜的离子选择通透性。结果表明质膜的离子选择通透性在盐胁迫下发生显著变化，对照的Ｋ＋ 、Ｎａ＋ 通透比（ＰＫ∶ＰＮａ）＝ ３．５，处理后ＰＫ∶ＰＮａ＝ １．５。讨论了这一变化的含义     

The Ultrastructure of Ion-Secreting and Non-Secreting Salt Glands of Limonium platyphyllum

The ultrastructure of salt glands in developing leaves of Limoniumplatyphyllum is described prior to exposure to 3% NaCl solution(with non-secreting glands) and after 4.5 and 18 h exposureto the salt solution. It is shown that in most glands, the transitionto active chloride transport was accompanied by the displacementof vacuoles toward the cell periphery and by the establishmentof plasmalemma contact sites with the tonoplast which appearedsimilar to gap junctions in animal epithelial cells. No evidencefor the exocytosis of vacuoles was found. It is suggested thatgland vacuoles may have a primary role in chloride secretionand that the tonoplast may be functionally asymmetrical, sothat the free part facing the hyaloplasm bears ion pumps, whereashighly permeable ion channels are active along the zone of contactwith the plasmalemma. It follows that the active step in chloridetransport in Limonium glands is the influx of ions into thevacuoles. Within the inner cup cells of the gland, vacuolescome into contact with the plasmalemma only at sites where thecell wall is adjacent to secretory and accessory cells. Suchan asymmetry appears to ensure the directed flux of ions intothis cell wall. Wall protuberances in the gland cells are rudimentaryand presumably not involved directly in NaCl secretion. Thenucleolus is activated during secretion and the frequency offree ribosomes is significantly increased, which is suggestiveof their involvement in the synthesis of membrane transportproteins. The ultrastructure of about one-third of the glandsremained unchanged in salt-treated leaves. Key words: Salt glands, ultrastructure, ion fluxes     

Plasmalemma abscisic acid perception leads to RAB18 expression via phospholipase D activation in Arabidopsis suspension cells

Abscisic acid (ABA) plays a key role in the control of stomatal aperture by regulating ion channel activities and water exchanges across the plasma membrane of guard cells. Changes in cytoplasmic calcium content and activation of anion and outward-rectifying K(+) channels are among the earliest cellular responses to ABA in guard cells. In Arabidopsis suspension cells, we have demonstrated that outer plasmalemma perception of ABA triggered similar early events. Furthermore, a Ca(2+) influx and the activation of anion channels are part of the ABA-signaling pathway leading to the specific expression of RAB18. Here, we determine whether phospholipases are involved in ABA-induced RAB18 expression. Phospholipase C is not implicated in this ABA pathway. Using a transphosphatidylation reaction, we show that ABA plasmalemma perception results in a transient stimulation of phospholipase D (PLD) activity, which is necessary for RAB18 expression. Further experiments showed that PLD activation was unlikely to be regulated by heterotrimeric G proteins. We also observed that ABA-dependent stimulation of PLD was necessary for the activation of plasma anion current. However, when ABA activation of plasma anion channels was inhibited, the ABA-dependent activation of PLD was unchanged. Thus, we conclude that in Arabidopsis suspension cells, ABA stimulation of PLD acts upstream from anion channels in the transduction pathway leading to RAB18 expression.     

Identification and characterization of inward K+ -channels in plasma membranes of Arabidopsis root cortex cells

Patch clamping whole-cell reeording techniques were apphed to study the inward K~ channels in Arabidopsis root cortex cells. The inward K~ -channels in the plasma membranes of the root cortex cell protoplasts were activated by hyperpolarized membrane potentials. The channels were highly selective tor K~ ions over Na~ ions. The channel activity was significantly inbibited by the external TEA(?) or Ba(?) The changes in cytoplasmic Ca~(2 ) concentrations did not affect the whole-cell inward K~ -currents. The possible asso(?)ation betw(?)en the channel selectivity to K~ and Na(?) ions and plant salt-tolerance was also discussed.     

Signal-transduction pathways that regulate smooth muscle function. II. Receptor-ion channel coupling mechanisms in gastrointestinal smooth muscle

Regulation of membrane ion channels by second messengers is an important mechanism by which gastrointestinal smooth muscle excitability is controlled. Receptor-mediated phosphorylation of Ca(2+) channels has been known for some time; however, recent findings indicate that these channels may also modulate intracellular signaling. The plasmalemma ion channels may also function as a point of convergence between different receptor types. In this review, the molecular mechanisms that link channel function and signal transduction are discussed. Emerging evidence also indicates altered second-messenger modulation of the Ca(2+) channel in the pathophysiology of smooth muscle dysmotility.     

Melittin inhibition of the gastric (H+ + K+) ATPase and photoaffinity labeling with [125I]azidosalicylyl melittin

Melittin is a 26-amino acid amphipathic polypeptide toxin from bee venom which forms anion-selective ion channels in bilayers and biological membranes under the influence of membrane potential. Melittin has been shown to interact with a number of membrane proteins. We found that melittin inhibited K+-stimulated ATP hydrolysis by the (H+ + K+) ATPase in parietal cell apical membrane vesicles derived from histamine-stimulated rabbit gastric mucosa with a KIapp of 0.5 micron. Melittin also inhibited K+-stimulated p-nitrophenyl hydrolysis activity which is associated with the gastric (H+ + K+) ATPase in a dose-dependent manner with a KIapp of 0.95 micron. ATP-driven, K+-dependent H+ transport was inhibited over this same concentration range, even in the absence of a membrane potential. Melittin did not appear to increase the H+ leak from vesicle with preformed H+ gradients when the H+ pump was arrested by Mg2+ chelation, but all possible membrane perturbation effects were difficult to rule out. However, the data suggest that melittin exerts its inhibitory effect through interaction with the (H+ + K+) ATPase. In order to determine whether direct interactions between the (H+ + K+) ATPase and melittin occurred, a radioactive derivative of melittin, [125I]azidosalicylyl melittin, was prepared and photoreacted with sealed rabbit gastric membranes and highly purified hog gastric membrane containing the (H+ + K+) ATPase. In the purified hog preparation only a 95,000-Da band, the (H+ + K+) ATPase was labeled, while in the rabbit preparation a 95,000-Da band and one other membrane protein of 70,000 Da were labeled with this reagent. Label incorporation into the (H+ + K+) ATPase and the 70,000-Da band was greatly reduced by addition of excess unlabeled melittin, suggesting specificity of the interaction. Label incorporation occurred in the absence of ATP or added salts and was not reduced by SCH28080 (a K+ site inhibitor) suggesting that the melittin binding site was distinct from the luminal K+ site of action of SCH28080.     

Shaker K(+)-channels are predicted to reduce the metabolic cost of neural information in Drosophila photoreceptors

Shaker K(+)-channels are one of several voltage-activated K(+)-channels expressed in Drosophila photoreceptors. We have shown recently that Shaker channels act as selective amplifiers, attenuating some signals while boosting others. Loss of these channels reduces the photoreceptor information capacity (bits s(-1)) and induces compensatory changes in photoreceptors enabling them to minimize the impact of this loss upon coding natural-like stimuli. Energy as well as coding is also an important consideration in understanding the role of ion channels in neural processing. Here, we use a simple circuit model that incorporates the major ion channels, pumps and exchangers of the photoreceptors to derive experimentally based estimates of the metabolic cost of neural information in wild-type (WT) and Shaker mutant photoreceptors. We show that in WT photoreceptors, which contain Shaker K(+)-channels, each bit of information costs approximately half the number of ATP molecules than each bit in Shaker photoreceptors, in which lack of the Shaker K(+)-channels is compensated by increased leak conductance. Additionally, using a Hodgkin-Huxley-type model coupled to the circuit model we show that the amount of leak present in both WT and Shaker photoreceptors is optimized to both maximize the available voltage range and minimize the metabolic cost.     

The effect of acetylcholine on Characeae K+ channels at rest and during action potential generation

The role of acetylcholine (ACh) as a signalling molecule in plants was investigated using a model system of Characeae cells. The effect of ACh on conductance of K⁺ channels in Nitella flexilis cells and on the action potential generation in Nitellopsis obtusa cells after H⁺-ATPase inhibition, where repolarization occurs after the opening of outward rectifying K⁺ channels, was investigated. Voltage-clamp method based on only one electrode impalement was used to evaluate the activity of separate potassium ion transport system at rest. We found that ACh at high concentrations (1 mM and 5 mM) activates K⁺ channels as the main membrane transport system at the resting state involved in electrogenesis of Characeaen membrane potential. We observed that ACh caused an increase in duration of AP repolarization of cells in K⁺ state when plasmalemma electrical characteristics are determined by large conductance K⁺ channels irrespective of whether AP were spontaneous or electrically evoked. These results indicate interference of ACh with electrical cellular signalling pathway in plants.     

Electrophysiological and other aspects of excitation-contraction coupling and uncoupling in mammalian airway smooth muscle

In this article the electrophysiological events which are believed to underly agonist-induced contraction and relaxation of airway smooth muscle are reviewed, with special emphasis on the indispensable role of the Ca ion. The contribution made by Na, K, Ca and Cl to, and the role that the electrogenic Na:K-dependent ATPase plays in, the maintenance of the resting membrane potential in both normal and sensitised airway smooth muscle cells is described together with the permeability changes that occur in the plasmalemma in response to excitatory and inhibitory agonists. In addition, the currently available evidence for the existence of potential-sensitive and receptor-operated Ca channels in respiratory smooth muscle, and how such channels may be involved in the regulation of airway calibre, is critically assessed.     

Orientation of membrane-bound melittin studied by a combination of HPLC and liquid secondary ion mass spectrometry (LSIMS)

Combining two analytical techniques, HPLC and liquid secondary ion mass spectrometry, the orientation of liposomal membrane-bound melittin was analyzed through its trypsin-digested products. We found that trypsin can access all proteolytic sites of the membrane-bound melittin when the liposomes have no transmembrane potential, whereas the proteolytic site near the N terminus of melittin is blocked when the liposomes have a negative transmembrane potential. The results suggest that the negative transmembrane potential may induce the melittin molecules to insert into the membrane perpendicularly, whereas melittin lies flat on the membrane surface in the absence of a negative potential.     

Effect of melittin on ion currents in heart cell membranes

It has been shown on auricle fibres of the frog that neurotoxin from bee poison melittin suppresses the ionic currents entering the cell through calcium and sodium channels of the membrane, increases the background potassium current, suppresses phasic and tonic contraction of the fibres. Toxin modifies the kinetics of calcium channels, but does not affect activation and desensitization of beta adrenoreceptors. Effects of melittin are not decreased when adding the inhibitor of phospholipase A2 indomethacin. The results show that melittin directly affects the protein components of the membrane-ionic channels, probably binding with them.     

Osmotic swelling of hepatocytes increases membrane conductance but not membrane capacitance.

We have used the whole-cell patch-clamp technique to study changes in membrane conductance and membrane capacitance after osmotic swelling in rat hepatocytes. Hypoosmotic solutions induced an instantaneous increase in the volume of patch-clamped cells that was followed by a slow decline reminiscent of regulatory volume decrease as seen in intact cells. These morphological changes were associated with a transient increase in membrane conductance. The rise in conductance was not correlated with changes in capacitance, neither in time after the initiation of cell swelling nor in magnitude. Therefore we conclude that an osmotically induced increase in conductance is probably a result of the activation of existent channels in the plasmalemma and not a result of the fusion of vesicle membrane containing ionic channels.     

Turgor regulation in a brackish water charophyte,Lamprothamnium succinctum

Internodal cells of a brackish water charophyte,Lamprothamnium succinctum (A. Br. in Ash.) R.D.W. regulate the turgor pressure in response to changes in both the cellular and the external osmotic pressures. During turgor regulation upon hypotonic treatment, net effluxes of K⁺ and Cl⁻ from the vacuole, membrane depolarization, a transient increase in the electrical membrane conductance and a transient increase in concentration of cytoplasmic Ca²⁺ are induced. Activation of the plasmalemma Ca²⁺ channels and the Ca²⁺-controlled passive effluxes of K⁺ and Cl⁻ through the plasmalemma ion channels are postulated.     

Control of Ion Fluxes in Stomatal Guard Cells

Opening and closing of the stomatal pore is associated with very large changes in K-salt accumulation in stomatal guard cells. This review discusses the ionic relations of guard cells in relation to the general pattern of transport processes in plant cells, in plasmalemma and tonoplast, involving primary active transport of protons, proton-linked secondary active transport, and a number of gated ion channels. The evidence available suggests that the initiation of stomatal opening is regulated through the uptake mechanisms, whereas initiation of stomatal closing is regulated by control of ion efflux at the plasmalemma, and of fluxes to and from the vacuole. In response to a closing signal there are large transient increases in efflux of both Cl^? (or Br^?) and Rb⁺ (K⁺) at the plasmalemma, with also a probable increase in anion flux from vacuole to cytoplasm and decrease in anion flux from cytoplasm to vacuole. A speculative hypothetical sequence of events is discussed, by which the primary response to a closing signal is an increase in Ca²⁺ influx at the plasmalemma, producing depolarisation and increase in cytoplasmic Ca²⁺. The consequent opening of Ca²⁺-sensitive Cl^? channels, and voltage-sensitive K⁺ channels (also Ca²⁺-sensitive?) in the plasmalemma, and of a Ca²⁺-sensitive nonspecific channel in the tonoplast, could produce the flux effects identified by the tracer work; this speculation is also consistent with the Ca²⁺-sensitivity of the response to closing signals and with evidence from patch clamping that such channels exist in at least some plant cells, though not yet all shown in guard cells.     

Haloperidol modulates ion transport in <Emphasis Type="Italic">Chara corallina</Emphasis> cells

The effects of haloperidol, an antagonist of D2 dopamine receptors, on the functioning of Ca²⁺, K⁺, and Cl^? ion channels in the membrane of Chara corallina cells and on the functional properties of their cytoskeleton was studied. Haloperidol blocked Ca²⁺ channels of the plasmalemma. In addition to bringing about a decrease in the amplitude of the calcium current, exposure to haloperidol decelerated the activation and inactivation of calcium channels. The effect of haloperidol was reversible; after it was removed, the characteristics of calcium current were restored. Haloperidol did not affect Ca²⁺-activated chloride channels. Haloperidol also inhibited microfilament-dependent motion of the cytoplasm. Cytoplasmic streaming was restored after haloperidol was removed from the extracellular solution. These results suggest that the concentration of free Ca²⁺ ions in the cytoplasm increases in the presence of haloperidol, and that Ca²⁺ channels of C. corallina plasmalemma possess specific binding sites both for dopamine receptors and for their antagonists.     

Electrophysiological study of frog eggs at different stages of development. II) Outward rectification in oocytes at the final stage of vitellogenesis

Voltage-clamp experiments in full-grown frog oocytes, in a range of membrane potentials from 90 mV negative to 30 mV positive, have revealed the presence of voltage-dependent channels selective for K+, blocked by extracellular TEA. The percentage of open K+-channels increases with membrane depolarizations over a range from -40 mV to +10 mV, thus supporting the outward rectification in the I/V relationship. The current transport through the K+-channels open at different potential levels and in various [K+]o takes place in accordance with the constant-field assumptions. The leakage current of the oocyte membrane was found to be considerable large.     

Wall-to-membrane linkers in onion epidermis: some hypotheses

Wall-to-wall linkage may help maintain cell integrity and polarity, and focus mechanical stress from wall to mech-anotransductive ion channels within the plasm a lemma. When cells of onion bulb scale epidermis shrink during plasmolysis with CaCl₂, the plasmalemma remains attached to the cell wall by Hechtian strands which we hypothesize might possibly be drawn out from linkages fulfilling the above functions. We show that at least many of the attachment loci are independent of the plasmodesmata. A priori, wall glycoproteins seem good candidates for the wall-to-membrane linkers; therefore, we investigated the distribution in wall and plasmalemma of antigen recognized by antibody to hydroxyproline-rich glycoprotein (HRGP). Using fluorescent secondary antibodies, we showed that polyclonal antibodies prepared against wall HRGP from soybean bind to the onion walls (following mild depectination), but also bind to the plasmalemma after the wall is enzymatically digested. The distribution of the antibodies is punctate. On the plasmalemma, the points tend to be scattered more or less uniformly, but can cluster at termini of large streaming strands (which rarely form in wall-constrained cells.) These streaming strands can be seen to exert tension on the membrane. We hypothesize that (1) the antigen on the surface of the protoplast may correspond to the antigen in the walls, (2) such antigen may be responsible for adhesion of membrane to wall at the linkage sites visualized by CaCl₂ plasmolysis, and (3) the linkage sites may be transmembrane proteins to which cytoskeleton can attach at the inner surface.