Inhibition of the Uptake and Long-Distance Transport of Calcium by Aluminium and Other Polyvalent Cations

Aluminium, scandium, and iron inhibit the uptake of calciumby week-old barley plants from acid culture solutions (pH 4.0–4.2).The inhibition by scandium can be detected when its ratio tocalcium is 1:1000. The onset of the inhibition may be quit rapidand will persist for at least. 24 h in the absence of the polyvalentcation. The inhibition caused by 25 and 50 µM aiuminiumsulphate may be overcome if the calicum chloride concentrationin the medium is raised to 15mM, but in this situation aluminiumstill inhibits root growth by more than 50 per cent. Elutionexperiments show that polyvalent cations reduce the amount ofcalcium held in the water free space (WFS) and the Donnan freespace (DFS) but increase both the exchangeable and absorbedchloide content of the root. Aluminium-treated roots transportedmuch less calcium to the shoot system than untreated plants.Autoradiographs showed that this difference was reflected ina greatly reduced labelled-calcium concentration over the tissuesof the stele. By contrast the non-exchangeable fraction of labelledcalicum in the cortex was similar in both treatments. Autoradiographsof ⁴⁶Sc showed that it was restricted to the epidermis and outerrank of cortical cells from whence it controls calicum movementthroughout the root. A theory to account for this control isoutlined.     

Membrane Adenosine Triphosphatase of Escherichia coli: Activation by Calcium Ion and Inhibition by Monovalent Cations

Membrane ghost preparations of Escherichia coli K-12 obtained by osmotic lysis of lysozyme-induced spheroplasts were found to possess both Mg(++)- and Ca(++)-activated adenosine 5'-triphosphatase (ATPase, EC 3.6.1.3) activities. Maximal activities of 1.0 to 1.5 mumoles of orthophosphate released per min per mg of protein were obtained at pH 9.0 with a molar Mg(++) to adenosine 5'triphosphate (ATP) ratio of 2:5 and at pH 9.9 with a molar Ca(++) to ATP ratio of 1:5. These ATPase activities were not altered by ouabain, fluoride, N-ethylmaleimide, 2,4-dinitrophenol, cyanide, or dithionite, but were inhibited by low concentrations of azide, p-chloromercuribenzoate, and pentachlorophenol. Mg(++) ATPase was more susceptible to inhibition by azide than was Ca(++) ATPase. Fifty per cent inactivation of both activities was observed when membrane ghost preparations were preincubated at 66 C for 10 min. The Mg(++) and Ca(++) ATPase activities of these preparations were not additive, but did respond independently to inhibition by monovalent cations. Ca(++) ATPase was found to be very sensitive to inhibition by K(+), Na(+), Li(+), Rb(+), and Cs(+); Mg(++) ATPase was relatively insensitive to these ions. One possible interpretation of the results presented in this paper is that the membrane of E. coli possesses an ATPase which is activated by either Mg(++) or Ca(++) and that activation by Ca(++) increases the susceptibility of this enzyme to inhibition by monovalent cations. Increased susceptibility of E. coli membrane ATPase to inhibition by monovalent cations such as Na(+) and K(+) as a consequence of Ca(++) activation could represent a regulatory mechanism.     

Inhibition of Cav3.2 T-type Calcium Channels by Its Intracellular I-II Loop

Voltage-dependent calcium channels (Cav) of the T-type family (Cav3.1, Cav3.2, and Cav3.3) are activated by low threshold membrane depolarization and contribute greatly to neuronal network excitability. Enhanced T-type channel activity, especially Cav3.2, contributes to disease states, including absence epilepsy. Interestingly, the intracellular loop connecting domains I and II (I-II loop) of Cav3.2 channels is implicated in the control of both surface expression and channel gating, indicating that this I-II loop plays an important regulatory role in T-type current. Here we describe that co-expression of this I-II loop or its proximal region (Δ1-Cav3.2; Ser⁴²³–Pro⁵⁴²) together with recombinant full-length Cav3.2 channel inhibited T-type current without affecting channel expression and membrane incorporation. Similar T-type current inhibition was obtained in NG 108-15 neuroblastoma cells that constitutively express Cav3.2 channels. Of interest, Δ1-Cav3.2 inhibited both Cav3.2 and Cav3.1 but not Cav3.3 currents. Efficacy of Δ1-Cav3.2 to inhibit native T-type channels was assessed in thalamic neurons using viral transduction. We describe that T-type current was significantly inhibited in the ventrobasal neurons that express Cav3.1, whereas in nucleus reticularis thalami neurons that express Cav3.2 and Cav3.3 channels, only the fast inactivating T-type current (Cav3.2 component) was significantly inhibited. Altogether, these data describe a new strategy to differentially inhibit Cav3 isoforms of the T-type calcium channels.     

Effect of Alkali Metal Cations on Slow Inactivation of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. The kinetics of decaying outward Na⁺ current in response to 1-s depolarizations in the F1485Q mutant depends on the predominant cation in the extracellular solution, suggesting an effect on slow inactivation. The decay rate is lower for the alkali metal cations Li⁺, Na⁺, K⁺, Rb⁺, and Cs⁺ than for the organic cations Tris, tetramethylammonium, N-methylglucamine, and choline. In whole cell recordings, raising [Na⁺]_o from 10 to 150 mM increases the rate of recovery from slow inactivation at −140 mV, decreases the rate of slow inactivation at relatively depolarized voltages, and shifts steady-state slow inactivation in a depolarized direction. Single channel recordings of F1485Q show a decrease in the number of blank (i.e., null) records when [Na⁺]_o is increased. Significant clustering of blank records when depolarizing at a frequency of 0.5 Hz suggests that periods of inactivity represent the sojourn of a channel in a slow-inactivated state. Examination of the single channel kinetics at +60 mV during 90-ms depolarizations shows that neither open time, closed time, nor first latency is significantly affected by [Na⁺]_o. However raising [Na⁺]_o decreases the duration of the last closed interval terminated by the end of the depolarization, leading to an increased number of openings at the depolarized voltage. Analysis of single channel data indicates that at a depolarized voltage a single rate constant for entry into a slow-inactivated state is reduced in high [Na⁺]_o, suggesting that the binding of an alkali metal cation, perhaps in the ion-conducting pore, inhibits the closing of the slow inactivation gate.     

Cyclic AMP-Independent Inhibition of Voltage-Sensitive Calcium Channels by Forskolin in PC12 Cells

Abstract: Forskolin has been used to stimulate adenylyl cyclase. However, we found that forskolin inhibited voltage-sensitive Ca²⁺ channels (VSCCs) in a cyclic AMP (cAMP)-independent manner in PC12 cells. Ca²⁺ influx induced by membrane depolarization with 70 m M K⁺ was inhibited when cells were preincubated with 10 µ M forskolin. Almost maximum inhibitory effect on Ca²⁺ influx without any significant increase in cellular cAMP level was observed in PC12 cells exposed to forskolin for 1 min. In addition, the forskolin effect on Ca²⁺ influx was not affected by the presence of 2',5'-dideoxyadenosine, an inhibitor of adenylyl cyclase that reduces dramatically forskolin-induced cAMP production. 1,9-Dideoxyforskolin, an inactive analogue of forskolin, also inhibited ∼80% of Ca²⁺ influx induced by 70 m M K⁺ without any increase in cAMP. The data suggest that forskolin and its analogue inhibit VSCCs in PC12 cells and that the inhibition is independent of cAMP generation.     

Nonspecific, Reversible Inhibition of Voltage-Gated Calcium Channels by CaMKII Inhibitor CK59

Investigation of kinase-related processes often uses pharmacological inhibition to reveal pathways in which kinases are involved. However, one concern about using such kinase inhibitors is their potential lack of specificity. Here, we report that the calcium–calmodulin-dependent kinase II (CaMKII) inhibitor CK59 inhibited multiple voltage-gated calcium channels, including the L-type channel during depolarization in a dose-dependent manner. The use of another CaMKII inhibitor, cell-permeable autocamtide-2 related inhibitory peptide II (Ant-AIP-II), failed to similarly decrease calcium current or entry in hippocampal cultures, as shown by ratiometric calcium imaging and whole-cell patch clamp electrophysiology. Notably, inhibition due to CK59 was reversible; washout of the drug brought calcium levels back to control values upon depolarization. Furthermore, the IC₅₀ for CK59 was approximately 50 μM, which is only fivefold larger than the reported IC₅₀ values for CaMKII inhibition. Similar nonspecific actions of other CaMKII inhibitors KN93 and KN62 have previously been reported. In the case of all three kinase inhibitors, the IC₅₀ for calcium current inhibition falls near that of CaMKII inhibition. Our findings demonstrate that CK59 attenuates activity of voltage-gated calcium channels, and thus provide more evidence for caution when relying on pharmacological inhibition to examine kinase-dependent phenomena.     

Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells

L-type calcium currents (I_Ca) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I_Ca and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca²⁺ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from ∼75%–80% to ∼50% by omitting β subunits but unaffected by omitting α₂δ subunits. Similarly, gluconate inhibition was reduced to ∼50% by deleting an α1 subunit N-terminal region of 15 residues critical for β subunit interactions regulating open probability. Omitting β subunits with this mutant α1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different β subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from ∼75%–80% to ∼50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to ∼60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ∼25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving β subunit interactions with the N terminus and a short C terminal region.     

Activation of Calcineurin by the Trivalent Metal Terbium

As a possible probe for metal activation of calcineurin, Tb³⁺ was tested for effects on calcineurin activity. Calcineurin was activated by Tb³⁺ with the following kinetic parameters estimated: k _cat = 0.78 ± 0.02 sec^–1, K _m(pNPP) = 32.6 ± 1.8 mM, and K _act(Tb³⁺) = 0.08 ± 0.03 mM. Terbium luminescence was demonstrated in the presence of the heterodimer of calcineurin and exploited to localize the binding of exogenous metal to the enzyme active site. Exogenous Mn²⁺ reduced luminescence, although the affinity of calcineurin for Tb³⁺ seemed to be greater. Putative active-site ligands, such as para-nitrophenol and a synthetic peptide from the autoinhibitory region, reduced the luminescence of terbium. Collectively, these data suggested that Tb³⁺ was binding directly at the active site of calcineurin, with the corollary that exogenous activating metal (Mn²⁺) binds at the active site of the enzyme. These data support the hypothesis that activating, exogenous divalent metal participates directly in catalysis.     

The Effect of Aluminium and some other Trivalent Metal Cations on Cell Division in the Root Apices of Allium cepa

The morphological abnormalities of root systems treated withaluminium salts are such that they may be explained by an inhibitoryeffect of aluminium on either cell division or cell extension.Elongation of onion roots was completely inhibited after 6–8hours' treatment with 10^–3and 10^–4M aluminium sulphatesolutions. Examination of aceto-carmine squashes of root apicesshowed that the cessation of root elongation was closely correlatedwith the disappearance of mitotic figures. The time taken forcomplete inhibition of cell division and root elongation wasdependent on the ambient temperature. Abnormalities of the mitoticapparatus were not seen. Treatment of onion roots with othertrivalent metals, gallium, indium, and lanthanum, produced similarresults. It is concluded that some mechanism associated with cell divisionis highly sensitive to aluminium and is permanently damagedby short exposures. The results are not satisfactorily explainedby the well-known effect of aluminium on phosphorus uptake.     

Magnesium Inhibition of Ryanodine-Receptor Calcium Channels: Evidence for Two Independent Mechanisms

The gating of ryanodine receptor calcium release channels (RyRs) depends on myoplasmic Ca²⁺ and Mg²⁺ concentrations. RyRs from skeletal and cardiac muscle are activated by μm Ca²⁺ and inhibited by mm Ca²⁺ and Mg²⁺. ⁴⁵Ca²⁺ release from skeletal SR vesicles suggests two mechanisms for Mg²⁺-inhibition (Meissner, Darling & Eveleth, 1986, Biochemistry 25:236–244). The present study investigates the nature of these mechanisms using measurements of single-channel activity from cardiac- and skeletal RyRs incorporated into planar lipid bilayers. Our measurements of Mg²⁺- and Ca²⁺-dependent gating kinetics confirm that there are two mechanisms for Mg²⁺ inhibition (Type I and II inhibition) in skeletal and cardiac RyRs. The mechanisms operate concurrently, are independent and are associated with different parts of the channel protein. Mg²⁺ reduces P _o by competing with Ca²⁺ for the activation site (Type-I) or binding to more than one, and probably two low affinity inhibition sites which do not discriminate between Ca²⁺ and Mg²⁺ (Type-II). The relative contributions of the two inhibition mechanisms to the total Mg²⁺ effect depend on cytoplasmic [Ca²⁺] in such a way that Mg²⁺ inhibition has the properties of Types-I and II inhibition at low and high [Ca²⁺] respectively. Both mechanisms are equally important when [Ca²⁺] = 10 μm in cardiac RyRs or 1 μm in skeletal RyRs. We show that Type-I inhibition is not the sole mechanism responsible for Mg²⁺ inhibition, as is often assumed, and we discuss the physiological implications of this finding. Received: 1 January 1996/Revised: 14 November 1996     

Activation and Inhibition of Histone Deacetylase 8 by Monovalent Cations

The metal-dependent histone deacetylases (HDACs) catalyze hydrolysis of acetyl groups from acetyllysine side chains and are targets of cancer therapeutics. Two bound monovalent cations (MVCs) of unknown function have been previously observed in crystal structures of HDAC8; site 1 is near the active site, whereas site 2 is located >20 Å from the catalytic metal ion. Here we demonstrate that one bound MVC activates catalytic activity (K_1/2 = 3.4 mm for K⁺), whereas the second, weaker-binding MVC (K_1/2 = 26 mm for K⁺) decreases catalytic activity by 11-fold. The weaker binding MVC also enhances the affinity of the HDAC inhibitor suberoylanilide hydroxamic acid by 5-fold. The site 1 MVC is coordinated by the side chain of Asp-176 that also forms a hydrogen bond with His-142, one of two histidines important for catalytic activity. The D176A and H142A mutants each increase the K_1/2 for potassium inhibition by ≥40-fold, demonstrating that the inhibitory cation binds to site 1. Furthermore, the MVC inhibition is mediated by His-142, suggesting that this residue is protonated for maximal HDAC8 activity. Therefore, His-142 functions either as an electrostatic catalyst or a general acid. The activating MVC binds in the distal site and causes a time-dependent increase in activity, suggesting that the site 2 MVC stabilizes an active conformation of the enzyme. Sodium binds more weakly to both sites and activates HDAC8 to a lesser extent than potassium. Therefore, it is likely that potassium is the predominant MVC bound to HDAC8 in vivo.     

Regulation of Sodium and Calcium Channels by Signaling Complexes

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.     

Inhibition of Cancer Cell Growth by Exposure to a Specific Time-Varying Electromagnetic Field Involves T-Type Calcium Channels

Electromagnetic field (EMF) exposures affect many biological systems. The reproducibility of these effects is related to the intensity, duration, frequency, and pattern of the EMF. We have shown that exposure to a specific time-varying EMF can inhibit the growth of malignant cells. Thomas-EMF is a low-intensity, frequency-modulated (25-6 Hz) EMF pattern. Daily, 1 h, exposures to Thomas-EMF inhibited the growth of malignant cell lines including B16-BL6, MDA-MB-231, MCF-7, and HeLa cells but did not affect the growth of non-malignant cells. Thomas-EMF also inhibited B16-BL6 cell proliferation in vivo. B16-BL6 cells implanted in syngeneic C57b mice and exposed daily to Thomas-EMF produced smaller tumours than in sham-treated controls. In vitro studies showed that exposure of malignant cells to Thomas-EMF for > 15 min promoted Ca²⁺ influx which could be blocked by inhibitors of voltage-gated T-type Ca²⁺ channels. Blocking Ca²⁺ uptake also blocked Thomas-EMF-dependent inhibition of cell proliferation. Exposure to Thomas-EMF delayed cell cycle progression and altered cyclin expression consistent with the decrease in cell proliferation. Non-malignant cells did not show any EMF-dependent changes in Ca²⁺ influx or cell growth. These data confirm that exposure to a specific EMF pattern can affect cellular processes and that exposure to Thomas-EMF may provide a potential anti-cancer therapy.     

Inhibition of Vacuolar Ion Channels by Polyamines

In this work, direct effects of cytosolic polyamines on the two principle vacuolar ion channels were studied by means of patch-clamp technique. Fast and slow activating vacuolar channels were analyzed on membrane patches isolated from vacuoles of the red beet taproot. The potency of the fast and of the slow vacuolar channel blockage by polyamines decreased with a decrease of the polycation charge, spermine⁴⁺ > spermidine³⁺ > putrescine²⁺. In contrast to the inhibition of the fast vacuolar channel, the blockage of the slow vacuolar channel by polyamines displayed a pronounced voltage-dependence. Hence, in the presence of high concentration of polyamines the slow vacuolar channel was converted into a strong inward rectifier as evidenced by its unitary current-voltage characteristic. The blockage of the slow vacuolar channel by polyamines was relieved at a large depolarization, in line with the permeation of polyamines through this channel. The voltage-dependence of blockage was analyzed in terms of the conventional model, assuming a single binding site for polyamines within the channel pore. Taking advantage of a simple linear structure of naturally occurring polyamines, conclusions on a possible architecture of the slow vacuolar channel pore were drawn. The role of common polyamines in regulation of vacuolar ion transport was discussed. Received: 1 May 1998/Revised: 25 September 1998     

MIC Channels Are Inhibited by Internal Divalent Cations but Not ATP

TRPM7 channels are nonselective cation channels that possess a functional α-kinase domain. It has been proposed that heterologously expressed TRPM7 channels are activated (Runnels et al., 2001) or inhibited (Nadler et al., 2001) by dialyzing the cell with millimolar levels of ATP. The endogenous correlate of TRPM7 has been identified in T-lymphocytes and RBL (rat basophilic leukemia) cells and named MagNuM (for Mg²⁺-nucleotide-inhibited metal) or MIC (for Mg²⁺-inhibited cation). Here, we report that internal Mg²⁺ rather than MgATP inhibits this current. Cytoplasmic MgATP, supplied by dialysis at millimolar concentrations, effectively inhibits only when a weak Mg²⁺ chelator is present in the pipette solution. Thus, MgATP acts as a source of Mg²⁺ rather than a source of ATP. Using an externally accessible site within the pore of the MIC channel itself as a bioassay, we show that equimolar MgCl₂ and MgATP solutions contain similar amounts of free Mg²⁺, explaining the fact that numeric values of Mg²⁺ and MgATP concentrations necessary for complete inhibition are the same. Furthermore, we demonstrate that Mg²⁺ is not unique in its inhibitory action, as Ba²⁺, Sr²⁺, Zn²⁺, and Mn²⁺ can substitute for Mg²⁺, causing complete inhibition. We conclude that MIC current inhibition occurs simply by divalent cations.     

Fluorescence Resonance Energy Transfer-Sensitized Emission of Yellow Cameleon 3.60 Reveals Root Zone-Specific Calcium Signatures in Arabidopsis in Response to Aluminum and Other Trivalent Cations

Fluorescence resonance energy transfer-sensitized emission of the yellow cameleon 3.60 was used to study the dynamics of cytoplasmic calcium ([Ca²⁺]_cyt) in different zones of living Arabidopsis (Arabidopsis thaliana) roots. Transient elevations of [Ca²⁺]_cyt were observed in response to glutamic acid (Glu), ATP, and aluminum (Al³⁺). Each chemical induced a [Ca²⁺]_cyt signature that differed among the three treatments in regard to the onset, duration, and shape of the response. Glu and ATP triggered patterns of [Ca²⁺]_cyt increases that were similar among the different root zones, whereas Al³⁺ evoked [Ca²⁺]_cyt transients that had monophasic and biphasic shapes, most notably in the root transition zone. The Al³⁺-induced [Ca²⁺]_cyt increases generally started in the maturation zone and propagated toward the cap, while the earliest [Ca²⁺]_cyt response after Glu or ATP treatment occurred in an area that encompassed the meristem and elongation zone. The biphasic [Ca²⁺]_cyt signature resulting from Al³⁺ treatment originated mostly from cortical cells located at 300 to 500 μ m from the root tip, which could be triggered in part through ligand-gated Glu receptors. Lanthanum and gadolinium, cations commonly used as Ca²⁺ channel blockers, elicited [Ca²⁺]_cyt responses similar to those induced by Al³⁺. The trivalent ion-induced [Ca²⁺]_cyt signatures in roots of an Al³⁺-resistant and an Al³⁺-sensitive mutant were similar to those of wild-type plants, indicating that the early [Ca²⁺]_cyt changes we report here may not be tightly linked to Al³⁺ toxicity but rather to a general response to trivalent cations.The role of calcium ions (Ca²⁺) as a ubiquitous cellular messenger in animal and plant cells is well established (Berridge et al., 2000; Sanders et al., 2002; Ng and McAinsh, 2003). Cellular signal transduction pathways are elicited as a result of fluctuations of free Ca²⁺ in the cytoplasm ([Ca²⁺]_cyt) in response to external and intracellular signals. These changes in [Ca²⁺]_cyt influence numerous cellular processes, including vesicle trafficking, cell metabolism, cell proliferation and elongation, stomatal opening and closure, seed and pollen grain germination, fertilization, ion transport, and cytoskeletal organization (Hepler, 2005). [Ca²⁺]_cyt fluctuations occur because cells have a Ca²⁺ signaling “toolkit” (Berridge et al., 2000) composed of on/off switches and a multitude of Ca²⁺-binding proteins. The on switches depend on membrane-localized Ca²⁺ channels that control the entry of Ca²⁺ into the cytosol (Piñeros and Tester, 1995, 1997; Thion et al., 1998; Kiegle et al., 2000a; White et al., 2000; Demidchik et al., 2002; Miedema et al., 2008). On the other hand, the off switches consist of a family of Ca²⁺-ATPases and Ca²⁺/H⁺ exchangers in the plasma membrane or endomembrane that remove Ca²⁺ from the cytosol, bringing the [Ca²⁺]_cyt down to the initial resting level (Lee et al., 2007; Li et al., 2008).The numerous cellular processes regulated by Ca²⁺ have led investigators to ask how specificity in Ca²⁺ signaling is maintained. It has been proposed that specificity in Ca²⁺ signaling is achieved because a particular stimulus elicits a distinct Ca²⁺ signature, which is defined by the timing, magnitude, and frequency of [Ca²⁺]_cyt changes. For instance, tip-growing plant cells such as root hairs and pollen tubes exhibit oscillatory elevations in [Ca²⁺]_cyt that partly mirror the oscillatory nature of growth in these cell types (Cárdenas et al., 2008; Monshausen et al., 2008). Another example is nuclear Ca²⁺ spiking in root hairs of legumes exposed to NOD factors (Oldroyd and Downie, 2006; Peiter et al., 2007). Recently, it was shown that mechanical forces applied to an Arabidopsis (Arabidopsis thaliana) root can trigger a stimulus-specific [Ca²⁺]_cyt response (Monshausen et al., 2009). Translating the Ca²⁺ signature into a defined cellular response is governed by a number of Ca²⁺-binding proteins such as calreticulin that act as [Ca²⁺]_cyt buffers, which shape both the amplitude and duration of the Ca²⁺ signal or Ca²⁺ sensors such as calmodulin that impact other downstream cellular effectors (Berridge et al., 2000; White and Broadley, 2003; Hepler, 2005).A deeper understanding of Ca²⁺ signaling mechanisms in plants has been driven in large part by our ability to monitor dynamic changes in [Ca²⁺]_cyt in the cell. Such measurements have been conducted using Ca²⁺-sensitive fluorescent indicator dyes (e.g. Indo and Fura), the luminescent protein aequorin (Knight et al., 1991, 1996; Legué et al., 1997; Wymer et al., 1997; Cárdenas et al., 2008), and more recently the yellow cameleon (YC) Ca²⁺ sensor, a chimeric protein that relies on fluorescence resonance energy transfer (FRET) as an indicator of [Ca²⁺]_cyt changes in the cell (Allen et al., 1999; Miwa et al., 2006; Qi et al., 2006; Tang et al., 2007; Haruta et al., 2008). The YC reporter is composed of cyan fluorescent protein (CFP), the C terminus of calmodulin (CaM), a Gly-Gly linker, the CaM-binding domain of myosin light chain kinase (M13), and a yellow fluorescent protein (YFP; Miyawaki et al., 1997, 1999). The increased interaction between M13 and CaM upon binding of Ca²⁺ to CaM triggers a conformational change in the protein that brings the CFP and YFP in close proximity, resulting in enhanced FRET efficiency between the two fluorophores (Miyawaki, 2003). Thus, changes in FRET efficiency between CFP and YFP in the cameleon reporter are correlated with changes in [Ca²⁺]_cyt.Since it was first introduced, improved versions of the cameleon reporter have been selected to more accurately report [Ca²⁺]_cyt levels in the cell. For instance, the YC3.60 version was selected because of its resistance to cytoplasmic acidification and its higher dynamic range compared with the earlier cameleons. The higher dynamic range of YC3.60 is due to the use of a circularly permutated YFP called Venus (cpVenus) that is capable of absorbing a greater amount of energy from CFP (Nagai et al., 2004). Recently, the utility of YC3.60 for monitoring [Ca²⁺]_cyt was demonstrated in Arabidopsis roots and pollen tubes using ratiometric imaging approaches (Monshausen et al., 2007, 2008, 2009; Haruta et al., 2008; Iwano et al., 2009). Here, we further evaluated YC3.60 as a [Ca²⁺]_cyt sensor in plants using confocal microscopy and FRET-sensitized emission imaging. Unlike the direct ratiometric measurement of cpVenus and CFP reported in previous studies using YC3.60-expressing plants (Monshausen et al., 2008, 2009), the sensitized FRET approach we describe here involves the use of donor-only (CFP) and acceptor-only (YFP) controls, allowing us to correct for bleed-through and background signals from the FRET specimen (van Rheenen et al., 2004; Feige et al., 2005).For this study, we focused on monitoring [Ca²⁺]_cyt changes in Arabidopsis seedling roots after aluminum (Al³⁺) exposure. Although Ca²⁺ signaling has long been implicated in mediating Al³⁺ responses in plants (Rengel and Zhang, 2003), the [Ca²⁺]_cyt changes evoked by Al³⁺ reported in the literature have been inconsistent, and as such, the significance of these [Ca²⁺]_cyt responses to mechanisms of Al³⁺ toxicity are not very clear. For instance, some studies reported that Al³⁺ caused a decrease in [Ca²⁺]_cyt in plants (Jones et al., 1998b; Kawano et al., 2004), and others demonstrated elevated [Ca²⁺]_cyt upon Al³⁺ treatment (Nichol and Oliveira, 1995; Lindberg and Strid, 1997; Jones et al., 1998a; Zhang and Rengel, 1999; Ma et al., 2002; Bhuja et al., 2004).Here, we report that Arabidopsis roots expressing the YC3.60 reporter exhibited transient elevations in [Ca²⁺]_cyt within seconds of Al³⁺ exposure. The general pattern of [Ca²⁺]_cyt changes observed after Al³⁺ treatment were distinct from those elicited by ATP or Glu, reinforcing the concept of specificity in [Ca²⁺]_cyt signaling. We also observed root zone-dependent variations in the [Ca²⁺]_cyt signatures evoked by Al³⁺ in regard to the shape, duration, and timing of the [Ca²⁺]_cyt response. Other trivalent ions such as lanthanum (La³⁺) and gadolinium (Ga³⁺), which have been widely used as Ca²⁺ channel blockers (Monshausen et al., 2009), also induced a rapid rise in [Ca²⁺]_cyt in root cells that were similar to those elicited by Al³⁺. Al³⁺, La³⁺, and Gd³⁺ elicited similar [Ca²⁺]_cyt signatures in the Al³⁺-tolerant mutant alr104 (Larsen et al., 1998) and the Al³⁺-sensitive mutant als3-1 (Larsen et al., 2005), indicating that the early [Ca²⁺]_cyt increases we report here may not be tightly linked to mechanisms of Al³⁺ toxicity but rather to a general trivalent cation response. Our study further shows that FRET-sensitized emission imaging of Arabidopsis roots expressing YC3.60 provides a robust method for documenting [Ca²⁺]_cyt signatures in different root developmental zones that should be useful for future studies on Ca²⁺ signaling mechanisms in plants.     

Permeant Cations and Blockers Modulate pH Gating of ROMK Channels

External potassium (K) activates the inward rectifier ROMK (K_ir1.1) by altering the pH gating of the channel. The present study examines this link between external K and internal pH sensitivity using both the two-electrode voltage clamp and the perfused, cut-open Xenopus oocyte preparation. Elevating extracellular K from 1 mM to 10 mM to 100 mM activated ROMK channels by shifting their apparent pK_a from 7.2 ± 0.1 (n = 6) in 1 mM K, to 6.9 ± 0.02 (n = 5) in 10 mM K, and to 6.6 ± 0.03 (n = 5) in 100 mM K. At any given internal pH, the number of active ROMK channels is a saturating function of external [K]. Extracellular Cs (which blocks almost all inward K current) also stimulated outward ROMK conductance (at constant 1 mM external K) by shifting the apparent pK_a of ROMK from 7.2 ± 0.1 (n = 6) in 1 mM K to 6.8 ± 0.01 (n = 4) in 1 mM K + 104 mM Cs. Surprisingly, the binding and washout of the specific blocker, Tertiapin-Q, also activated ROMK in 1 mM K and caused a comparable shift in apparent pK_a. These results are interpreted in terms of both a three-state kinetic model and a two-gate structural model that is based on results with KcsA in which the selectivity filter can assume either a high or low K conformation. In this context, external K, Cs, and Tertiapin-Q activate ROMK by destabilizing the low-K (collapsed) configuration of the selectivity filter.