Transmembrane transport of K<Superscript>+</Superscript> and Cl<Superscript>−</Superscript> during pollen grain activation in vivo and in vitro

We studied the possibility of K⁺ and Cl⁻ efflux from tobacco pollen grains during their activation in vitro or on the stigma of a pistil. For this purpose the X-ray microanalysis and spectrofluorometry were applied. We found that the relative content of potassium and chlorine in the microvolume of pollen grain decreases during its hydration and activation on stigma. Efflux of these ions was found both in vivo and in vitro. In model in vitro experiments anion channel inhibitor NPPB ((5-nitro-2-(3-phenylpropylamino) benzoic acid) in the concentration that was blocking pollen germination, reduced Cl⁻ efflux; potassium channel inhibitor TEA (tetraethylammonium chloride) partially reduced K⁺ efflux and lowered the percent of activated cells. Another blocker of potassium channels Ba²⁺ caused severe decrease in cell volume and blocked the activation. In general, the obtained data demonstrates that the initiation of pollen germination both in vivo and in vitro involves the activation of K⁺ and Cl⁻ release. An important role in these processes is played by NPPB-, TEA- and Ba²⁺-sensitive plasmalemma ion channels.     

Transport of Cl– across the Plasma Membrane during Pollen Grain Germination in Tobacco

The role of Cl- transport across the plasma membrane was studied in an early step of pollen grain germination in tobacco Nicotiana tabacum L. The Cl- channel blockers, 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) and niflumic acid, completely suppress the germination with IC(50) approximately 8 micro M. At this concentration NPPB reduces the rate of Cl- efflux out of pollen grain by 1.8-fold in the interval 5-12 min, and niflumic acid reduces the rate 1.2-fold. 4,4;-Diisothiocyanatostilbene-2,2;-disulfonic acid, a known inhibitor of Cl- channels and antiporters, completely suppresses germination as well (IC(50) = 240 micro M), but has no effect on the rate of Cl- efflux. Inhibitors of chloride co-transporters, such as furosemide, bumetanide, and bis(1,3-dibutylbarbituric acid)pentamethine oxonol, suppress the germination by less than 50%. This set of data suggests that NPPB-sensitive anion channels are involved in the activation of pollen grains in the early stage of germination.     

Barium mimics the effect of D-glucose on 86Rb+ fluxes in mouse pancreatic beta-cells

The interaction between Ba2+, furosemide and D-glucose on 86Rb+ fluxes in ob/ob mouse islets was investigated. Ba2+ (2 mM) significantly reduced the ouabain-resistant 86Rb+ influx, without affecting the ouabain-sensitive influx. D-Glucose (20 mM) reduced the 86Rb+ influx in the absence of Ba2+ (2 mM) but not in the presence of the cation. Furosemide, an inhibitor of Na+, K+, Cl- co-transport, reduced the 86Rb+ influx and the effect was partly additive to the effect of 2 mM Ba2+. When the islets were preincubated with Ba2+ (2 mM) the specific effect of 1 mM furosemide on the 86Rb+ influx was reduced, whereas, in acute experiments, Ba2+ (2 mM) did not affect the specific effect of furosemide on 86Rb+ influx. 86Rb+ efflux from preloaded islets was significantly reduced by 2 mM Ba2+ and during the first 5 min of ion efflux the effect of the combination of 2 mM Ba2+ and 1 mM furosemide was stronger than the effect of Ba2+ alone. The data show that Ba2+ reduces 86Rb+ fluxes in the beta-cells and suggest that this is mainly mediated by inhibition of K+ channels in the beta-cell plasma membrane. Long-term exposure to Ba2+ may also reduce the activity of the Na+, K+, Cl- co-transport system. The effect of Ba2+ on K+ channels may help to explain the stimulatory effect on insulin release in the absence of nutrient secretagogues.     

T淋巴细胞上的离子通道

近年的研究证明,淋巴细胞上的离子通道,在免疫功能调节中具有重要的作用。T淋巴细胞上主要有三类离子通道,即Ca2 、K 和C1-通道。Ca2 通过T淋巴细胞膜上的Ca2 通道(CRAC)进入细胞内,可作为第二信使激活T淋巴细胞。通过K 通道的K 外流是T淋巴细胞膜电位形成的基础。由于膜电位水平可以影响钙离子的内流,因此,K 通道可以间接调节T淋巴细胞的活化和功能。T淋巴细胞上的Cl-通道是新近发现的一种离子通道,可能与细胞的体积调节有关。本文扼要总结了T淋巴细胞上离子通道的新近进展。     

Odd behaviour of Li+ in frog skin ion transport

1. Frog skin epithelium has basolateral K+ channels that normally define the basolateral membrane potential between 80 and 100 mV. 2. The membrane mentioned also has almost silent chloride channels and a [Na+, K+, 2Cl-] cotransport, the latter probably maintains the high Cl- in the capital (also called syncytium) cells. 3. If the K+ channels are blocked by Ba2+ (or Li+) it is possible to demonstrate potential gating of the chloride channels of the basolateral membrane. 4. When the normal K+ channels are blocked, a potential-dependent K+ conductance slowly emerges. 5. If Li+ is substituted for outside Na+ the skin shows potential oscillations of about 40 mV at a frequency of about six per hour. 6. The anion channel inhibitor Indacrinone stops these oscillations. 7. The role of Cl- and K+ channels in these oscillations is discussed. 8. The transepithelial inward transport of Li+ requires the presence of Na+ and seems to be due to exchange of cellular Li+ against inside Na+ via the basolateral Na+/H+ exchanger.     

Anion Selectivity of Slow Anion Channels in the Plasma Membrane of Guard Cells (Large Nitrate Permeability)

Closing of stomatal pores in the leaf epidermis of higher plants is mediated by long-term release of potassium and the anions chloride and malate from guard cells and by parallel metabolism of malate. Previous studies have shown that slowly activating anion channels in the plasma membrane of guard cells can provide a major pathway for anion efflux while also controlling K+ efflux during stomatal closing: Anion efflux produces depolarization of the guard cell plasma membrane that drives K+ efflux required for stomatal closing. The patch-clamp technique was applied to Vicia faba guard cells to determine the permeability of physiologically significant anions and halides through slow anion channels to assess the contribution of these anion channels to anion efflux during stomatal closing. Permeability ratio measurements showed that all tested anions were permeable with the selectivity sequence relative to Cl- of NO3- > Br- > F- ~ Cl- ~ I- > malate. Large malate concentrations in the cytosol (150 mM) produced a slow down-regulation of slow anion channel currents. Single anion channel currents were recorded that correlated with whole-cell anion currents. Single slow anion channels confirmed the large permeability ratio for nitrate over chloride ions. Furthermore, single-channel studies support previous indications of multiple conductance states of slow anion channels, suggesting cooperativity among anion channels. Anion conductances showed that slow anion channels can mediate physiological rates of Cl- and initial malate efflux required for mediation of stomatal closure. The large NO3- permeability as well as the significant permeabilities of all anions tested indicates that slow anion channels do not discriminate strongly among anions. Furthermore, these data suggest that slow anion channels can provide an efficient pathway for efflux of physiologically important anions from guard cells and possibly also from other higher plant cells that express slow anion channels.     

Membrane proteins involved in potassium shifts during muscle activity and fatigue

Muscle activity is associated with potassium displacements, which may cause fatigue. It was reported previously that the density of the large-conductance Ca2+-dependent K+ (BK(Ca)) channel is higher in the T tubule membrane than in the sarcolemmal membrane and that the opposite is the case for the ATP-sensitive K+ (K(ATP)) channel. In the present experiments, we investigated the subcellular localizations of the strong inward rectifier 2.1 K+ (Kir2.1) channel and the Na+-K+-2Cl- (NKCC)1 cotransporter with Western blot analysis of different muscle fractions. Furthermore, muscle function was studied while trying to manipulate the opening probability or transport capacity of these proteins during electrical stimulation of isolated soleus muscles. All experiments were made with excised muscle from male Wistar rats. Kir2.1 channels were almost undetectable in the sarcolemmal membrane but present in the T tubule membrane, whereas NKCC1 cotransporters were present in the sarcolemmal membrane. For muscles incubated in a buffer containing pinacidil, NS1619, Ba2+, or bumetanide, there was a faster reduction in peak force (P < 0.05). Furthermore, bumetanide incubation reduced the peak force at the onset of electrical stimulation (P < 0.05). Thus the effects on muscle force indicate that these drugs can affect K+-transporting proteins and thereby influence K+ accumulation, especially in the T tubules, suggesting that K(ATP) and BK(Ca) channels are responsible for K+ release and decrease in force during repeated muscle contractions, whereas Kir2.1 and NKCC1 may have a role in K+ reuptake.     

Effects of extracellular ATP on ion transport systems and [Ca2+]i in rat parotid acinar cells. Comparison with the muscarinic agonist carbachol

The effects of extracellular ATP on ion fluxes and the intracellular free Ca2+ concentration ([Ca2+]i) were examined using a suspension of rat parotid acinar cells and were contrasted with the effects of the muscarinic agonist carbachol. Although ATP and carbachol both rapidly increased [Ca2+]i about threefold above the resting level (200-250 nM), the effect of ATP was due primarily to an influx of Ca2+ across the plasma membrane, while the initial response to carbachol was due to a release of Ca2+ from intracellular stores. Within 10 s, ATP (1 mM) and carbachol (20 microM) reduced the cellular Cl- content by 39-50% and cell volume by 15-25%. Both stimuli reduced the cytosolic K+ content by 57-65%, but there were marked differences in the rate and pattern of net K+ movement as well as the effects of K+ channel inhibitors on the effluxes initiated by the two stimuli. The maximum rate of the ATP-stimulated K+ efflux (approximately 2,200 nmol K+/mg protein per min) was about two-thirds that of the carbachol-initiated efflux rate, and was reduced by approximately 30% (vs. 60% for the carbachol-stimulated K+ efflux) by TEA (tetraethylammonium), an inhibitor of the large conductance (BK) K+ channel. Charybdotoxin, another K+ channel blocker, was markedly more effective than TEA on the effects of both agonists, and reduced the rate of K+ efflux initiated by both ATP and carbachol by approximately 80%. The removal of extracellular Ca2+ reduced the ATP- and the carbachol-stimulated rates of K+ efflux by 55 and 17%, respectively. The rate of K+ efflux initiated by either agonist was reduced by 78-95% in cells that were loaded with BAPTA to slow the elevation of [Ca2+]i. These results indicated that ATP and carbachol stimulated the efflux of K+ through multiple types of K(+)-permeable channels, and demonstrated that the relative proportion of efflux through the different pathways was different for the two stimuli. ATP and carbachol also stimulated the rapid entry of Na+ into the parotid cell, and elevated the intracellular Na+ content to 4.4 and 2.6 times the normal level, respectively. The rate of Na+ entry through Na(+)-K(+)-2Cl- cotransport and Na(+)-H+ exchange was similar whether stimulated by ATP, carbachol, or ionomycin, and uptake through these two carrier-mediated transporters accounted for 50% of the ATP-promoted Na+ influx. The remainder may be due to a nonselective cation channel and an ATP-gated cation channel that is also permeable to Ca2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Identification and characterization of stretch-activated ion channels in pollen protoplasts

Pollen tube growth requires a Ca2+ gradient, with elevated levels of cytosolic Ca2+ at the growing tip. This gradient's magnitude oscillates with growth oscillation but is always maintained. Ca2+ influx into the growing tip is necessary, and its magnitude also oscillates with growth. It has been widely assumed that stretch-activated Ca2+ channels underlie this influx, but such channels have never been reported in either pollen grains or pollen tubes. We have identified and characterized stretch-activated Ca2+ channels from Lilium longiflorum pollen grain and tube tip protoplasts. The channels were localized to a small region of the grain protoplasts associated with the site of tube germination. In addition, we find a stretch-activated K+ channel as well as a spontaneous K+ channel distributed over the entire grain surface, but neither was present at the germination site or at the tip. Neither stretch-activated channel was detected in the grain protoplasts unless the grains were left in germination medium for at least 1 h before protoplast preparation. The stretch-activated channels were inhibited by a spider venom that is known to block stretch-activated channels in animal cells, but the spontaneous channel was unaffected by the venom. The venom also stopped pollen tube germination and elongation and blocked Ca2+ entry into the growing tip, suggesting that channel function is necessary for growth.     

Cardiac calcium channels in planar lipid bilayers. L-type channels and calcium-permeable channels open at negative membrane potentials

Planar lipid bilayer recordings were used to study Ca channels from bovine cardiac sarcolemmal membranes. Ca channel activity was recorded in the absence of nucleotides or soluble enzymes, over a range of membrane potentials and ionic conditions that cannot be achieved in intact cells. The dihydropyridine-sensitive L-type Ca channel, studied in the presence of Bay K 8644, was identified by a detailed comparison of its properties in artificial membranes and in intact cells. L-type Ca channels in bilayers showed voltage dependence of channel activation and inactivation, open and closed times, and single-channel conductances in Ba2+ and Ca2+ very similar to those found in cell-attached patch recordings. Open channels were blocked by micromolar concentrations of external Cd2+. In this cell-free system, channel activity tended to decrease during the course of an experiment, reminiscent of Ca2+ channel "rundown" in whole-cell and excised-patch recordings. A purely voltage-dependent component of inactivation was observed in the absence of Ca2+ stores or changes in intracellular Ca2+. Millimolar internal Ca2+ reduced unitary Ba2+ influx but did not greatly increase the rate or extent of inactivation or the rate of channel rundown. In symmetrical Ba2+ solutions, unitary conductance saturated as the Ba2+ concentration was increased up to 500 mM. The bilayer recordings also revealed activity of a novel Ca2+-permeable channel, termed "B-type" because it may contribute a steady background current at negative membrane potentials, which is distinct from L-type or T-type Ca channels previously reported. Unlike L-type channels, B-type channels have a small unitary Ba2+ conductance (7 pS), but do not discriminate between Ba2+ and Ca2+, show no obvious sensitivity to Bay K 8644, and do not run down. Unlike either L- or T-type channels, B-type channels did not require a depolarization for activation and displayed mean open times of greater than 100 ms.     

Role of Ca2+-activated Cl- channels in the mechanism of apoptosis induced by cyclosporin A in a human hepatoma cell line

The mechanism of apoptosis induced by cyclosporin A (CsA) in a human hepatoma cell line was investigated. CsA induced apoptosis in a dose- and time-dependent manner in HepG2 human hepatoma cells. CsA induced Cl- efflux, which was significantly blocked by niflumic acid (NA), a specific inhibitor, and flufenamic acid (FA), 5-nitro-2-(3-phenyl-propylamino)-benzoate (NPPB), and 4,4'-diisothiocyanoto-stibene-2,2'-disulfonic acid (DIDS), non-specific inhibitors of Ca2+-activated Cl- channels (CaCCs), not by calyculin A, an inhibitor of K+,Cl- -cotransport. In addition, CsA did not alter intracellular K+ concentration. Moreover, CsA increased intracellular Ca2+ concentration, and treatment with BAPTA/AM, an intracellular Ca2+ chelator, significantly inhibited the CsA-induced Cl- efflux, indicating that CsA induced Cl- efflux through the activation of CaCCs. Treatment with these CaCC inhibitors (NA, FA, NPPB, and DIDS) markedly prevented the CsA-induced apoptosis. Taken together, these results suggest that CaCCs may mediate apoptosis induced by CsA in HepG2 cells. Furthermore, these results provide a new insight into the novel function of CaCCs in the regulation of cancer cell apoptosis associated with perturbation of intracellular Ca2+ signal.     

In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts

The focus of this study is to investigate the regulatory role of K(+) influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 microm after a 6 h incubation. A decrease in external K(+) concentration from 1 mM to 35 microM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K(+) concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K(+) influx is associated with pollen germination and tube growth, regulation of the inward K(+) channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K(+) currents were first identified in Arabidopsis pollen protoplasts. The inward K(+) currents were insensitive to changes in cytoplasmic Ca(2+) but were inhibited by a high concentration of external Ca(2+). A decrease of external Ca(2+) concentration from 10 mM (control) to 1 mM had no significant effect on the inward K(+) currents, while an increase of external Ca(2+) concentration from 10 mM to 50 mM inhibited the inward K(+) currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K(+) currents. The physiological importance of potassium influx mediated by the inward K(+)-channels during Arabidopsis pollen germination and tube growth is discussed.     

Ionic mechanisms of histamine-induced responses in guinea pig intracardiac neurons

Histamine, released from mast cells, can modulate the activity of intrinsic neurons in the guinea pig cardiac plexus. The present study examined the ionic mechanisms underlying the histamine-induced responses in these cells. Histamine evokes a small membrane depolarization and an increase in neuronal excitability. Using intracellular voltage recording from individual intracardiac neurons, we were able to demonstrate that removal of extracellular sodium reduced the membrane depolarization, whereas inhibition of K+ channels by 1 mM Ba2+, 2 mM Cs+, or 5 mM tetraethylammonium had no effect. The depolarization was also not inhibited by either 10 microM Gd3+ or a reduced Cl- solution. The histamine-induced increase in excitability was unaffected by K+ channel inhibitors; however, it was reduced by either blockage of voltage-gated Ca2+ channels with 200 microM Cd2+ or replacement of extracellular Ca2+ with Mg2+. Conversely, alterations in intracellular calcium with thapsigargin or caffeine did not inhibit the histamine-induced effects. However, in cells treated with both thapsigargin and caffeine to deplete internal calcium stores, the histamine-induced increase in excitability was decreased. Treatment with the phospholipase C inhibitor U73122 also prevented both the depolarization and the increase in excitability. From these data, we conclude that histamine, via activation of H1 receptors, activates phospholipase C, which results in 1) the opening of a nonspecific cation channel, such as a transient receptor potential channel 4 or 5; and 2) in combination with either the influx of Ca2+ through voltage-gated channels or the release of internal calcium stores leads to an increase in excitability.     

Ion channel modifying agents influence the electrical activity generated by canine intrinsic cardiac neurons in situ

This study was designed to establish whether agents known to modify neuronal ion channels influence the behavior of mammalian intrinsic cardiac neurons in situ and, if so, in a manner consistent with that found previously in vitro. The activity generated by right atrial neurons was recorded extracellularly in varying numbers of anesthetized dogs before and during continuous local arterial infusion of several neuronal ion channel modifying agents. Veratridine (7.5 microM), the specific modifier of Na+-selective channels, increased neuronal activity (95% above control) in 80% of dogs tested (n = 25). The membrane depolarizing agent potassium chloride (40 mM) reduced neuronal activity (43% below control) in 84% of dogs tested (n = 19). The inhibitor of voltage-sensitive K+ channels, tetraethylammonium (10 mM), decreased neuronal activity (42% below control) in 73% of dogs tested (n = 11). The nonspecific potassium channel inhibitor barium chloride (5 mM) excited neurons (47% above control) in 13 of 19 animals tested. Cadmium chloride (200 microM), which inhibits Ca2+-selective channels and Ca2+-dependent K+ channels, increased neuronal activity (65% above control) in 79% of dogs tested (n = 14). The specific L-type Ca2+ channel blocking agent nifedipine (5 microM) reduced neuronal activity (52% blow control in 72% of 11 dogs tested), as did the nonspecific inhibitor of L-type Ca2+ channels, nickel chloride (5 mM) (36% below control in 69% of 13 dogs tested). Each agent induced either excitatory or inhibitory responses, depending on the agent tested. It is concluded that specific ion channels (I(Na), I(CaL), I(Kv), and I(KCa)) that have been associated with intrinsic cardiac neurons in vitro are involved in their capacity to generate action potentials in situ.     

Ba2+-sensitive K+ channels in luminal-membrane vesicles from pars convoluta of rabbit proximal tubule

This paper describes properties of 86Rb+ fluxes through a novel K+ channel in luminal-membrane vesicles isolated from pars convoluta of rabbit proximal tubule. The uptake of 86Rb+ into potassium salt loaded vesicles was specifically inhibited by Ba2+. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using these membrane vesicles loaded with anions of different membrane permeability and was as follows: gluconate greater than SO4(2-) greater than Cl-. Furthermore, the vesicles containing the channels show a cation selectivity with the order K+ greater than Rb+ greater than Li+ greater than Na+ = choline+.     

Signal transduction and ion channels in guard cells.

Our understanding of the signalling mechanisms involved in the process of stomatal closure is reviewed. Work has concentrated on the mechanisms by which abscisic acid (ABA) induces changes in specific ion channels at both the plasmalemma and the tonoplast, leading to efflux of both K+ and anions at both membranes, requiring four essential changes. For each we need to identify the specific channels concerned, and the detailed signalling chains by which each is linked through signalling intermediates to ABA. There are two global changes that are identified following ABA treatment: an increase in cytoplasmic pH and an increase in cytoplasmic Ca2+, although stomata can close without any measurable global increase in cytoplasmic Ca2+. There is also evidence for the importance of several protein phosphatases and protein kinases in the regulation of channel activity. At the plasmalemma, loss of K+ requires depolarization of the membrane potential into the range at which the outward K+ channel is open. ABA-induced activation of a non-specific cation channel, permeable to Ca2+, may contribute to the necessary depolarization, together with ABA-induced activation of S-type anion channels in the plasmalemma, which are then responsible for the necessary anion efflux. The anion channels are activated by Ca2+ and by phosphorylation, but the precise mechanism of their activation by ABA is not yet clear. ABA also up-regulates the outward K+ current at any given membrane potential; this activation is Ca(2+)-independent and is attributed to the increase in cytoplasmic pH, perhaps through the marked pH-sensitivity of protein phosphatase type 2C. Our understanding of mechanisms at the tonoplast is much less complete. A total of two channels, both Ca(2+)-activated, have been identified which are capable of K+ efflux; these are the voltage-independent VK channel specific to K+, and the slow vacuolar (SV) channel which opens only at non-physiological tonoplast potentials (cytoplasm positive). The SV channel is permeable to K+ and Ca2+, and although it has been argued that it could be responsible for Ca(2+)-induced Ca2+ release, it now seems likely that it opens only under conditions where Ca2+ will flow from cytoplasm to vacuole. Although tracer measurements show unequivocally that ABA does activate efflux of Cl- from vacuole to cytoplasm, no vacuolar anion channel has yet been identified. There is clear evidence that ABA activates release of Ca2+ from internal stores, but the source and trigger for ABA-induced increase in cytoplasmic Ca2+ are uncertain. The tonoplast and another membrane, probably ER, have IP3-sensitive Ca2+ release channels, and the tonoplast has also cADPR-activated Ca2+ channels. Their relative contributions to ABA-induced release of Ca2+ from internal stores remain to be established. There is some evidence for activation of phospholipase C by ABA, by an unknown mechanism; plant phospholipase C may be activated by Ca2+ rather than by the G-proteins used in many animal cell signalling systems. A further ABA-induced channel modulation is the inhibition of the inward K+ channel, which is not essential for closing but will prevent opening. It is suggested that this is mediated through the Ca(2+)-activated protein phosphatase, calcineurin. The question of Ca(2+)-independent stomatal closure remains controversial. At the plasmalemma the stimulation of K+ efflux is Ca(2+)-independent and, at least in Arabidopsis, activation of anion efflux by ABA may also be Ca(2+)-independent. But there are no indications of Ca(2+)-independent mechanisms for K+ efflux at the tonoplast, and the appropriate anion channel at the tonoplast is still to be found. There is also evidence that ABA interferes with a control system in the guard cell, resetting its set-point to lower contents, suggesting that stretch-activated channels also feature in the regulation of guard cell ion channels, perhaps through interactions with cytoskeletal proteins. (ABSTRACT TRUN     

K+ currents through SV-type vacuolar channels are sensitive to elevated luminal sodium levels

Non-selective slow vacuolar (SV) channels mediate uptake of K+ and Na+ into vacuolar compartment. Under salt stress plant cells accumulate Na+ in the vacuole and release vacuolar K+ into the cytoplasm. It is, however, unclear how plants mediate transport of K+ from the vacuole without concomitant efflux of toxic Na+. Here we show by patch-clamp studies on isolated Arabidopsis thaliana cell culture vacuoles that SV channels do not mediate Na+ release from the vacuole as luminal Na+ blocks this channel. Gating of the SV channel is dependent on the K+ gradient across the vacuolar membrane. Under symmetrical K+ concentrations on both sides of the vacuolar membrane, SV channels mediate potassium uptake. When cytoplasmic K+ decreases, SV channels allow K+ release from the vacuole. In contrast to potassium, Na+ can be taken up by SV channels, but not released even in the presence of a 150-fold gradient (lumen to cytoplasm). Accumulation of Na+ in the vacuole shifts the activation potential of SV channels to more positive voltages and prevents gradient-driven efflux of K+. Similar to sodium, under physiological conditions, vacuolar Ca2+ is not released from vacuoles via SV channels. We suggest that a major Arabidopsis SV channel is equipped with a positively charged intrinsic gate located at the luminal side, which prevents release of Na+ and Ca2+, but permits efflux of K+. This property of the SV channel guarantees that K+ can shuttle across the vacuolar membrane while maintaining Na+ and Ca2+ stored in this organelle.     

Ion channels in rabbit cultured fibroblasts

Large outward currents are recorded with the whole-cell patch-clamp technique on depolarization of rabbit cultured fibroblasts. Our findings suggest that these outward currents consist of two voltage-dependent components, one of which also depends on cytoplasmic calcium concentration. Total replacement of external Cl- by the large anion ascorbate does not affect the amplitude of the currents, indicating that both components must be carried by K+. Consistent with these findings with whole-cell currents, in single channel recordings from fibroblasts we found that most patches contain high-conductance potassium-selective channels whose activation depends on both membrane potential and the calcium concentration at the cytoplasmic surface of the membrane. In a smaller number of patches, a second population of high-conductance calcium-independent potassium channels is observed having different voltage-dependence. The calcium- and voltage-dependence suggest that these two channels correspond with the two components of outward current seen in the whole-cell recordings. The single channel conductance of both channels in symmetrical KCl (150 mM) is 260-270 pS. Both channels are highly selective for K+ over both Na+ and Cl-. The conductance of the channels when outward current is carried by Rb+ is considerably smaller than when it is carried by K+. Some evidence is adduced to support the hypothesis that these potassium channel populations may be involved in the control of cell proliferation.     

Maxi K+ channel mediates regulatory volume decrease response in a human bronchial epithelial cell line

The cell regulatory volume decrease (RVD) response triggered by hypotonic solutions is mainly achieved by the coordinated activity of Cl- and K+ channels. We now describe the molecular nature of the K(+) channels involved in the RVD response of the human bronchial epithelial (HBE) cell line 16HBE14o-. These cells, under isotonic conditions, present a K+ current consistent with the activity of maxi K+ channels, confirmed by RT-PCR and Western blot. Single-channel and whole cell maxi K+ currents were readily and reversibly activated following the exposure of HBE cells to a 28% hypotonic solution. Both maxi K+ current activation and RVD response showed calcium dependency, inhibition by TEA, Ba2+, iberiotoxin, and the cationic channel blocker Gd3+ but were insensitive to clofilium, clotrimazole, and apamin. The presence of the recently cloned swelling-activated, Gd3+-sensitive cation channels (TRPV4, also known as OTRPC4, TRP12, or VR-OAC) was detected by RT-PCR in HBE cells. This channel, TRPV4, which senses changes in volume, might provide the pathway for Ca2+ influx under hypotonic solutions and, consequently, for the activation of maxi K+ channels.     

Inward rectifier K+-channel kinetics from analysis of the complex conductance of Aplysia neuronal membrane.

Conduction in inward rectifier, K+-channels in Aplysia neuron and Ba++ blockade of these channels were studied by rapid measurement of the membrane complex admittance in the frequency range 0.05 to 200 Hz during voltage clamps to membrane potentials in the range -90 to -40 mV. Complex ionic conductances of K+ and Cl- rectifiers were extracted from complex admittances of other membrane conduction processes and capacitance by vector subtraction of the membrane complex admittance during suppressed inward K+ current (near zero-mean current and in zero [K+]0) from complex admittances determined at other [K+]0 and membrane potentials. The contribution of the K+ rectifier to the admittance is distinguishable in the frequency domain above 1 Hz from the contribution of the Cl- rectifier, which is only apparent at frequencies less than 0.1 Hz. The voltage dependence (-90 to -40 mV) of the chord conductance (0.2 to 0.05 microS) and the relaxation time (4-8 ms) of K+ rectifier channels at [K+]0 = 40 mM were determined by curve fits of admittance data by a membrane admittance model based on the linearized Hodgkin-Huxley equations. The conductance of inward rectifier, K+ channels at a membrane potential of -80 mV had a square-root dependence on external K+ concentration, and the relaxation time increased from 2 to 7.5 ms for [K+]0 = 20 and 100 mM, respectively. The complex conductance of the inward K+ rectifier, affected by Ba++, was obtained by complex vector subtraction of the membrane admittance during blockage of inward rectifier, K+ channels (at -35 mV and [Ba++]0 = 5 mM) from admittances determined at -80 mV and at other Ba++ concentrations. The relaxation time of the blockade process decreased with increases in Ba++ concentration. An open-closed channel state model produces the inductive-like kinetic behavior in the complex conductance of inward rectifier, K+ channels and the addition of a blocked channel state accounts for the capacitive-like kinetic behavior of the Ba++ blockade process.