A novel FK506- and rapamycin-binding protein (FPR3 gene product) in the yeast Saccharomyces cerevisiae is a proline rotamase localized to the nucleolus

The gene (FPR3) encoding a novel type of peptidylpropyl-cis-trans- isomerase (PPIase) was isolated during a search for previously unidentified nuclear proteins in Saccharomyces cerevisiae. PPIases are thought to act in conjunction with protein chaperones because they accelerate the rate of conformational interconversions around proline residues in polypeptides. The FPR3 gene product (Fpr3) is 413 amino acids long. The 111 COOH-terminal residues of Fpr3 share greater than 40% amino acid identity with a particular class of PPIases, termed FK506-binding proteins (FKBPs) because they are the intracellular receptors for two immunosuppressive compounds, rapamycin and FK506. When expressed in and purified from Escherichia coli, both full-length Fpr3 and its isolated COOH-terminal domain exhibit readily detectable PPIase activity. Both fpr3 delta null mutants and cells expressing FPR3 from its own promoter on a multicopy plasmid have no discernible growth phenotype and do not display any alteration in sensitivity to the growth-inhibitory effects of either FK506 or rapamycin. In S. cerevisiae, the gene for a 112-residue cytosolic FKBP (FPR1) and the gene for a 135-residue ER-associated FKBP (FPR2) have been described before. Even fpr1 fpr2 fpr3 triple mutants are viable. However, in cells carrying an fpr1 delta mutation (which confers resistance to rapamycin), overexpression from the GAL1 promoter of the C-terminal domain of Fpr3, but not full-length Fpr3, restored sensitivity to rapamycin. Conversely, overproduction from the GAL1 promoter of full- length Fpr3, but not its COOH-terminal domain, is growth inhibitory in both normal cells and fpr1 delta mutants. In fpr1 delta cells, the toxic effect of Fpr3 overproduction can be reversed by rapamycin. Overproduction of the NH2-terminal domain of Fpr3 is also growth inhibitory in normal cells and fpr1 delta mutants, but this toxicity is not ameliorated in fpr1 delta cells by rapamycin. The NH2-terminal domain of Fpr3 contains long stretches of acidic residues alternating with blocks of basic residues, a structure that resembles sequences found in nucleolar proteins, including S. cerevisiae NSR1 and mammalian nucleolin. Indirect immunofluorescence with polyclonal antibodies raised against either the NH2- or the COOH-terminal segments of Fpr3 expressed in E. coli demonstrated that Fpr3 is located exclusively in the nucleolus.     

Splice Cassette II of Na+,HCO3? Cotransporter NBCn1 (slc4a7) Interacts with Calcineurin A: IMPLICATIONS FOR TRANSPORTER ACTIVITY AND INTRACELLULAR pH CONTROL DURING RAT ARTERY CONTRACTIONS*

Activation of Na⁺,HCO₃⁻ cotransport in vascular smooth muscle cells (VSMCs) contributes to intracellular pH (pH_i) control during artery contraction, but the signaling pathways involved have been unknown. We investigated whether physical and functional interactions between the Na⁺,HCO₃⁻ cotransporter NBCn1 (slc4a7) and the Ca²⁺/calmodulin-activated serine/threonine phosphatase calcineurin exist and play a role for pH_i control in VSMCs. Using a yeast two-hybrid screen, we found that splice cassette II from the N terminus of NBCn1 interacts with calcineurin Aβ. When cassette II was truncated or mutated to disrupt the putative calcineurin binding motif PTVVIH, the interaction was abolished. Native NBCn1 and calcineurin Aβ co-immunoprecipitated from A7r5 rat VSMCs. A peptide (acetyl-DDIPTVVIH-amide), which mimics the putative calcineurin binding motif, inhibited the co-immunoprecipitation whereas a mutated peptide (acetyl-DDIATAVAA-amide) did not. Na⁺,HCO₃⁻ cotransport activity was investigated in VSMCs of mesenteric arteries after an NH₄⁺ prepulse. During depolarization with 50 mm extracellular K⁺ to raise intracellular [Ca²⁺], Na⁺,HCO₃⁻ cotransport activity was inhibited 20–30% by calcineurin inhibitors (FK506 and cyclosporine A). FK506 did not affect Na⁺,HCO₃⁻ cotransport activity in VSMCs when cytosolic [Ca²⁺] was lowered by buffering, nor did it disrupt binding between NBCn1 and calcineurin Aβ. FK506 augmented the intracellular acidification of VSMCs during norepinephrine-induced artery contractions. No physical or functional interactions between calcineurin Aβ and the Na⁺/H⁺ exchanger NHE1 were observed in VSMCs. In conclusion, we demonstrate a physical interaction between calcineurin Aβ and cassette II of NBCn1. Intracellular Ca²⁺ activates Na⁺,HCO₃⁻ cotransport activity in VSMCs in a calcineurin-dependent manner which is important for protection against intracellular acidification.     

Sodium sensing induces different changes in free cytosolic calcium concentration and pH in salt-tolerant and -sensitive rice (Oryza sativa) cultivars

Perception of salt stress in plant cells induces a change in the free cytosolic Ca²⁺, [Ca²⁺]_cyt, which transfers downstream reactions toward salt tolerance. Changes in cytosolic H⁺ concentration, [H⁺]_cyt, are closely linked to the [Ca²⁺]_cyt dynamics under various stress signals. In this study, salt‐induced changes in [Ca²⁺]_cyt, and [H⁺]_cyt and vacuolar [H⁺] concentrations were monitored in single protoplasts of rice (Oryza sativa L. indica cvs. Pokkali and BRRI Dhan29) by fluorescence microscopy. Changes in cytosolic [Ca²⁺] and [H⁺] were detected by use of the fluorescent dyes acetoxy methyl ester of calcium‐binding benzofuran and acetoxy methyl ester of 2′, 7′‐bis‐(2‐carboxyethyl)‐5‐(and‐6) carboxyfluorescein, respectively, and for vacuolar pH, fluorescent 6‐carboxyfluorescein and confocal microscopy were used. Addition of NaCl induced a higher increase in [Ca²⁺]_cyt in the salt‐tolerant cv. Pokkali than in the salt‐sensitive cv. BRRI Dhan29. From inhibitor studies, we conclude that the internal stores appear to be the major source for [Ca²⁺]_cyt increase in Pokkali, although the apoplast is more important in BRRI Dhan29. The [Ca²⁺]_cyt measurements in rice also suggest that Na⁺ should be sensed inside the cytosol, before any increase in [Ca²⁺]_cyt occurs. Moreover, our results with individual mesophyll protoplasts suggest that ionic stress causes an increase in [Ca²⁺]_cyt and that osmotic stress sharply decreases [Ca²⁺]_cyt in rice. The [pH]_cyt was differently shifted in the two rice cultivars in response to salt stress and may be coupled to different activities of the H⁺‐ATPases. The changes in vacuolar pH were correlated with the expressional analysis of rice vacuolar H⁺‐ATPase in these two rice cultivars.     

Role of calcineurin and actin dynamics in regulated secretion of microneme proteins in Plasmodium falciparum merozoites during erythrocyte invasion

Plasmodium falciparum invades host erythrocytes by multiple invasion pathways. The invasion of erythrocytes by P. falciparum merozoites is a complex process that requires multiple interactions between host receptors and parasite ligands. A number of parasite proteins that mediate interaction with host receptors during invasion are localized to membrane‐bound apical organelles referred to as micronemes and rhoptries. The timely release of these proteins to the merozoite surface is crucial for receptor engagement and invasion. It has been demonstrated previously that exposure of merozoites to a low potassium (K⁺) ionic environment as found in blood plasma leads to a rise in cytosolic calcium (Ca²⁺), which triggers microneme secretion. The signalling pathways that regulate microneme discharge in response to rise in cytosolic Ca²⁺ are not completely understood. Here, we show that a P. falciparum Ca²⁺‐dependent protein phosphatase, calcineurin (PfCN), is an essential regulator of Ca²⁺‐dependent microneme exocytosis. An increase in PfCN activity was observed in merozoites following exposure to a low K⁺ environment. Treatment of merozoites with calcineurin inhibitors such as FK506 and cyclosporin A prior to transfer to a low K⁺ environment resulted in inhibition of secretion of microneme protein apical merozoite antigen‐1 (PfAMA‐1). Inhibition of PfCN was shown to result in reduced dephosphorylation and depolymerization of apical actin, which appears to be criticalfor microneme secretion. PfCN thus serves as an effector of Ca²⁺‐dependent microneme exocytosis by regulating depolymerization of apical actin. Inhibitors that target PfCN block microneme exocytosis and limit growth of P. falciparum blood‐stage parasites providing a novel approach towards development of new therapeutic strategies against malaria.     

Calcium supply effects on wheat cultivars differing in salt resistance with special reference to leaf cytosol ion homeostasis

Salinity causes changes in cytosolic Ca²⁺, [Ca²⁺]_cyt, Na⁺, [Na⁺]_cyt and pH, pH_cyt, which induce specific reactions and signals. Reactions causing a rebalancing of the physiological homeostasis of the cytosol could result in plant resistance and growth. Two wheat cultivars, Triticum aestivum, Seds1 and Vinjett, were grown in nutrient solution for 7 days under moderate salinity (0 and 50 mM NaCl) with and without extra addition of 5 mM CaSO₄ to investigate the seedling‐ion homeostasis under salinity. In the leaf protoplasts [Ca²⁺]_cyt, [Na⁺]_cyt and pH_cyt were detected using acetoxymethyl esters of the ion‐specific dyes, Fura 2, SBFI and BCECF, respectively, and fluorescence microscopy. In addition, both cultivars were grown for 3 weeks at 0, 50 and 125 mM NaCl with, or without, extra addition of 5 mM CaSO₄ to detect overall Na⁺ and Ca²⁺ concentrations in leaves and salinity effects on dry weights. In both cultivars, salinity decreased [Ca²⁺]_cyt, while at extra Ca²⁺ supplied, [Ca²⁺]_cyt increased. The [Ca²⁺]_cyt increase was accompanied by increase in the overall Ca²⁺ concentrations in leaves and decrease in the overall Na⁺ concentration. Moreover, irrespective of Ca²⁺ treatment under salinity, the cultivars reacted in different ways; [Na⁺]_cyt significantly increased only in cv. Vinjett, while pH_cyt increased only in cv. Seds1. Even at rather high total Na⁺ concentrations, the cytosolic concentrations were kept low in both cultivars. It is discussed whether the increase of [Ca²⁺]_cyt and pH_cyt can contribute to salt tolerance and if the cytosolic changes are due to changes in overall Ca²⁺ and Na⁺ concentrations.     

Mechanism of Ca2+-dependent Inactivation of L-type Ca2+ Channels in GH3 Cells: Direct Evidence Against Dephosphorylation by Calcineurin

Dephosphorylation of Ca²⁺ channels by the Ca²⁺-activated phosphatase 2B (calcineurin) has been previously suggested as a mechanism of Ca²⁺-dependent inactivation of Ca²⁺ current in rat pituitary tumor (GH₃) cells. Although recent evidence favors an inactivation mechanism involving direct binding of Ca²⁺ to the channel protein, the alternative ``calcineurin hypothesis' has not been critically tested using the specific calcineurin inhibitors cyclosporine A (CsA) or FK506 in GH₃ cells. To determine if calcineurin plays a part in the voltage- and/or Ca²⁺-dependent components of dihydropyridine-sensitive Ca²⁺ current decay, we rapidly altered the intracellular Ca²⁺ buffering capacity of GH₃ cells by flash photolysis of DM-nitrophen, a high affinity Ca²⁺ chelator. Flash photolysis induced a highly reproducible increase in the extent of Ca²⁺ current inactivation in a two-pulse voltage protocol with Ca²⁺ as the charge carrier, but had no effect when Ba²⁺ was substituted for Ca²⁺. Despite confirmation of the abundance of calcineurin in the GH₃ cells by biochemical assays, acute application of CsA or FK506 after photolysis had no effect on Ca²⁺-dependent inactivation of Ca²⁺ current, even when excess cyclophilin or FK binding protein were included in the internal solution. Prolonged preincubation of the cells with FK506 or CsA did not inhibit Ca²⁺-dependent inactivation. Similarly, blocking calmodulin activation with calmidazolium or blocking calcineurin with fenvalerate did not influence the extent of Ca²⁺-dependent inactivation after photolysis. The results provide strong evidence against Ca²⁺-dependent dephosphorylation as the mechanism of Ca²⁺ current inactivation in GH₃ cells, but support the alternative idea that Ca²⁺-dependent inactivation reflects a direct effect of intracellular Ca²⁺ on channel gating. Received: 12 August 1996/Revised: 21 October 1996     

Trimethyltin chloride induced chloride secretion across rat distal colon

TMT (trimethyltin chloride), an organotin, is ubiquitous in the environment. The consumption of contaminated food may cause exposure of the human diet to this toxic compound. The present study was to investigate the effects of TMT on the regulation of ion transport across the rat distal colon. The rat colonic mucosa was mounted in Ussing chambers. The effects of TMT were assessed using the I_sc (short‐circuit current). Both apical and basolateral TMT induced, dose‐dependently, an increase in I_sc, which was due to a stimulation of Cl^? secretion as measured using ion substitution experiments and pharmacological manoeuvres. The secretion was also inhibited by several K⁺ channel blockers administrated at the basolateral side. When the apical side was permeabilized by nystatin, the TMT‐induced K⁺ conductance was effectively blocked by tetrapentylammonium, a Ca²⁺‐sensitive K⁺ channel blocker. The response of TMT was sensitive to the basolateral Ca²⁺ and the intracellular Ca²⁺ store, which could be disclosed by applying the inhibitors of ryanodine receptors and inositol 1,4,5‐trisphosphate receptors. In conclusion, TMT led to Cl^? secretion, which was essentially regulated by basolateral Ca²⁺‐sensitive K⁺ channels. These results suggest the importance of K⁺ channels in the toxicity hazard of TMT.     

H2O2 and cytosolic Ca2+ signals triggered by the PM H+‐coupled transport system mediate K+/Na+ homeostasis in NaCl‐stressed Populus euphratica cells

Using confocal microscopy, X‐ray microanalysis and the scanning ion‐selective electrode technique, we investigated the signalling of H₂O₂, cytosolic Ca²⁺ ([Ca²⁺]_cyt) and the PM H⁺‐coupled transport system in K⁺/Na⁺ homeostasis control in NaCl‐stressed calluses of Populus euphratica. An obvious Na⁺/H⁺ antiport was seen in salinized cells; however, NaCl stress caused a net K⁺ efflux, because of the salt‐induced membrane depolarization. H₂O₂ levels, regulated upwards by salinity, contributed to ionic homeostasis, because H₂O₂ restrictions by DPI or DMTU caused enhanced K⁺ efflux and decreased Na⁺/H⁺ antiport activity. NaCl induced a net Ca²⁺ influx and a subsequent rise of [Ca²⁺]_cyt, which is involved in H₂O₂‐mediated K⁺/Na⁺ homeostasis in salinized P. euphratica cells. When callus cells were pretreated with inhibitors of the Na⁺/H⁺ antiport system, the NaCl‐induced elevation of H₂O₂ and [Ca²⁺]_cyt was correspondingly restricted, leading to a greater K⁺ efflux and a more pronounced reduction in Na⁺/H⁺ antiport activity. Results suggest that the PM H⁺‐coupled transport system mediates H⁺ translocation and triggers the stress signalling of H₂O₂ and Ca²⁺, which results in a K⁺/Na⁺ homeostasis via mediations of K⁺ channels and the Na⁺/H⁺ antiport system in the PM of NaCl‐stressed cells. Accordingly, a salt stress signalling pathway of P. euphratica cells is proposed.     

Chimeras of P-type ATPases and their transcriptional regulators: contributions of a cytosolic amino-terminal domain to metal specificity

Zn²⁺‐responsive repressor ZiaR and Co²⁺‐responsive activator CoaR modulate production of P₁‐type Zn²⁺‐ (ZiaA) and Co²⁺‐ (CoaT) ATPases respectively. What dictates metal selectivity? We show that Δ ziaΔcoa double mutants had similar Zn²⁺ resistance to Δzia single mutants and similar Co²⁺ resistance to Δcoa single mutants. Controlling either ziaA or coaT with opposing regulators restored no resistance to metals sensed by the regulators, but coincident replacement of the deduced cytosolic amino‐terminal domain CoaT_N with ZiaA_N (in ziaR‐_p ziaA‐ziaA_NcoaT) conferred Zn²⁺ resistance to ΔziaΔcoa, Zn²⁺ content was lowered and residual Co²⁺ resistance lost. Metal‐dependent molar absorptivity under anaerobic conditions revealed that purified ZiaA_N binds Co²⁺ in a pseudotetrahedral two‐thiol site, and Co²⁺ was displaced by Zn²⁺. Thus, the amino‐terminal domain of ZiaA inverts the metals exported by zinc‐regulated CoaT from Co²⁺ to Zn²⁺, and this correlates simplistically with metal‐binding preferences; K_ZiaAN Zn²⁺ tighter than Co²⁺. However, Zn²⁺ did not bleach Cu⁺‐ZiaA_N, and only Cu⁺ co‐migrated with ZiaA_N after competitive binding versus Zn²⁺. Bacterial two‐hybrid assays that detected interaction between the Cu⁺‐metallochaperone Atx1 and the amino‐terminal domain of Cu⁺‐transporter PacS_N detected no interaction with the analogous, deduced, ferredoxin‐fold subdomain of ZiaA_N. Provided that there is no freely exchangeable cytosolic Cu⁺, restricted contact with the Cu⁺‐metallochaperone can impose a barrier impairing the formation of otherwise favoured Cu⁺–ZiaA_N complexes.     

Ca2+‐regulated and diurnal rhythm‐regulated Na+/Ca2+ exchanger AtNCL affects flowering time and auxin signalling in Arabidopsis

Calcium (Ca²⁺) is vital for plant growth, development, hormone response and adaptation to environmental stresses, yet the mechanisms regulating plant cytosolic Ca²⁺ homeostasis are not fully understood. Here, we characterize an Arabidopsis Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger AtNCL that regulates Ca²⁺ and multiple physiological processes. AtNCL was localized to the tonoplast in yeast and plant cells. AtNCL appeared to mediate sodium (Na⁺) vacuolar sequestration and meanwhile Ca²⁺ release. The EF‐hand domains within AtNCL regulated Ca²⁺ binding and transport of Ca²⁺ and Na⁺. Plants with diminished AtNCL expression were more tolerant to high CaCl₂ but more sensitive to both NaCl and auxin; heightened expression of AtNCL rendered plants more sensitive to CaCl₂ but tolerant to NaCl. AtNCL expression appeared to be regulated by the diurnal rhythm and suppressed by auxin. DR5::GUS expression and root responses to auxin were altered in AtNCL mutants. The auxin‐induced suppression of AtNCL was attenuated in SLR/IAA14 and ARF6/8 mutants. The mutants with altered AtNCL expression also altered flowering time and FT and CO expression; FT may mediate AtNCL‐regulated flowering time change. Therefore, AtNCL is a vacuolar Ca²⁺‐regulated Na⁺/Ca²⁺ exchanger that regulates auxin responses and flowering time.     

Calcium elevation-dependent and attenuated resting calcium-dependent abscisic acid induction of stomatal closure and abscisic acid-induced enhancement of calcium sensitivities of S-type anion and inward-rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca²⁺] ([Ca²⁺]_i), and also on mechanisms that are independent of [Ca²⁺]_i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)‐induced stomatal closure response in the [Ca²⁺]_i‐elevation‐independent pathway? (ii) How do ABA‐insensitive mutants affect the [Ca²⁺]_i‐elevation‐independent pathway? (iii) Does ABA enhance (prime) the Ca²⁺ sensitivity of anion and inward‐rectifying K⁺ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca²⁺]_i elevations and clamping [Ca²⁺]_i to resting levels. The absence of [Ca²⁺]_i elevations was confirmed by ratiometric [Ca²⁺]_i imaging experiments. ABA‐induced stomatal closure in the absence of [Ca²⁺]_i elevations above the physiological resting [Ca²⁺]_i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca²⁺]_i elevations. The ABA‐insensitive mutants ost1‐2, abi2‐1 and gca2 showed partial stomatal closure responses that correlate with [Ca²⁺]_i‐dependent ABA signaling. Interestingly, patch‐clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca²⁺ to activate S‐type anion channels and down‐regulate inward‐rectifying K⁺ channels, providing strong evidence for a Ca²⁺ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca²⁺]_i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca²⁺]_i sensitivity of stomatal closure mechanisms.     

Annexin 1 regulates the H2O2‐induced calcium signature in Arabidopsis thaliana roots

Hydrogen peroxide is the most stable of the reactive oxygen species (ROS) and is a regulator of development, immunity and adaptation to stress. It frequently acts by elevating cytosolic free Ca²⁺ ([Ca²⁺]_cyt) as a second messenger, with activation of plasma membrane Ca²⁺‐permeable influx channels as a fundamental part of this process. At the genetic level, to date only the Ca²⁺‐permeable Stelar K⁺ Outward Rectifier (SKOR) channel has been identified as being responsive to hydrogen peroxide. We show here that the ROS‐regulated Ca²⁺ transport protein Annexin 1 in Arabidopsis thaliana (AtANN1) is involved in regulating the root epidermal [Ca²⁺]_cyt response to stress levels of extracellular hydrogen peroxide. Peroxide‐stimulated [Ca²⁺]_cyt elevation (determined using aequorin luminometry) was aberrant in roots and root epidermal protoplasts of the Atann1 knockout mutant. Similarly, peroxide‐stimulated net Ca²⁺ influx and K⁺ efflux were aberrant in Atann1 root mature epidermis, determined using extracellular vibrating ion‐selective microelectrodes. Peroxide induction of GSTU1 (Glutathione‐S‐Transferase1 Tau 1), which is known to be [Ca²⁺]_cyt‐dependent was impaired in mutant roots, consistent with a lesion in signalling. Expression of AtANN1 in roots was suppressed by peroxide, consistent with the need to restrict further Ca²⁺ influx. Differential regulation of annexin expression was evident, with AtANN2 down‐regulation but up‐regulation of AtANN3 and AtANN4. Overall the results point to involvement of AtANN1 in shaping the root peroxide‐induced [Ca²⁺]_cyt signature and downstream signalling.     

Mechanism of the persistent sodium current activator veratridine-evoked Ca2+ elevation: implication for epilepsy

Although the role of Na⁺ in several aspects of Ca²⁺ regulation has already been shown, the exact mechanism of intracellular Ca²⁺ concentration ([Ca²⁺]_i) increase resulting from an enhancement in the persistent, non‐inactivating Na⁺ current (I_Na,P), a decisive factor in certain forms of epilepsy, has yet to be resolved. Persistent Na⁺ current, evoked by veratridine, induced bursts of action potentials and sustained membrane depolarization with monophasic intracellular Na⁺ concentration ([Na⁺]_i) and biphasic [Ca²⁺]_i increase in CA1 pyramidal cells in acute hippocampal slices. The Ca²⁺ response was tetrodotoxin‐ and extracellular Ca²⁺‐dependent and ionotropic glutamate receptor‐independent. The first phase of [Ca²⁺]_i rise was the net result of Ca²⁺ influx through voltage‐gated Ca²⁺ channels and mitochondrial Ca²⁺ sequestration. The robust second phase in addition involved reverse operation of the Na⁺–Ca²⁺ exchanger and mitochondrial Ca²⁺ release. We excluded contribution of the endoplasmic reticulum. These results demonstrate a complex interaction between persistent, non‐inactivating Na⁺ current and [Ca²⁺]_i regulation in CA1 pyramidal cells. The described cellular mechanisms are most likely part of the pathomechanism of certain forms of epilepsy that are associated with I_Na,P. Describing the magnitude, temporal pattern and sources of Ca²⁺ increase induced by I_Na,P may provide novel targets for antiepileptic drug therapy.     

Distinct roles of Drosophila cacophony and Dmca1D Ca2+ channels in synaptic homeostasis: Genetic interactions with slowpoke Ca2+‐activated BK channels in presynaptic excitability and postsynaptic response

Ca²⁺ influx through voltage‐activated Ca²⁺ channels and its feedback regulation by Ca²⁺‐activated K⁺ (BK) channels is critical in Ca²⁺‐dependent cellular processes, including synaptic transmission, growth and homeostasis. Here we report differential roles of cacophony (Ca_V2) and Dmca1D (Ca_V1) Ca²⁺ channels in synaptic transmission and in synaptic homeostatic regulations induced by slowpoke (slo) BK channel mutations. At Drosophila larval neuromuscular junctions (NMJs), a well‐established homeostatic mechanism of transmitter release enhancement is triggered by experimentally suppressing postsynaptic receptor response. In contrast, a distinct homeostatic adjustment is induced by slo mutations. To compensate for the loss of BK channel control presynaptic Sh K⁺ current is upregulated to suppress transmitter release, coupled with a reduction in quantal size. We demonstrate contrasting effects of cac and Dmca1D channels in decreasing transmitter release and muscle excitability, respectively, consistent with their predominant pre‐ vs. postsynaptic localization. Antibody staining indicated reduced postsynaptic GluRII receptor subunit density and altered ratio of GluRII A and B subunits in slo NMJs, leading to quantal size reduction. Such slo‐triggered modifications were suppressed in cac;;slo larvae, correlated with a quantal size reversion to normal in double mutants, indicating a role of cac Ca²⁺ channels in slo‐triggered homeostatic processes. In Dmca1D;slo double mutants, the quantal size and quantal content were not drastically different from those of slo, although Dmca1D suppressed the slo‐induced satellite bouton overgrowth. Taken together, cac and Dmca1D Ca²⁺ channels differentially contribute to functional and structural aspects of slo‐induced synaptic modifications. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 1–15, 2014     

The role of plasma membrane H+‐ATPase in jasmonate‐induced ion fluxes and stomatal closure in Arabidopsis thaliana

Methyl jasmonate (MeJA) elicits stomatal closure in many plant species. Stomatal closure is accompanied by large ion fluxes across the plasma membrane (PM). Here, we recorded the transmembrane ion fluxes of H⁺, Ca²⁺ and K⁺ in guard cells of wild‐type (Col‐0) Arabidopsis, the CORONATINE INSENSITIVE1 (COI1) mutant coi1‐1 and the PM H⁺‐ATPase mutants aha1‐6 and aha1‐7, using a non‐invasive micro‐test technique. We showed that MeJA induced transmembrane H⁺ efflux, Ca²⁺ influx and K⁺ efflux across the PM of Col‐0 guard cells. However, this ion transport was abolished in coi1‐1 guard cells, suggesting that MeJA‐induced transmembrane ion flux requires COI1. Furthermore, the H⁺ efflux and Ca²⁺ influx in Col‐0 guard cells was impaired by vanadate pre‐treatment or PM H⁺‐ATPase mutation, suggesting that the rapid H⁺ efflux mediated by PM H⁺‐ATPases could function upstream of the Ca²⁺ flux. After the rapid H⁺ efflux, the Col‐0 guard cells had a longer oscillation period than before MeJA treatment, indicating that the activity of the PM H⁺‐ATPase was reduced. Finally, the elevation of cytosolic Ca²⁺ concentration and the depolarized PM drive the efflux of K⁺ from the cell, resulting in loss of turgor and closure of the stomata.     

Development of depolarization‐induced calcium transients in insect glial cells is dependent on the presence of afferent axons

Changes in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by depolarization have been measured in glial cells acutely isolated from antennal lobes of the moth Manduca sexta at different postembryonic developmental stages. Depolarization of the glial cell membrane was elicited by increasing the external K⁺ concentration from 4 to 25 mM. At midstage 5 and earlier stages, less than 20% of the cells responded to 25 mM K⁺ (1 min) with a transient increase in [Ca²⁺]_i of approximately 40 nM. One day later, at late stage 5, 68% of the cells responded to 25 mM K⁺, the amplitude of the [Ca²⁺]_i transients averaging 592 nM. At later stages, all cells responded to 25 mM K⁺ with [Ca²⁺]_i transients with amplitudes not significantly different from those at late stage 5. In stage 6 glial cells isolated from deafferented antennal lobes, i.e., from antennal lobes chronically deprived of olfactory receptor axons, only 30% of the cells responded with [Ca²⁺]_i transients. The amplitudes of these [Ca²⁺]_i transients averaged 93 nM and were significantly smaller than those in normal stage 6 glial cells. [Ca²⁺]_i transients were greatly reduced in Ca²⁺‐free, EGTA‐buffered saline, and in the presence of the Ca²⁺ channel blockers cadmium and verapamil. The results suggest that depolarization of the cell membrane induces Ca²⁺ influx through voltage‐activated Ca²⁺ channels into antennal lobe glial cells. The development of the depolarization‐induced Ca²⁺ transients is rapid between midstage 5 and stage 6, and depends on the presence of afferent axons from the olfactory receptor cells in the antenna. © 2002 Wiley Periodicals, Inc. J Neurobiol 52: 85–98, 2002