Current-voltage relationships of a sodium-sensitive potassium channel in the tonoplast ofChara corallina

Summary The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m^–2 creating a voltage across the tonoplast of about –7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K⁺-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K⁺ with limiting currents of about +15 and –30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na⁺ and Cl^–. However, addition of Na⁺ decreases the K⁺ currents. TheI–V relationships of the open channel as measured at various K⁺ concentrations with or without Na⁺ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.     

Transport ofl-histidine by human diploid fibroblasts in culture

Summary The transport ofl-histidine has been characterized in skin derived diploid human fibroblasts, cultured under strictly controlled conditions. The transport measurements were made on cells grown to subconfluency after 60 to 90 min timed preincubation. The data, at substrate concentrations ranging from 0.050 to 10 mmol/l, were analyzed by a computer program. A saturable transport system (K _m =0.25 mmol/l, V _max =17 nmol/mg protein per min) and a nonsaturable component of influx (K _d =1.6±0.4 nmol/mg protein/min per mmol) were found.l-Histidine displayed no Na⁺ requirement at either low or high concentrations. Inhibition analysis demonstrated thatl-histidine uptake at low concentration was poorly inhibited by amino acids known to be effective inhibitors of system A. The largest fraction ofl-histidine uptake was inhibited by 2-amino-bicyclo (2,2,1)-heptane-2-carboxylic acid (BCH), leucine, and tryptophan. These results indicated thatl-histidine is transported in human fibroblasts, mainly by the Na⁺ independent system L. The differences between this cell type and others studied previously are discussed. This work was supported in part by Grant 773 from UER de Médecine, Université Paris XI (France).     

ATP-sensitive potassium channels in adult mouse skeletal muscle: Characterization of the ATP-binding site

Summary Single K⁺-selective channels were studied in excised inside-out membrane patches from dissociated mouse toe muscle fibers. Channels of 74 pS conductance in symmetrical 160mm KCl solutions were blocked reversibly by 10 m internal ATP and thus identified as ATP-sensitive K⁺ channels. The channels were also blocked reversibly bymm concentrations of internal adenosine, adenine and thymine, but not by cytosine and uracil. The efficacy of the reversible channel blockers was higher when they were present in internal NaCl instead of KCl solutions. An irreversible inhibition of ATP-sensitive K⁺ channels was observed after application of several sulphydryl-modifying substances in the internal solution: 0.5mm chloramine-T, 50mm hydrogen peroxide or 2mm n-ethylmaleimide (NEM). Largeconductance Ca-activated K⁺ channels were not affected by these reagents. The presence of 1mm internal ATP prevents the irreversible inhibition of ATP-sensitive K⁺ channels by NEM. The results suggest that internal Na⁺ ions increase the affinity of the ATP-sensitive K⁺ channel to ATP and to other reversible channel blockers and that a functionally important SH-group is located at or near the ATP-binding site.     

An evolvant ofEscherichia coli that employs thel-fucose pathway also for growth onl-galactose andd-arabinose

Summary l-Galactose,d-arabinose, andl-fucose form six-membered rings with identical stereoconfigurations. However, onlyl-fucose can serve as the sole carbon and energy source of wild-typeEscherichia coli K-12. A mutant that can grow onl-galactose andd-arabinose was isolated by alternate selection on the two sugars. Thel-fucose pathway became inducible by all three sugars. Transduction into the mutant of the wild-type fuc⁺ region containing both the regulatory and structural genes abolished the novel growth abilities onl-galactose andd-arabinose, whereas transduction into the mutant of a fuc deletion abolished the growth abilities on all three sugars. Introduction of the wild-type fucR⁺ (which encodes the activator protein for the fuc regulon) on a multicopy plasmid depressed the growth abilities of the mutant onl-galactose andd-arabinose, but not onl-fucose. The results suggest that the effector specificity of the activator protein in the mutant was broadened. It is proposed that an adaptive response of an activator-controlled system is more likely than that of a repressor-controlled system to achieve fixation in a population, because the first variant to emerge in response to a novel metabolic demand has a good chance of having an altered specificity of regulation. Such a change entails little or no metabolic liability during the absence of the novel substrate. In contrast, the first variant of a negatively controlled system to emerge has an overwhelming chance of being the result of a random mutation that destroys repressor function. Although negatively controlled systems can be more opportunistic in exploiting new conditions than positively controlled systems, an adaptive change is less likely to become fixed because of the cost associated with gratuitous constitutive gene expression in the absence of the substrate.     

l-Fucose residues on cellulose-based dialysis membranes: Quantification of membrane-associatedl-fucose and analysis of specific lectin binding

Contact of mononuclear human leukocytes with cellulose dialysis membranes may result in complement-independent cell activation, i.e. enhanced synthesis of cytokines, prostaglandins and an increase in 2-microglobulin synthesis. Cellular contact activation is specifically inhibited by the monosaccharidel-fucose suggesting that dialysis membrane associatedl-fucose residues are involved in leukocyte activation. In this study we have detected and quantitatedl-fucose on commercially-available cellulose dialysis membranes using two approaches. A sensitive enzymatic fluorescence assay detectedl-fucose after acid hydrolysis of flat sheet membranes. Values ranged from 79.3±3.6 to 90.2±5.0 pmol cm^–2 for Hemophan® or Cuprophan® respectively. Enzymatic cleavage of terminal -l-fucopyranoses with -l-fucosidase yielded 7.7±3.3 pmoll-fucose per cm² for Cuprophan. Enzymatic hydrolysis of the synthetic polymer membranes AN-69 and PC-PE did not yield detectable amounts ofl-fucose. In a second approach, binding of the fucose specific lectins ofLotus tetragonolobus andUlex europaeus (UEAI) demonstrated the presence of biologically accessiblel-fucose on the surface of cellulose membranes. Specific binding was observed with Cuprophan®, and up to 2.6±0.3 pmoll-fucose per cm² was calculated to be present from Langmuir-type adsorption isotherms. The data presented are in line with the hypothesis that surface-associatedl-fucose residues on cellulose dialysis membranes participate in leukocyte contact activation.     

Kinetics and specificity of the renal Na+/myo-inositol cotransporter expressed in Xenopus Oocytes

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na⁺/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V _m ), the external myo-inositol concentration and the external Na⁺ concentration, yielding the kinetic parameters: K _0.5 ^MI , K _0.5 ^Na , and the Hill coefficient n. At 100 mM NaCl, K _0.5 ^MI was about 50 m and was independent of V _m . At 0.5 mm myo-inositol, K _0.5 ^Na ranged from 76 mm at V _m =–50 mV to 40 mm at V _m =–150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na⁺ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K _I of 64 m at V _m =–50 mV and 130 m at V _m = –150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V _m =–150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, -methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na⁺/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V _m =–150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, -methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na⁺-dependent uptake of ³H-d-glucose and ³H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na⁺ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V _m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model. Present address: W.M. Keck Biotechnology Resource Laboratory, Boyer Center for Molecular Medicine, Rm, 305A, Yale University, 295 Congress Ave., New Haven, Connecticut 06536-0812 Present address: National Institute for Physiological Sciences, Department of Cell Physiology, Okazaka, 444, JapanContributed equally to this workWe thank John Welborn for the HPLC analysis of the sugar substrates. This work was supported by grants from the National Institutes of Health DK19567, DK42479 and NS25554.     

Redistribution of hepatocyte chloride during l-alanine uptake

We used ion-sensitive, double-barrel microelectrodes to measure changes in hepatocyte transmembrane potential (V _m), intracellular K⁺, Cl^-, and Na⁺ activities (a ⁱ _k, a _Cl ⁱ and a _Na ⁱ ), and water volume during l-alanine uptake. Mouse liver slices were superfused with control and experimental Krebs physiological salt solutions. The experimental solution contained 20 m l-alanine, and the control solution was adjusted to the same osmolality (305 mOsm) with added sucrose. Hepatocytes also were loaded with 50 mm tetramethylammonium ion (TMA⁺) for 10 min. Changes in cell water volume during l-alanine uptake were determined by changes in intracellular, steady-state TMA⁺ activity measured with the K⁺ electrode. Hepatocyte control V _m was -33±1 mV. l-alanine uptake first depolarized V _m by 2±0.2 mV and then hyperpolarized V _m by 5 mV to-38±1 mV (n = 16) over 6 to 13 min. During this hyperpolarization, a _Na ⁱ increased by 30% from 19±2 to 25±3 mm (P < 0.01), and a _K ⁱ did not change significantly from 83±3 mm. However, with added ouabain (1 mm) l-alanine caused only a 2-mV increase in V _m, but now a _K ⁱ decreased from 61±3 to 54±5 mm (P < 0.05). Hyperpolarization of V _m by l-alanine uptake also resulted in a 38% decrease of a _Cl ⁱ from 20±2 to 12±3 mm (P < 0.001). Changes in V _m and V _Cl — V _m voltage traces were parallel during the time of l-alanine hyperpolarization, which is consistent with passive distribution of intracellular Cl^– with the V _m in hepatocytes. Added Ba²⁺ abolished the l-alanineinduced hyperpolarization, and a _Cl ⁱ remained unchanged. Hepatocyte water volume during l-alanine uptake increased by 12±3%. This swelling did not account for any changes in ion activities following l-alanine uptake. We conclude that hepatocyte a _K ⁱ is regulated by increased Na⁺-K⁺ pump activity during l-alanine uptake in spite of cell swelling and increased V _m due to increased K⁺ conductance. The hyperpolarization of V _m during l-alanine uptake provides electromotive force to decrease a _Cl ⁱ . The latter may contribute to hepatocyte volume regulation during organic solute transport.This work was supported by grant AA-08867 from the Alcohol, Drug Abuse, and Mental Health Association.     

Block of Persistent Late Na+ Currents by Antidepressant Sertraline and Paroxetine

Antidepressants, such as traditional tricyclic antidepressants (TCAs), are the first-line treatment for various pain syndromes. Available evidence indicates that TCAs may target Na⁺ channels for their analgesic action. In this report, we examined the effects of contemporary antidepressants sertraline and paroxetine on (1) neuronal Na⁺ channels expressed in GH₃ cells and (2) muscle rNav1.4 Na⁺ channels heterologously expressed in Hek293t cells. Our results showed that both antidepressants blocked Na⁺ channels in a highly state-dependent manner. The 50% inhibitory concentrations (IC₅₀) for sertraline and paroxetine ranged ∼18–28 μm for resting block and ∼2–8 μm for inactivated block of neuronal and rNav1.4 Na⁺ channels. Surprisingly, the IC₅₀ values for both drugs were about 0.6–0.7 μm for the open channel block of persistent late Na⁺ currents generated through inactivation-deficient rNav1.4 mutant Na⁺ channels. For comparison, the open channel block in neuronal hNav1.7 counterparts yielded IC₅₀ values around 0.3–0.4 μm for both drugs. Receptor mapping using fast inactivation-deficient rNav1.4-F1579A/K mutants with reduced affinities toward local anesthetics (LAs) and TCAs indicated that the F1579 residue is not involved in the binding of sertraline and paroxetine. Thus, sertraline and paroxetine are potent open channel blockers that target persistent late Na⁺ currents preferentially, but their block is not mediated via the phenylalanine residue at the known LA/TCA receptor site.     

The characteristics of arginine transport by rat cerebellar and cortical synaptosomes

The uptake ofl-[³H]arginine into synaptosomes prepared from rat cerebellum and cortex occurred by a high-affinity carrier-mediated process. The uptake of arginine appeared to be potentiated by removal of extracellular Na⁺, inhibited by high levels of extracellular K⁺, but not by depolarization with veratridine or 4-amino pyridine. The effect of Na⁺ removal or K⁺ elevation did not seem to be due to changes in intracellular Ca²⁺ or pH. In both brain regions, uptake was significantly inhibited byl-arginine,l-lysine,l-ornithine, andl-homoarginine, but not byd-arginine norl-citrulline. Uptake was also inhibited by N^G-monomethyl-l-arginine acetate, but not by N^G-nitro-l-arginine methyl ester nor N^G-nitro-l-arginine except in the cortex at a concentration of 1 mM. The results indicate that the carrier system operating in synaptosomes showed many of the characteristics of the ubiquitous y⁺ system seen in many other tissues, although its apparent sensitivity to variations in extracellular Na⁺ was unusual.     

HERG1 Currents in Native K562 Leukemic Cells

The human ether-a-go-go related gene (HERG1) K⁺ channel is expressed in neoplastic cells, in which it was proposed to play a role in proliferation, differentiation and/or apoptosis. K562 cells (a chronic myeloid leukemic human cell line) express both the full-length (herg1a) and the N-terminally truncated (herg1b) isoforms of the gene, and this was confirmed with Western blots and coimmunoprecipitation experiments. Whole-cell currents were studied with a tail protocol. Seventy-eight percent of cells showed a HERG1-like current: repolarization to voltages negative to −40 mV produced a transient peak inward tail current, characteristic of HERG1 channels. Cells were exposed to a HERG-specific channel blocker, E4031. Half-maximal inhibitory concentration (IC₅₀) of the blocker was 4.69 nm. The kinetics of the HERG1 current in K562 cells resembled the rapid component of the native cardiac delayed rectifier current, known to be conducted by heterotetrameric HERG1 channels. Fast and slow deactivation time constants at −120 mV were 27.5 and 239.5 ms, respectively. Our results in K562 cells suggest the assembling of heterotetrameric channels, with some parameters being dominated by one of the isoforms and other parameters being intermediate. Hydrogen peroxide was shown to increase HERG1a K⁺ current in heterologous expression systems, which constitutes an apoptotic signal. However, we found that K562 HERG1 whole-cell currents were not activated by H₂O₂.     

Characterization of Regulatory Volume Behavior by Fluorescence Quenching in Human Corneal Epithelial Cells

An in-depth understanding of the mechanisms underlying regulatory volume behavior in corneal epithelial cells has been in part hampered by the lack of adequate methodology for characterizing this phenomenon. Accordingly, we developed a novel approach to characterize time-dependent changes in relative cell volume induced by anisosmotic challenges in calcein-loaded SV40-immortalized human corneal epithelial (HCE) cells with a fluorescence microplate analyzer. During a hypertonic challenge, cells shrank rapidly, followed by a temperature-dependent regulatory volume increase (RVI), τ_c = 19 min. In contrast, a hypotonic challenge induced a rapid (τ_c = 2.5 min) regulatory volume decrease (RVD). Temperature decline from 37 to 24°C reduced RVI by 59%, but did not affect RVD. Bumetanide (50 μM), ouabain (1 mM), DIDS (1 mM), EIPA (100 μM), or Na⁺-free solution reduced the RVI by 60, 61, 39, 32, and 69%, respectively. K⁺, Cl⁻ channel and K⁺-Cl⁻ cotransporter (KCC) inhibition obtained with either 4-AP (1 mM), DIDS (1 mM), DIOA (100 μM), high K⁺ (20 mM) or Cl⁻-free solution, suppressed RVD by 42, 47, 34, 52 and 58%, respectively. KCC activity also affects steady-state cell volume, since its inhibition or stimulation induced relative volume alterations under isotonic conditions. Taken together, K⁺ and Cl⁻ channels in parallel with KCC activity are important mediators of RVD, whereas RVI is temperature-dependent and is essentially mediated by the Na⁺-K⁺-2Cl⁻ cotransporter (Na⁺-K⁺-2Cl⁻) and the Na⁺-K⁺ pump. Inhibition of K⁺ and Cl⁻ channels and KCC but not Na⁺-K⁺-2Cl⁻ affect steady-state cell volume under isotonic conditions. This is the first report that KCC activity is required for HCE cell volume regulation and maintenance of steady-state cell volume.     

Modification of single cardiac Na+ channels by DPI 201-106

Summary In inside-out patches from cultured neonatal rat heart cells, single Na⁺ channel currents were analyzed under the influence of the cardiotonic compound DPI 201-106 (DPI), a putative novel channel modifier. In absence of DPI, normal cardiac single Na⁺ channels studied at –30 mV have one open state which is rapidly left with a rate constant of 826.5 sec^–1 at 20°C during sustained depolarization., Reconstructed macroscopic currents relax completely with 7 to 10 msec. The current decay fits a single exponential. A considerable percentage of openings may occur during relaxation of the macroscopic current. In patches treated with 3×10^–6 m DPI in the pipette solution, stepping to –30 mV results in drastically prolonged and usually repetitive openings. This channel activity mostly persists over the whole depolarization (usually 160 msec in duration) but is abruptly terminated on clamping back the patch to the holding potential. Besides these modified events, apparently normal openings occur. The open time distribution of DPI-treated Na⁺ channels is the sum of two exponentials characterized by time constants of 0.85 msec (which is close to the time constant found in the control patches, 1.21 msec) and 12 msec. Moreover, DPI-modified Na⁺ channels exhibit a sustained high, time-independent open probability. Similar to normal Na⁺ channels, the mean number of open DPI-modified Na⁺ channels is voltage-dependent and increases on shifting the holding potential in the hyperpolarizing direction. These kinetic changes suggest an elimination of Na⁺ channel inactivation as it may follow from an interaction of DPI with Na⁺ channels.     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Evidence for coupling between Na+ pump activity and TEA-sensitive K+ currents in Xenopus laevis oocytes

Using the two-microelectrode voltage clamp technique in Xenopus laevis oocytes, we estimated Na⁺-K⁺-ATPase activity from the dihydroouabain-sensitive current (I _DHO) in the presence of increasing concentrations of tetraethylammonium (TEA⁺; 0, 5, 10, 20, 40 mm), a well-known blocker of K⁺ channels. The effects of TEA⁺ on the total oocyte currents could be separated into two distinct parts: generation of a nonsaturating inward current increasing with negative membrane potentials (V _M) and a saturable inhibitory component affecting an outward current easily detectable at positive V _M. The nonsaturating component appears to be a barium-sensitive electrodiffusion of TEA⁺ which can be described by the Goldman-Hodgkin-Katz equation, while the saturating component is consistent with the expected blocking effect of TEA⁺ on K⁺ channels. Interestingly, this latter component disappears when the Na⁺-K⁺-ATPase is inhibited by 10 m DHO. Conversely, TEA⁺ inhibits a component of I _DHO with a k _d of 25±4 mm at +50 mV. As the TEA⁺-sensitive current present in I _DHO reversed at –75 mV, we hypothesized that it could come from an inhibition of K⁺ channels whose activity varies in parallel with the Na⁺-K⁺-ATPase activity. Supporting this hypothesis, the inward portion of this TEA⁺-sensitive current can be completely abolished by the addition of 1 mm Ba²⁺ to the bath. This study suggests that, in X. laevis oocytes, a close link exists between the Na-K-ATPase activity and TEA⁺-sensitive K⁺ currents and indicates that, in the absence of effective K⁺ channel inhibitors, I _DHO does not exclusively represent the Na⁺-K⁺-ATPase-generated current.     

Cell swelling activates K+ and Cl− channels as well as nonselective,stretch-activated cation channels in ehrlich ascites tumor cells

Summary Cell-attached patch-clamp recordings from Ehrlich ascites tumor cells reveal nonselective cation channels which are activated by mechanical deformation of the membrane. These channels are seen when suction is applied to the patch pipette or after osmotic cell swelling. The channel activation does not occur instantaneously but within a time delay of 1/2 to 1 min. The channel is permeable to Ba²⁺ and hence presumably to Ca²⁺. It seems likely that the function of the nonselective, stretch-activated channels is correlated with their inferred Ca²⁺ permeability, as part of the volume-activated signal system. In isolated insideout patches a Ca²⁺-dependent, inwardly rectifying K⁺ channel is demonstrated. The single-channel conductance recorded with symmetrical 150 mm K⁺ solutions is for inward current estimated at 40 pS and for outward current at 15 pS. Activation of the K⁺ channel takes place after an increase in Ca²⁺ from 10^–7 to 10^–6 m which is in the physiological range. Patch-clamp studies in cellattached mode show K⁺ channels with spontaneous activity and with characteristics similar to those of the K⁺ channel seen in excised patches. The single-channel conductance for outward current at 5 mm external K⁺ is estimated at about 7 pS. A K⁺ channel with similar properties can be activated in the cellattached mode by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by cell swelling, within 1 min following hypotonic exposure. No evidence was found of channel activation by membrane stretch (suction). The time-averaged number of open K⁺ channels during regulatory volume decrease (RVD) can be estimated at 40 per cell. The number of open K⁺ channels following addition of Ca²⁺ plus ionophore A23187 was estimated at 250 per cell. Concurrent activation in cell-attached patches of stretch-activated, nonselective cation channels and K⁺ channels in the presence of 3 mm Ca²⁺ in the pipette suggests a close spatial relationship between the two channels. In excised inside-out patches (with NMDG chloride on both sides) a small 5-pS chloride channel with low spontaneous activity is observed. The channel activity was not dependent on Ca²⁺ and could not be activated by membrane stretch (suction). In cell-attached mode singlechannel currents with characteristics similar to the channels seen in isolated patches are seen. In contrast to the channels seen in isolated patches, the channels in the cell-attached mode could be activated by addition of Ca²⁺ plus ionophore A23187. The channel is also activated by hypotonic exposure with a single-channel conductance at 7 pS (or less) and with a time delay at about 1 min. The number of open channels during RVD is estimated at 80 per cell. Two other types of Cl^– channels were regularly recorded in excised inside-out patches: a voltage-activated 400-pS channel and a 34-pS Cl^– channel which show properties similar to the Cl^– channel in the apical membrane in human airway epithelial cells. There is no evidence for a role in RVD for either of these two channels.     

Voltage-dependent Conductance Changes in a Nonvoltage-activated Sodium Current from a Mast Cell Line

Nonexcitable cells do not express voltage-activated Na⁺ channels. Instead, selective Na⁺ influx is accomplished through GTP-activated Na⁺ channels, the best characterized of which are found in renal epithelia. We have described recently a GTP-dependent Na⁺ current in rat basophilic leukemia (RBL) cells that differs from previous reported Na⁺ channels in several ways including selectivity, pharmacology and mechanism of activation. In this report, we have investigated the biophysical properties of the RBL cell Na⁺ current using the whole cell patch-clamp technique. Following activation by 250–500 μm GTPγS, hyperpolarizing steps to a fixed potential (−100 mV) from a holding potential of 0 mV evoked transient inward Na⁺ currents that declined during the pulse. If the holding potential was made more positive (range 0 to +100 mV), then the amplitude of the transient inward current evoked by the hyperpolarization increased steeply, demonstrating that the conductance of the channels was voltage-dependent. Using a paired pulse protocol (500 msec pulses to −100 mV from a holding potential of 0 mV), it was found that the peak amplitude of the current during the second pulse became larger as the interpulse potential became more positive. In addition, increasing the time at which the cells were held at positive potentials also resulted in larger currents, indicating a time-dependent conductance change. With symmetrical Na⁺ solutions, outward currents were recorded at positive potentials and these demonstrated both a time- and voltage-dependent increase in conductance. The results show that a nonvoltage activated Na⁺ channel in an electrically nonexcitable cell undergoes prominent voltage-dependent transitions. Possible mechanisms underlying this voltage dependency are discussed. Received: 12 March 1998/Revised: 5 June 1998     

Sodium-selective channels in membranes of rat macrophages

With the use of the patch-clamp technique, highly selective nonvoltage-gated sodium channels were found in the membrane of rat peritoneal macrophages. The inward single channel currents were measured in cell-attached and outside-out mode experiments at different holding membrane potentials within the range of-60 to +40 mV. The channels had a unitary conductance of 10.2 ± 0.2 pS with 145 mm Na⁺ in the external solution at 23–24°C. The results of ion-substitution experiments confirmed that this novel type of cation channel in macrophages is characterized by high selectivity for Na⁺ over K⁺ (as for Cs⁺, NH₄ ⁺, Ca²⁺, Ba²⁺) ions, whose conduction through these sodium-permeable channels was not measurable. Lithium is the only other ion that is transported by this pathway; the unitary conductance was equal to 3.9 ± 0.2 pS in the Li⁺-containing external solution. Single channel currents and conductance were found to be linearly dependent on the external sodium concentration. Sodium channels in macrophage membrane patches were not blocked by tetrodotoxin (0.01–1 m). Single sodium currents were reversibly inhibited by the external application of amiloride (0.1–2 mm) and its derivative ethylisopropilamiloride (0.01–0.1 Mm). The mechanism of channel block by amiloride and its analogue seems to be different.We thank Dr. G.N. Mozhayeva and Dr. A.P. Naumov for useful discussions. This work has been supported by a grant from the Russian Basic Research Foundation, 93-04-21722.     

Kinetic studies on the stimulation of Na+−H+ exchange activity in renal brush border membranes isolated from thyroid hormone-treated rats

Summary Na⁺–H⁺ exchange activity in renal brush border membrane vesicles isolated from hyperthyroid rats was increased. When examined as a function of [Na⁺], treatment altered the initial rate of Na⁺ uptake by increasingV _m (hyperthyroid, 18.9±1.1 nmol Na⁺ · mg^–1 · 2 sec^–1; normal, 8.9±0.3 nmol Na⁺ · mg^–1 · 2 sec^–1), and not the apparent affinityK _Na ⁺ (hyperthyroid, 7.3±1.7mm; normal, 6.5±0.9mm). When examined as a function of [H⁺] and at a subsaturating [Na⁺] (1mm), hyperthyroidism resulted in the proportional increase in Na⁺ uptake at every intravesicular pH measured. A positive cooperative effect on Na⁺ uptake was found with increased intravesicular acidity in vesicles from both normal and hyperthyroid rats. When the data were analyzed by the Hill equation, it was found that hyperthyroidism did not change then (hyperthyroid, 1.2±0.06; normal, 1.2±0.07) or the [H⁺]_0.5 (hyperthyroid, 0.39±0.08 m; normal, 0.44±0.07 m) but increased the apparentV _m (hyperthyroid, 1.68±0.14 nmol Na⁺ · mg^–1 · 2 sec^–1; normal 0.96±0.10 nmol Na⁺ · mg^–1 · 2 sec^–1). The uptake of Na⁺ in exchange for H⁺ in membrane vesicles from normal and hyperthyroid animals was not influenced by membrane potential. H⁺ translocation or debinding was rate limiting for Na⁺–H⁺ exchange since Na⁺–Na⁺ exchange activity was greater than Na⁺–H⁺ exchange activity. Hyperthyroidism caused a proportional increase and hypothyroidism caused a proportional decrease in Na⁺–Na⁺ and Na⁺–H⁺ exchange. We conclude that hyperthyroidism leads to either an increase in the number of functional exchangers in the membrane or exactly proportional increases in the rate-limiting steps for Na⁺–Na⁺ and Na⁺–H⁺ exchange activity.     

Properties of the K+ inward rectifier in the plasma membrane of xylem parenchyma cells from barley roots: Effects of TEA+, Ca2+, Ba2+ and La3+

Xylem parenchyma cells are situated around the (apoplastic) xylem vessels and are involved in the control of the composition of the xylem sap by exporting and resorbing solutes. We investigated properties of the K⁺ inward rectifier in the plasma membrane of these cells by performing patch clamp experiments on protoplasts in the whole-cell configuration. Inward currents were sensitive to the K⁺ channel blocker TEA⁺ at a high concentration (20 mm). Barium, another classical K⁺ channel blocker, inhibited K⁺ currents with a K _i of about 1.3 mm. In contrast to guard cells, the cytosolic Ca²⁺ level proved to be ineffective in regulating the K⁺ conductance at hyperpolarization. External Ca²⁺ blocked currents weakly in a voltage-dependent manner. From instantaneous current-voltage curves, we identified a binding site in the channel pore with an electrical distance of about 0.2 to 0.5. Lanthanum ions reduced the inward current in a voltage-dependent manner and simultaneously displaced the voltage at which half of the channels are in the open state to more positive values. This finding was interpreted as resulting from a sum of two molecular effects, an interaction with the mouth of the channel that causes a reduction of current, and a binding to the voltage sensor, leading to a shielding of surface charges and, subsequently, a modulation of channel gating.A comparison between the K⁺ inward rectifier in xylem parenchyma cells, guard cells and KAT1 from Arabidopsis leads to the conclusion that these rectifiers form subtypes within one class of ion channels. The ineffectiveness of Ca²⁺ to control K⁺ influx in xylem parenchyma cells is interpreted in physiological terms.     

Paramecium Na+ channels activated by Ca2+-calmodulin: Calmodulin is the Ca2+ sensor in the channel gating mechanism

Paramecium Na⁺ channels, which were Ca²⁺-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10^–5 to 10^–3 m Ca²⁺ in the bath. They became much less active in the presence of 10^–6 m Ca²⁺, and their activity subsided completely at 10^–8 m Ca²⁺. A Hill plot showed a dissociation constant of 6 m for Ca²⁺ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mm Mg²⁺. The channels also exhibited a mild voltage dependence. When exposed to 10^–8 m Ca²⁺ for an extended period of 2–4 min, channels were further inactivated even after bath Ca²⁺ was restored to 10^–4 m. Whereas neither high voltage (+100 mV) nor high Ca²⁺ (10^–3 m) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca²⁺ to the bath restored channel activity without a requirement of additional Mg²⁺ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca²⁺ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na⁺ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na⁺ channel activation, and is the Ca²⁺ sensor of the Na⁺-channel gating machinery.We thank C. Kung for his kind support, and A. Boileau for critical reading. Supported by grants from National Institutes of Health GM 22714-20 and 36386-09.