Salt Adaptation of K+ Channels in the Plasma Membrane of Tobacco Cells in Suspension Culture

The patch-clamp technique was used to study and compare thecharacteristics of cation channels in the plasma membrane ofcultured lines of tobacco (Nicotiana tabacum L. cv. Bright Yellow-2)cells that were unadapted (NaCl-unadapted cells) and adaptedto 50 and 100 mM NaCl (Na50-adapted and Na100-adapted cells).In these three types of tobacco cell, the outward whole-cellcurrent activated by depolarization was dominated mainly bythe activity of the outward rectifying K⁺ channels with a single-channelconductance of 20 pS. The steady-state amplitude of the outwardwhole-cell currents at all the positive potentials examineddecreased in the following order: NaCl-unadapted cells>Na50-adaptedcells>Na100-adapted cells. There were no significant differencesbetween the NaCl-unadapted and the Na50-adapted cells in termsof the ratio of permeabilities of these channels to K⁺ and Na⁺ions. Furthermore, no significant differences in terms of thesingle-channel conductance of these channels were observed amongthe NaCl-unadapted, the Na50-adapted and the Na100-adapted cells.These observations suggest that adaptation to salinity of tobaccocells in suspension results in reduced permeability of the K⁺channels to both K⁺ and Na⁺ ions, without any change in theK⁺/Na⁺ selectivity and single-channel conductance of these channels. ¹Present address: Research Laboratory of Applied Biochemistry,Tanabe Seiyaku Co., Ltd.16-89 Kashima 3-chome, Yodogawaku, Osaka,532 Japan     

Small Inward Rectifying K+ Channels in Coleoptiles: Inhibition by External Ca2+ and Function in Cell Elongation

Plant growth requires a continuous supply of intracellular solutes in order to drive cell elongation. Ion fluxes through the plasma membrane provide a substantial portion of the required solutes. Here, patch clamp techniques have been used to investigate the electrical properties of the plasma membrane in protoplasts from the rapid growing tip of maize coleoptiles. Inward currents have been measured in the whole cell configuration from protoplasts of the outer epidermis and from the cortex. These currents are essentially mediated by K⁺ channels with a unitary conductance of about 12 pS. The activity of these channels was stimulated by negative membrane voltage and inhibited by extracellular Ca²⁺ and/or tetraethylammonium-CI (TEA). The kinetics of voltage- and Ca²⁺-gating of these channels have been determined experimentally in some detail (steady-state and relaxation kinetics). Various models have been tested for their ability to describe these experimental data in straightforward terms of mass action. As a first approach, the most appropriate model turned out to consist of an active state which can equilibrate with two inactive states via independent first order reactions: a fast inactivation/activation by Ca²⁺-binding and -release, respectively (rate constants >>10³ sec⁻¹) and a slower inactivation/activation by positive/negative voltage, respectively (voltage-dependent rate constants in the range of 10³ sec⁻¹). With 10 mm K⁺ and 1 mm Ca²⁺ in the external solution, intact coleoptile cells have a membrane voltage (V) of −105 ± 7 mV. At this V, the density and open probability of the inward-rectifying channels is sufficient to mediate K⁺ uptake required for cell elongation. Extracellular TEA or Ca²⁺, which inhibit the K⁺ inward conductance, also inhibit elongation of auxin-depleted coleoptile segments in acidic solution. The comparable effects of Ca²⁺ and TEA on both processes and the similar Ca²⁺ concentration required for half maximal inhibition of growth (4.3 mm Ca²⁺) and for conductance (1.2 mm Ca²⁺) suggest that K⁺ uptake through the inward rectifier provides essential amounts of solute for osmotic driven elongation of maize coleoptiles. Received: 6 June 1995/Revised: 12 September 1995     

Reduced Permeability to K+ and Na+ Ions of K+ Channels in the Plasma Membrane of Tobacco Cells in Suspension after Adaptation to 50 mM NaCl

The whole-cell patch-clamp technique was used to study and comparethe characteristics of K⁺-and Na⁺-transport processes acrossthe plasma membrane in two types of protoplast isolated fromNaCl-adapted and -unadapted cells of tobacco (Nicotiana tabacumL. cv. Bright Yellow-2) in suspension culture. In both typesof protoplast, with 100 mM KCl in the bathing solution and inthe pipette solution, depolarization of the plasma membranefrom the holding potential of 0 mV to a positive potential resultedin a relatively large outward current which increased with increasingpositive potential, whereas hyperpolarization to negative potentialsup to –100 mV resulted in only a small inward current.The outward current activated by depolarization was predominantlycarried by K⁺ ions through K⁺ channels. Na⁺ ions also had afinite ability to pass through these K⁺ channels. The outwardK⁺ and Na⁺ currents of the NaCl-adapted cells were considerablysmaller than those of the NaCl-unadapted cells. These resultssuggest that adaptation to salinity results in reduced permeabilityof the plasma membrane to both K⁺ and Na⁺ ions. ¹Present address: Research Laboratory of Applied Biochemistry,Tanabe Seiyaku Co., Ltd., 16-89, Kashima 3-chome, Yodogawa-ku,Osaka, 532 Japan     

Hyperpolarization-activated Ca2+-permeable Channels in the Plasma Membrane of Tomato Cells

The hyperpolarization of the electrical plasma membrane potential difference has been identified as an early response of plant cells to various signals including fungal elicitors. The hyperpolarization-activated influx of Ca²⁺ into tomato cells was examined by the application of conventional patch clamp techniques. In both whole cell and single-channel recordings, clamped membrane voltages more negative than −120 mV resulted in time- and voltage-dependent current activation. Single-channel currents saturated with increasing activities of Ca²⁺ and Ba²⁺ from 3 to 26 mm and the single channel conductance increased from 4 pS to 11 pS in the presence of 20 mm Ca²⁺ or Ba²⁺, respectively. These channels were 20–25 and 10–13 times more permeable to Ca²⁺ than to K⁺ and to Cl⁻, respectively. Channel currents were strongly inhibited by 10 μm lanthanum and 50% inhibited by 100 μm nifedipine. This evidence suggests that hyperpolarization-activated Ca²⁺-permeable channels provide a mechanism for the influx of Ca²⁺ into tomato cells. Received: 13 February 1996/Revised: 12 August 1996     

Role of Calcineurin-Mediated Dephosphorylation in Modulation of an Inwardly Rectifying K+ Channel in Human Proximal Tubule Cells

Activity of an inwardly rectifying K⁺ channel with inward conductance of about 40 pS in cultured human renal proximal tubule epithelial cells (RPTECs) is regulated at least in part by protein phosphorylation and dephosphorylation. In this study, we examined involvement of calcineurin (CaN), a Ca²⁺/calmodulin (CaM)–dependent phosphatase, in modulating K⁺ channel activity. In cell-attached mode of the patch-clamp technique, application of a CaN inhibitor, cyclosporin A (CsA, 5 μM) or FK520 (5 μM), significantly suppressed channel activity. Intracellular Ca²⁺ concentration ([Ca²⁺] _i ) estimated by fura-2 imaging was elevated by these inhibitors. Since inhibition of CaN attenuates some dephosphorylation with increase in [Ca²⁺] _i , we speculated that inhibiting CaN enhances Ca²⁺-dependent phosphorylation, which might result in channel suppression. To verify this hypothesis, we examined effects of inhibitors of PKC and Ca²⁺/CaM-dependent protein kinase-II (CaMKII) on CsA-induced channel suppression. Although the PKC inhibitor GF109203X (500 nM) did not influence the CsA-induced channel suppression, the CaMKII inhibitor KN62 (20 μM) prevented channel suppression, suggesting that the channel suppression resulted from CaMKII-dependent processes. Indeed, Western blot analysis showed that CsA increased phospho-CaMKII (Thr286), an activated CaMKII in inside–out patches, application of CaM (0.6 μM) and CaMKII (0.15 U/ml) to the bath at 10^?6 M Ca²⁺ significantly suppressed channel activity, which was reactivated by subsequent application of CaN (800 U/ml). These results suggest that CaN plays an important role in supporting K⁺ channel activity in RPTECs by preventing CaMKII-dependent phosphorylation.     

Characterization of the High-Affinity Verapamil Binding Site in a Plant Plasma Membrane Ca2+-selective Channel

Despite biochemical evidence for the existence of high-affinity phenylalkylamine receptors in higher plants, their effects on channel activity have only been demonstrated at relatively high concentrations. We have performed a quantitative single-channel analysis of the changes induced by extracellular verapamil in the rca channel [a wheat root plasma membrane Ca²⁺-selective channel (Pi?eros & Tester, 1995. Planta 195:478–488)]. Concentrations as low as 0.5 μm verapamil induced a blockade of the inward current, with no evident reduction of the single-channel current amplitude. Blockade by verapamil was concentration and voltage dependent. Preliminary analysis suggested the blockade was due to a reduction in the maximum open state probability rather than a change in V_0.5. Further analysis of the association and dissociation rate constants revealed a binding site located 56 to 59% down the voltage drop from the extracellular face of the channel, with a K _d (0) of 24 to 26 μm. This results in a K _d at −100 mV of 2 μm. Methoxyverapamil had qualitatively the same effects. This intra-pore binding site can be accessed directly from the extracellular side of the rca channel, but apparently not from the cytosolic side. Received: 15 August 1996/Revised: 23 December 1996     

Role of the Plasma Membrane H+-ATPase in K+ Transport

The role of the plant plasma membrane H+-ATPase in K+ uptake was examined using red beet (Beta vulgaris L.) plasma membrane vesicles and a partially purified preparation of the red beet plasma membrane H+-ATPase reconstituted in proteoliposomes and planar bilayers. For plasma membrane vesicles, ATP-dependent K+ efflux was only partially inhibited by 100 [mu]M vanadate or 10 [mu]M carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. However, full inhibition of ATP-dependent K+ efflux by these reagents occurred when the red beet plasma membrane H+-ATPase was partially purified and reconstituted in proteoliposomes. When reconstituted in a planar bilayer membrane, the current/voltage relationship for the plasma membrane H+-ATPase showed little effect of K+ gradients imposed across the bilayer membrane. When taken together, the results of this study demonstrate that the plant plasma membrane H+-ATPase does not mediate direct K+ transport chemically linked to ATP hydrolysis. Rather, this enzyme provides a driving force for cellular K+ uptake by secondary mechanisms, such as K+ channels or H+/K+ symporters. Although the presence of a small, protonophore-insensitive component of ATP-dependent K+ transport in a plasma membrane fraction might be mediated by an ATP-activated K+ channel, the possibility of direct K+ transport by other ATPases (i.e. K+-ATPases) associated with either the plasma membrane or other cellular membranes cannot be ruled out.     

Homer2 Protein Regulates Plasma Membrane Ca2+-ATPase-mediated Ca2+ Signaling in Mouse Parotid Gland Acinar Cells

Homer proteins are scaffold molecules with a domain structure consisting of an N-terminal Ena/VASP homology 1 protein-binding domain and a C-terminal leucine zipper/coiled-coil domain. The Ena/VASP homology 1 domain recognizes proline-rich motifs and binds multiple Ca²⁺-signaling proteins, including G protein-coupled receptors, inositol 1,4,5-triphosphate receptors, ryanodine receptors, and transient receptor potential channels. However, their role in Ca²⁺ signaling in nonexcitable cells is not well understood. In this study, we investigated the role of Homer2 on Ca²⁺ signaling in parotid gland acinar cells using Homer2-deficient (Homer2^−/−) mice. Homer2 is localized at the apical pole in acinar cells. Deletion of Homer2 did not affect inositol 1,4,5-triphosphate receptor localization or channel activity and did not affect the expression and activity of sarco/endoplasmic reticulum Ca²⁺-ATPase pumps. In contrast, Homer2 deletion markedly increased expression of plasma membrane Ca²⁺-ATPase (PMCA) pumps, in particular PMCA4, at the apical pole. Accordingly, Homer2 deficiency increased Ca²⁺ extrusion by acinar cells. These findings were supported by co-immunoprecipitation of Homer2 and PMCA in wild-type parotid cells and transfected human embryonic kidney 293 (HEK293) cells. We identified a Homer-binding PPXXF-like motif in the N terminus of PMCA that is required for interaction with Homer2. Mutation of the PPXXF-like motif did not affect the interaction of PMCA with Homer1 but inhibited its interaction with Homer2 and increased Ca²⁺ clearance by PMCA. These findings reveal an important regulation of PMCA by Homer2 that has a central role on PMCA-mediated Ca²⁺ signaling in parotid acinar cells.     

Induction of Ca2+-Activated K+ Current and Transient Outward Currents in Human Capillary Endothelial Cells

Human capillary endothelial cells (HCEC) in normal media contain noninactivating outwardly rectifying chloride currents, TEA-sensitive delayed rectifier K⁺ currents and an inward rectifier K⁺ current. Two additional ionic currents are induced in HCEC when the media are allowed to become conditioned: A Ca²⁺-activated K⁺ current (BKCA) that is sensitive to iberiotoxin is induced in 23.5% of the cells, a transient 4-AP-sensitive K⁺ current (A current) is induced in 24.7% of the cells, and in 22.3% of the cells both the transient and BKCA currents are coinduced. The EC₅₀ for Ca²⁺ activation of the BKCA current in HCEC from conditioned media is 213 nM. RNA message for BKCA (hSlo clone) is undetecable after PCR amplification in control cells but is seen in those from conditioned cells. The induction of BKCA current is not blocked by conditioning with inhibitors of nitric oxide synthase, cyclo-oxgenase or lypo-oxygenase pathways. Apparently the characteristics of human endothelial cells are highly malleable and can be easily modified by their local environment. Received: 21 May 1998/Revised: 23 September 1998     

The Plasma Membrane Ca2+ ATPase and the Plasma Membrane Sodium Calcium Exchanger Cooperate in the Regulation of Cell Calcium

Calcium is an ambivalent signal: it is essential for the correct functioning of cell life, but may also become dangerous to it. The plasma membrane Ca²⁺ ATPase (PMCA) and the plasma membrane Na⁺/Ca²⁺ exchanger (NCX) are the two mechanisms responsible for Ca²⁺ extrusion. The NCX has low Ca²⁺ affinity but high capacity for Ca²⁺ transport, whereas the PMCA has a high Ca²⁺ affinity but low transport capacity for it. Thus, traditionally, the PMCA pump has been attributed a housekeeping role in maintaining cytosolic Ca²⁺, and the NCX the dynamic role of counteracting large cytosolic Ca²⁺ variations (especially in excitable cells). This view of the roles of the two Ca²⁺ extrusion systems has been recently revised, as the specific functional properties of the numerous PMCA isoforms and splicing variants suggests that they may have evolved to cover both the basal Ca²⁺ regulation (in the 100 nM range) and the Ca²⁺ transients generated by cell stimulation (in the μM range).Ca²⁺ controls critical cellular responses in all eukaryotic organisms. It controls both short-term biological processes that occur in milliseconds, such as muscle contraction, as well as long-term processes that require longer times, such as cell proliferation and organ development. The specificity of cellular Ca²⁺ signals is controlled by a sophisticated “toolkit” comprising numerous ion channels, pumps, and exchangers that drive the fluxes of Ca²⁺ ions across the plasma membrane and across the membranes of intracellular organelles (Berridge et al. 2003).The plasma membrane contains several types of channels that mediate Ca²⁺ entry from the extracellular ambient, and two systems for Ca²⁺ extrusion: a low affinity, high capacity Na⁺/Ca²⁺ exchanger (NCX), and a high-affinity, low-capacity Ca²⁺-ATPase (the plasma membrane Ca²⁺ pump (PMCA)) (Fig. 1). The type of channels and the relative proportions of NCX and PMCA vary with the cell type, the NCX being particularly abundant in excitable tissues, e.g., heart and brain. The regulated opening of the Ca²⁺ channels by either voltage gating, interaction with ligands or the emptying of intracellular stores, allows a limited amount of Ca²⁺ to enter the cell to transmit signals to its designated targets. Thereafter, the Ca²⁺ transients must be dissipated: its extrusion from the cell is mediated by the NCX and the PMCA pump, but Ca²⁺ is also restored to basal levels by sequestration in the endo/sarcoplasmic reticulum via the SERCA pump and in the mitochondria by the electrophoretic uniporter. The NCX has also been found at the inner membrane of the nuclear envelope (NE) and has been proposed to mediate Ca²⁺ flux between the nucleoplasm and the NE (Xie et al. 2002), and then to the ER (Wu et al. 2009) in neuronal and certain other cell types. Ca²⁺ binding proteins also contributed to Ca²⁺ buffering: In this review, we will not cover them, as we will only discuss the systems that extrude Ca²⁺ out of the cell.Open in a separate windowFigure 1.A schematic representation of the structures involved in cellular Ca²⁺ homeostasis. The model shows a cell with its Ca²⁺-transporting systems: Ca²⁺-ATPases (plasma membrane and sarco/endoplasmic reticulum, PMCA and SERCA), plasma membrane (PM) Ca²⁺ channels, Na⁺/Ca²⁺ exchangers (NCX and NCLX), 1,4,5-triphosphate receptor (IP₃R) and ryanodine receptor (RyR), the electrophoretic mitochondrial uptake uniporter (U). Mitochondria are drawn as yellow ellipses, nucleus as orange circle and endoplasmic reticulum is colored in red. The different Ca²⁺-transporting systems cooperate to maintain the Ca²⁺ concentration gradient between the extracellular and the intracellular ambient.The PMCA pump is a minor component of the total protein of the plasma membrane (less than 0.1% of it). Quantitatively, it is overshadowed by the more powerful NCX in excitable tissue like heart; however, even cells in which the NCX predominates, the PMCA pump is likely to be the fine tuner of cytosolic Ca²⁺, as it can operate in a concentration range in which the low affinity NCX is relatively very inefficient.The PMCA was discovered in erythrocytes (Schatzmann 1966), and was then described and characterized in numerous other cell types. It was purified in 1979 using a calmodulin affinity column (Niggli et al. 1979), and cloned about 10 years later (Shull and Greeb 1988; Verma et al. 1988). It shows the same essential membrane topology properties of the SERCA pump. Molecular modeling work using the structure of the SERCA pump as a template (Toyoshima et al. 2000) predicts the same general features of the latter, with 10 transmembrane domains and the large cytosolic headpiece divided into the three main cytosolic A, N, and P domains. The Na⁺/Ca²⁺ cotransport process was discovered at about the same time as PMCA by two independent groups working on heart (Reuter and Seitz 1968) and on the squid giant axon (Baker et al. 1969). The exchanger was cloned in 1990 (Nicoll et al. 1990). The sequence was initially predicted to correspond to a protein with 11 transmembrane domains and one large cytosolic loop linking transmembrane domain five and six but a revised model predicting only nine transmembrane domains is now generally accepted.     

Inward Rectifying K Channels in the Plasma Membrane of Arabidopsis thaliana

Ion channels in the plasma membrane of protoplasts isolated from cultured cells of Arabidopsis thaliana were studied by means of the patch-clamp technique applied in the whole-cell configuration. In some protoplasts, depolarizing pulses and, in other protoplasts, hyperpolarizing pulses elicited time-dependent currents; both kinds of current were only rarely observed in the same protoplast. The hyperpolarization-activated inward rectifying currents, the focus of this paper, appeared to be due to the relatively slow opening of channels (activation time constant = 150 to 300 milliseconds), which closed at positive potentials. The reversal potential of this current, measured in the presence of different ion concentrations (symmetrical or asymmetrical K⁺ and Cl⁻ or gluconate), was always close to the electrochemical equilibrium potential of K⁺. The currents were inhibited by 10 millimolar tetraethylammonium, a K⁺ channel blocker. These data show that the hyperpolarization-activated currents flow through K⁺ channels, which can provide a pathway for the passive diffusion of K⁺ down its electrochemical gradient.     

Modulation of the Plasma Membrane Ca2+ Pump

The plasma membrane calcium pump, which ejects Ca²⁺ from the cell, is regulated by calmodulin. In the absence of calmodulin, the pump is relatively inactive; binding of calmodulin to a specific domain stimulates its activity. Phosphorylation of the pump with protein kinase C or A may modify this regulation. Most of the regulatory functions of the enzyme are concentrated in a region at the carboxyl terminus. This region varies substantially between different isoforms of the pump, causing substantial differences in regulatory properties. The pump shares some motifs of the carboxyl terminus with otherwise unrelated proteins: The calmodulin-binding domain is a modified IQ motif (a motif which is present in myosins) and the last 3 residues of isoform 4b are a PDZ target domain. The pump is ubiquitous, with isoforms 1 and 4 of the pump being more widely distributed than 2 and 3. In some kinds of cells isoform 1 or 4 is missing, and is replaced by another isoform. Received: 26 January 1998/Revised: 6 April 1998     

The Plasma Membrane Ca2+-Pump of Plant Cells: A Radiation Inactivation Study

The functional molecular weight of the plasma membrane Ca²⁺-ATPase of radish (Raphanus sativus L.) seeds was determined by measuring the Ca²⁺-dependent ATPase activity and the MgATP-dependent Ca²⁺ transport activity of membrane samples irradiated, in the lyophilized state, with γ rays from [⁶⁰Co] source. The results gave a target size of about 270,000 dalton for both the measured activities, thus confirming (i) that both activities are catalyzed by the same enzyme and (ii) the similarity between the plasma membrane Ca²⁺-ATPase of higher plants and that of the erythrocytes.     

Role of the Plasma Membrane Ca2+-ATPase Pump in the Regulation of Rhythm Generation by an Interstitial Cell of Cajal: A Computational Study

Neurophysiology - Gastrointestinal motility is based on the rhythmic activity of interstitial cells of Cajal (ICCs). The ICC rhythm generation relies upon characteristic Ca2+-handling mechanisms...     

Regulation of Ca2+-activated K+ Channel from Rabbit Distal Colon Epithelium by Phosphorylation and Dephosphorylation

The Ca²⁺-activated maxi K⁺ channel is predominant in the basolateral membrane of the surface cells in the distal colon. It may play a role in the regulation of the aldosterone-stimulated Na⁺ reabsorption from the intestinal lumen. Previous measurements of these basolateral K⁺ channels in planar lipid bilayers and in plasma membrane vesicles have shown a very high sensitivity to Ca²⁺ with a K _0.5 ranging from 20 nm to 300 nm, whereas other studies have a much lower sensitivity to Ca²⁺. To investigate whether this difference could be due to modulation by second messenger systems, the effect of phosphorylation and dephosphorylation was examined. After addition of phosphatase, the K⁺ channels lost their high sensitivity to Ca²⁺, yet they could still be activated by high concentrations of Ca²⁺ (10 μm). Furthermore, the high sensitivity to Ca²⁺ could be restored after phosphorylation catalyzed by a cAMP dependent protein kinase. There was no effect of addition of protein kinase C. In agreement with the involvement of enzymatic processes, lag periods of 30–120 sec for dephosphorylation and of 10–280 sec for phosphorylation were observed. The phosphorylation state of the channel did not influence the single channel conductance. The results demonstrate that the high sensitivity to Ca²⁺ of the maxi K⁺ channel from rabbit distal colon is a property of the phosphorylated form of the channel protein, and that the difference in Ca²⁺ sensitivity between the dephosphorylated and phosphorylated forms of the channel protein is more than one order of magnitude. The variety in Ca²⁺ sensitivities for maxi K⁺ channels from tissue to tissue and from different studies on the same tissue could be due to modification by second messenger systems. Received: 28 February 1995/Revised: 22 December 1995     

Redox Regulation of Large Conductance Ca2+-activated K+ Channels in Smooth Muscle Cells

The effects of sulfhydryl reduction/oxidation on the gating of large-conductance, Ca²⁺-activated K⁺ (maxi-K) channels were examined in excised patches from tracheal myocytes. Channel activity was modified by sulfhydryl redox agents applied to the cytosolic surface, but not the extracellular surface, of membrane patches. Sulfhydryl reducing agents dithiothreitol, β-mercaptoethanol, and GSH augmented, whereas sulfhydryl oxidizing agents diamide, thimerosal, and 2,2′-dithiodipyridine inhibited, channel activity in a concentration-dependent manner. Channel stimulation by reduction and inhibition by oxidation persisted following washout of the compounds, but the effects of reduction were reversed by subsequent oxidation, and vice versa. The thiol-specific reagents N-ethylmaleimide and (2-aminoethyl)methanethiosulfonate inhibited channel activity and prevented the effect of subsequent sulfhydryl oxidation. Measurements of macroscopic currents in inside-out patches indicate that reduction only shifted the voltage/nP_o relationship without an effect on the maximum conductance of the patch, suggesting that the increase in nP_o following reduction did not result from recruitment of more functional channels but rather from changes of channel gating. We conclude that redox modulation of cysteine thiol groups, which probably involves thiol/disulfide exchange, alters maxi-K channel gating, and that this modulation likely affects channel activity under physiological conditions.