Plasma membrane-associated proline-rich extensin-like receptor kinase 4, a novel regulator of Ca2+ signalling, is required for abscisic acid responses in Arabidopsis thaliana

Plant roots respond to environmental stresses or the exogenous plant hormone abscisic acid (ABA) by undergoing marked physiological and morphological changes. We show here that PERK4 , a gene that encodes a member of the Arabidopsis thaliana proline-rich extensin-like receptor kinase family, plays an important role in ABA responses. Mutation of PERK4 by T-DNA insertion decreased sensitivity to ABA with respect to seed germination, seedling growth and primary root tip growth. The effect on root growth was due to enhanced cell elongation rather than cell division. The cytosolic free calcium concentration and Ca²⁺ channel currents were lower in perk4 root cells than in wild-type cells in the presence of ABA. Root growth was similar in wild-type and perk4 plants after the application of a Ca²⁺ channel blocker. PERK4 localised to the plasma membrane, and was shown to be an ABA- and Ca²⁺-activated protein kinase. Our data suggest that the receptor-like kinase encoded by PERK4 functions at an early stage of ABA signalling to inhibit root cell elongation by perturbing Ca²⁺ homeostasis.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

The fou2 mutation in the major vacuolar cation channel TPC1 confers tolerance to inhibitory luminal calcium

The SV channel encoded by the TPC1 gene represents a Ca²⁺- and voltage-dependent vacuolar cation channel. Point mutation D454N within TPC1 , named fou2 for fatty acid oxygenation upregulated 2 , results in increased synthesis of the stress hormone jasmonate. As wounding causes Ca²⁺ signals and cytosolic Ca²⁺ is required for SV channel function, we here studied the Ca²⁺-dependent properties of this major vacuolar cation channel with Arabidopsis thaliana mesophyll vacuoles. In patch clamp measurements, wild-type and fou2 SV channels did not exhibit differences in cytosolic Ca²⁺ sensitivity and Ca²⁺ impermeability. K⁺ fluxes through wild-type TPC1 were reduced or even completely faded away when vacuolar Ca²⁺ reached the 0.1-m m level. The fou2 protein under these conditions, however, remained active. Thus, D454N seems to be part of a luminal Ca²⁺ recognition site. Thereby the SV channel mutant gains tolerance towards elevated luminal Ca²⁺. A three-fold higher vacuolar Ca/K ratio in the fou2 mutant relative to wild-type plants seems to indicate that fou2 can accumulate higher levels of vacuolar Ca²⁺ before SV channel activity vanishes and K⁺ homeostasis is impaired. In response to wounding fou2 plants might thus elicit strong vacuole-derived cytosolic Ca²⁺ signals resulting in overproduction of jasmonate.     

Role of Ca2+ in Differentiation Mediated by Nerve Growth Factor and Dibutyryl Cyclic AMP in PC12 Cells

Abstract: Nerve growth factor (NGF) and dibutyryl cyclic AMP (dbcAMP) have synergistic effects on the neurite outgrowth of rat pheochromocytoma PC12 cells. The sites of interaction between NGF and dbcAMP have been studied extensively; however, the role of Ca²⁺ in differentiation induced by the two agents remains unclear. To understand whether intracellular Ca²⁺ is involved in the differentiation induced by the two agents, PC12 cells were treated with NGF, dbcAMP, or NGF plus dbcAMP for 2 days, and then effects on neurite outgrowth, ATP-induced Ca²⁺ influx, and Ca²⁺ mobilization from intracellular Ca²⁺ pools were examined. NGF or dbcAMP alone enhanced neurite outgrowth and Ca²⁺ accumulation by nonmitochondrial Ca²⁺ pools or the thapsigargin (TG)-sensitive Ca²⁺ pool. The dbcAMP acted synergistically with NGF to increase neurite outgrowth and to enlarge the TG-sensitive Ca²⁺ pool. The synergistic effect occurred within the first hour of treatment with dbcAMP plus NGF. On the other hand, dbcAMP abolished NGF's ability to enhance ATP-induced influx of extracellular Ca²⁺. Therefore, NGF and dbcAMP induced different effects on Ca²⁺ signaling pathways through two different but interacting pathways. In PC12 cells pretreated with TG to deplete the TG-sensitive Ca²⁺ pool, the dbcAMP- or dbcAMP plus NGF-mediated neurite outgrowth was significantly inhibited, whereas NGF-mediated neurite outgrowth was not affected by TG pretreatment. Our results suggest that the intracellular nonmitochondrial Ca²⁺ pools were changed in the differentiation process and were necessary for the synergistic effect of NGF and dbcAMP.     

Sweet taste receptor interacting protein CIB1 is a general inhibitor of InsP3-dependent Ca2+ release in vivo

In a search for sweet taste receptor interacting proteins, we have identified the calcium- and integrin-binding protein 1 (CIB1) as specific binding partner of the intracellular carboxyterminal domain of the rat sweet taste receptor subunit Tas1r2. In heterologous human embryonic kidney 293 (HEK293) cells, the G protein chimeras Gα_16gust44 and Gα_15i3 link the sweet taste receptor dimer TAS1R2/TAS1R3 to an inositol 1,4,5-trisphosphate (InsP₃)-dependent Ca²⁺ release pathway. To demonstrate the influence of CIB1 on the cytosolic Ca²⁺ concentration, we used sweet and umami compounds as well as other InsP₃-generating ligands in FURA-2-based Ca²⁺ assays in wild-type HEK293 cells and HEK293 cells expressing functional human sweet and umami taste receptor dimers. Stable and transient depletion of CIB1 by short-hairpin RNA increased the Ca²⁺ response of HEK293 cells to the InsP₃-generating ligands ATP, UTP and carbachol. Over-expression of CIB1 had the opposite effect as shown for the sweet ligand saccharin, the umami receptor ligand monosodium glutamate and UTP. The CIB1 effect was dependent on the thapsigargin-sensitive Ca²⁺ store of the endoplasmic reticulum (ER) and independent of extracellular Ca²⁺. The function of CIB1 on InsP₃-evoked Ca²⁺ release from the ER is most likely mediated by its interaction with the InsP₃ receptor. Thus, CIB1 seems to be an inhibitor of InsP₃-dependent Ca²⁺ release in vivo .     

Involvement of Calcineurin in Ca2+ Paradox-Like Injury of Cultured Rat Astrocytes

Abstract: The Ca²⁺/calmodulin-dependent phosphatase calcineurin may have physiological and pathological roles in neurons, but little is known about the roles of the enzyme in glial cells. We have previously reported that reperfusion of cultured astrocytes in Ca²⁺-containing medium after exposure to Ca²⁺-free medium caused Ca²⁺ influx followed by delayed cell death. In this study, we examined if calcineurin is involved in this Ca²⁺-mediated astrocytic injury. FK506, an inhibitor of calcineurin, protected cultured rat astrocytes against paradoxical Ca²⁺ challenge-induced injury in a dose-dependent manner (10⁻¹⁰–10⁻⁸ M ). Cyclosporin A at 1 µ M mimicked the effect of FK506. Rapamycin (1 µ M ) did not affect astrocyte injury, but it blocked the protective effect of FK506. Deltamethrin (20 n M ), another calcineurin inhibitor, had a similar protective effect, whereas okadaic acid did not. FK506 affected neither paradoxical Ca²⁺ challenge-induced increase in cytosolic Ca²⁺ level nor Na⁺-Ca²⁺ exchange activity in the cells, suggesting that the calcineurin is involved in processes downstream of increased cytosolic Ca²⁺ level. Immunochemical studies showed that both calcineurin A (probably the Aβ2 isoform) and B subunits were expressed in the cells. It is concluded that calcineurin is present in cultured astrocytes and it has a pathological role in the cells.     

Phosphorylation status of the NR2B subunit of NMDA receptor regulates its interaction with calcium/calmodulin-dependent protein kinase II

Ca²⁺ influx through NMDA-type glutamate receptor at excitatory synapses causes activation of post-synaptic Ca²⁺/calmodulin-dependent protein kinase type II (CaMKII) and its translocation to the NR2B subunit of NMDA receptor. The major binding site for CaMKII on NR2B undergoes phosphorylation at Ser1303, in vivo . Even though some regulatory effects of this phosphorylation are known, the mode of dephosphorylation of NR2B-Ser1303 is still unclear. We show that phosphorylation status at Ser1303 enables NR2B to distinguish between the Ca²⁺/calmodulin activated form and the autonomously active Thr286-autophosphorylated form of CaMKII. Green fluorescent protein–α-CaMKII co-expressed with NR2B sequence in human embryonic kidney 293 cells was used to study intracellular binding between the two proteins. Binding in vitro was studied by glutathione- S -transferase pull-down assay. Thr286-autophosphorylated α-CaMKII or the autophosphorylation mimicking mutant, T286D-α-CaMKII, binds NR2B sequence independent of Ca²⁺/calmodulin unlike native wild-type α-CaMKII. We show enhancement of this binding by Ca²⁺/calmodulin. Phosphorylation or a phosphorylation mimicking mutation on NR2B (NR2B-S1303D) abolishes the Ca²⁺/calmodulin-independent binding whereas it allows the Ca²⁺/calmodulin-dependent binding of α-CaMKII in vitro . Similarly, the autonomously active mutants, T286D-α-CaMKII and F293E/N294D-α-CaMKII, exhibited Ca²⁺-independent binding to non-phosphorylatable mutant of NR2B under intracellular conditions. We also show for the first time that phosphatases in the brain such as protein phosphatase 1 and protein phosphatase 2A dephosphorylate phospho-Ser1303 on NR2B.     

Regulation of synaptic vesicle accumulation and axon terminal remodeling during synapse formation by distinct Ca2+ signaling

The synaptic vesicle accumulation and subsequent morphological remodeling of axon terminals are characteristic features of presynaptic differentiation of zebrafish olfactory sensory neurons. The synaptic vesicle accumulation and axon terminal remodeling are regulated by protein kinase A and calcineurin signaling, respectively. To investigate upstream signals of presynaptic differentiation, we focused on Ca²⁺ signaling as Ca²⁺/calmodulin is required for the activation of both calcineurin and some adenylyl cyclases. We here showed that application of Ca²⁺/calmodulin inhibitor or olfactory sensory neuron-specific expression of calmodulin inhibitory peptide suppressed both synaptic vesicle accumulation and axon terminal remodeling. Thus, the trigger of presynaptic differentiation could be Ca²⁺ release from intracellular stores or Ca²⁺ influx. Application of a phospholipase C inhibitor or olfactory sensory neuron-specific expression of inositol 1,4,5-trisphosphate (IP₃) 5-phosphatase suppressed synaptic vesicle accumulation, but not morphological remodeling. In contrast, application of a voltage-gated Ca²⁺ channel blocker or expression of Kir2.1 inward rectifying potassium channel prevented the morphological remodeling. We also provided evidence that IP₃ signaling acted upstream of protein kinase A signaling. Our results suggest that IP₃-mediated Ca²⁺/calmodulin signaling stimulates synaptic vesicle accumulation and subsequent neuronal activity-dependent Ca²⁺/calmodulin signaling induces the morphological remodeling of axon terminals.     

Cellular Differentiation in Submerged Monolayers of Dictyostelium discoideum: Possible Functions of Cytoplasmic Ca2+and DIF

Cytoplasmic calcium ion (Ca²⁺) has generally been proposed to be a key factor of numerous cellular processes. Among several agents which might be expected to alter cytoplasmic Ca²⁺-concentration ([Ca²⁺]_i), unexpectedly Ca²⁺-antagonist TMB-8 was found to raise considerably [Ca²⁺]_i, and inhibited not only the formation of prespore cells, but also their maintenance in the monolayer cultures of Dictyostelium discoideum . This seems to indicate that higher [Ca²⁺]_i is unfavorable to the prespore differentiation. In this study, we adopted the monolayer culture technique to monitor cell differentiation. However, in high density monolayers there arised a number of unique cells which was highly vacuolated and morphologically intermediate between the stalk and spore cells. These vacuolated cells having both cellulosic wall and spore coat were also induced by differentiation inducing factor (DIF). Thus the monolayer culture system used might be not necessarily qualified to monitor the terminal differentiation of Dictyostelium cells. Nevertheless, the data presented here have strongly suggested that DIF have two physiologically valued roles: 1) Induction of the membrane fusion of vesicles and/or vacuoles (vacuolization), and 2) Induction of the fusion between the cell membrane and vacuole (or vesicle) membrane (exocytosis).     

α-Synuclein modulation of Ca2+ signaling in human neuroblastoma (SH-SY5Y) cells

Parkinson's disease (PD) is characterized in part by the presence of α-synuclein (α-syn) rich intracellular inclusions (Lewy bodies). Mutations and multiplication of the α-synuclein gene ( SNCA ) are associated with familial PD. Since Ca²⁺ dyshomeostasis may play an important role in the pathogenesis of PD, we used fluorimetry in fura-2 loaded SH-SY5Y cells to monitor Ca²⁺ homeostasis in cells stably transfected with either wild-type α-syn, the A53T mutant form, the S129D phosphomimetic mutant or with empty vector (which served as control). Voltage-gated Ca²⁺ influx evoked by exposure of cells to 50 mM K⁺ was enhanced in cells expressing all three forms of α-syn, an effect which was due specifically to increased Ca²⁺ entry via L-type Ca²⁺ channels. Mobilization of Ca²⁺ by muscarine was not strikingly modified by any of the α-syn forms, but they all reduced capacitative Ca²⁺ entry following store depletion caused either by muscarine or thapsigargin. Emptying of stores with cyclopiazonic acid caused similar rises of [Ca²⁺]_i in all cells tested (with the exception of the S129D mutant), and mitochondrial Ca²⁺ content was unaffected by any form of α-synuclein. However, only WT α-syn transfected cells displayed significantly impaired viability. Our findings suggest that α-syn regulates Ca²⁺ entry pathways and, consequently, that abnormal α-syn levels may promote neuronal damage through dysregulation of Ca²⁺ homeostasis.     

Ca2+/Calmodulin-Mediated Regulation of Agonist-Induced Sequestration of Gq Protein-Coupled Histamine H1 Receptors in Human U373 MG Astrocytoma Cells

Abstract: We investigated the regulation by intracellular Ca²⁺ of agonist-induced sequestration of G_q protein-coupled histamine H₁ receptors in human U373 MG astrocytoma cells. Histamine-induced sequestration of H₁ receptors from the cell surface membrane was detected as the loss of [³H]mepyramine binding sites on intact cells accessible to the hydrophilic H₁-receptor antagonist pirdonium. The changes in the pirdonium-sensitive binding of [³H]mepyramine were mirrored by changes in the subcellular distribution of H₁ receptors detected by sucrose density gradient centrifugation. The histamine-induced sequestration of H₁ receptors did not occur in hypertonic medium, in which clathrin-mediated endocytosis is known to be inhibited, but was significantly accelerated in the absence of extracellular Ca²⁺ or in the presence of the calmodulin antagonists W-7 and calmidazolium. Inhibitors of protein kinase C (H-7 and GF109203X), Ca²⁺/calmodulin-dependent protein kinase II (KN-62), or protein phosphatase 2B (FK506) did not alter the time course of H₁-receptor sequestration. These results provide the first evidence that agonist-induced, clathrin-mediated sequestration of G_q protein-coupled receptors is transiently inhibited by Ca²⁺/calmodulin, with the result that receptors remain on the cell surface membrane during the early stage of agonist stimulation.     

Recombinant stigmatic self-incompatibility (S-) protein elicits a Ca2+ transient in pollen of Papaver rhoeas

Evidence for Ca²⁺ signalling in pollen during the self-incompatibility (SI) response in Papaver rhoeas L. has been presented previously. However, it was not known whether the S-protein alone could act as an elicitor of the response or whether the presence of other stigmatic components was required, since relatively crude stigmatic extracts had been used. The S ₁ gene has since been cloned and its product expressed in Escherichia coli has been shown to exhibit biological activity. In this paper it is reported that the recombinant protein (S₁e) elicits a transient rise in [Ca²⁺]_i in incompatible pollen. The Ca²⁺ signal appears indistinguishable from that elicited by S-gene products partially purified from plant extracts in terms of both its timing and spatial distribution. Pollen tube growth is arrested directly after the rise in [Ca²⁺]_i.
The results provide direct evidence that the S-protein alone acts as an elicitor which triggers the Ca²⁺ signal for the pollen SI response. In addition, it is now clear that the recombinant S-protein does not require several post-translational processing events which take place in the plant to act as an elicitor. With respect to the spatial distribution of the Ca²⁺ transient, data are presented which correlate the localized rise in intracellular Ca²⁺ ([Ca²⁺]_i) with the 'nuclear complex' and the endoplasmic reticulum which is associated with this region.     

Involvement of Protein Kinase C in Ca2+-Signaling Pathways to Activation of AP-1 DNA-Binding Activity Evoked via NMDA- and Voltage-Gated Ca2+ Channels

Abstract: Stimulation of cultured cerebellar granule cells with N -methyl- d -aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein-1 (AP-1) DNA-binding activity, which can be monitored by an increase in 12- O -tetradecanoylphorbol 13-acetate (TPA)-responsive element (TRE)-binding activity, in concert with c- fos induction. For this increase in TRE-binding activity, Ca²⁺ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca²⁺ chelator, BAPTA-AM, abolished this increase. Close correspondence between the dose-response curves of ⁴⁵Ca²⁺ uptake and TRE-binding activity by NMDA or KA suggested that Ca²⁺ influx not only triggered sequential activation of Ca²⁺-signaling processes leading to the increase in TRE-binding activity, but also controlled its increased level. Stimulation of non-NMDA receptors by KA mainly caused Ca²⁺ influx through voltage-gated Ca²⁺ channels, whereas stimulation of NMDA receptors caused Ca²⁺ influx through NMDA-gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE-binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down-regulation of PKC inhibited the increase in TRE-binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca²⁺-signaling pathways for the increase in TRE-binding activity coupled with the activation of NMDA- and non-NMDA receptors.     

Catecholamine Secretion from Bovine Adrenal Chromaffin Cells: The Role of the Na+/Ca2+ Exchanger and the Intracellular Ca2+ Pool

Abstract: The role of the Na⁺/Ca²⁺ exchanger and intracellular nonmitochondrial Ca²⁺ pool in the regulation of cytosolic free calcium concentration ([Ca²⁺]_i) during catecholamine secretion was investigated. Catecholamine secretion and [Ca²⁺]_i were simultaneously monitored in a single chromaffin cell. After high-K⁺ stimulation, control cells and cells in which the Na⁺/Ca²⁺ exchange activity was inhibited showed similar rates of [Ca²⁺]_i elevation. However, the recovery of [Ca²⁺]_i to resting levels was slower in the inhibited cells. Inhibition of the exchanger increased the total catecholamine secretion by prolonging the secretion. Inhibition of the Ca²⁺ pump of the intracellular Ca²⁺ pool with thapsigargin caused a significant delay in the recovery of [Ca²⁺]_i and greatly enhanced the secretory events. These data suggest that both the Na⁺/Ca²⁺ exchanger and the thapsigargin-sensitive Ca²⁺ pool are important in the regulation of [Ca²⁺]_i and, by modulating the time course of secretion, are important in determining the extent of secretion.     

Involvement of Ca2+ and cGMP in bacterial taxis

Abstract The level of cGMP in a suspension of Escherichia coli cells increased transiently upon the addition of chemoattractants. Ca²⁺ (1 mM), but not Mg²⁺, produced constant tumbling of cells in the presence of the ionophore A23187. The effect was observed either in stationary-state cells, or in a logarithmic culture treated with EDTA to increase permeability by A23187. Under the same conditions, Ca²⁺ decreased the cytoplasmic level of cGMP. In Phormidium uncinatum , rapid ⁴⁵Ca²⁺ accumulation followed a light-dark stimulus, or the addition of tetramethylquinone (TMQ), a chemorepellent. La³⁺, which increases the reversal rate, also enhanced the level of cytoplasmic Ca²⁺, presumably by blocking the outward Ca²⁺ flux. In both E. coli and P. uncinatum Ca²⁺ inhibited methylaccepting chemotaxis protein (MCP) methylation. It is concluded that cGMP and Ca²⁺ are secondary messengers in taxis information-processing.     

Ca2+-Dependent Inactivation of P-Type Calcium Channels in Nerve Terminals

Abstract: Rapid Ca²⁺ signals evoked by K⁺ depolarization of rat cerebral cortical synaptosomes were measured by dual-channel Ca²⁺ spectrofluorometry coupled to a stopped-flow device. Kinetic analysis of the signal rise phase at various extracellular Ca²⁺ concentrations revealed that the responsible voltage-dependent Ca²⁺ channels, previously identified as P-type Ca²⁺ channels, inactivate owing to the rise in intracellular Ca²⁺ levels. At millimolar extracellular Ca²⁺ concentrations the channels were inactivated very rapidly and the rate was dependent on the high influx rate of Ca²⁺, thus limiting the Ca²⁺ signal amplitudes to 500–600 n M. A slower, probably voltage-dependent regulation appears to be effective at lower Ca²⁺ influx rates, leading to submaximal Ca²⁺ signal amplitudes. The functional feedback regulation of calcium channels via a sensor for intracellular Ca²⁺ levels appears to be responsible for the different inhibition characteristics of Cd²⁺ versus ω-agatoxin IVa.     

The Metabotropic Glutamate Receptor mGluR5 Induces Calcium Oscillations in Cultured Astrocytes via Protein Kinase C Phosphorylation

Abstract: The metabotropic glutamate receptor mGluR5, but not the closely related mGluR1, is expressed in cultured astrocytes, and this expression is up-regulated by specific growth factors. We investigated the capability and underlying mechanisms of mGluR5 to induce oscillatory responses of intracellular calcium concentration ([Ca²⁺]_i) in cultured rat astrocytes. Single-cell [Ca²⁺]_i recordings indicated that an mGluR-selective agonist, (1 S ,3 R )-1-aminocyclopentane-1,3-dicarboxylate (1 S ,3 R -ACPD), elicits [Ca²⁺]_i oscillations in good agreement with the growth factor-induced up-regulation of mGluR5 in cultured astrocytes. A protein kinase C (PKC) inhibitor, bisindolylmaleimide I, converted a 1 S ,3 R -ACPD-mediated oscillatory response into a nonoscillatory response. In addition, the PKC activator phorbol 12-myristate 13-acetate completely abolished the [Ca²⁺]_i increase. These and other pharmacological properties of 1 S ,3 R -ACPD-induced [Ca²⁺]_i oscillations correlate well with those of the cloned mGluR5 characterized in heterologous expression systems. Furthermore, the potential involvement of protein phosphatases in [Ca²⁺]_i oscillations is suggested. The present study demonstrates that mGluR5 is capable of inducing [Ca²⁺]_i oscillations in cultured astrocytes and that phosphorylation/dephosphorylation of mGluR5 is critical in [Ca²⁺]_i oscillations, analogous to the cloned mGluR5 expressed in heterologous cell lines.     

Regulation of the Ca2+ Sensitivity of Exocytosis by Rab3a

Abstract: Ca²⁺ ions trigger the release of hormones and neurotransmitters and contribute to making the secretory vesicles competent for fusion. Here, we present evidence for the involvement of the GTP-binding protein Rab3a in the sensitivity of the exocytotic process to internal [Ca²⁺]. The secretory activity of bovine adrenal chromaffin cells was elicited by Ca²⁺ dialysis through a patch-clamp pipette and assayed by monitoring changes in cell membrane capacitance. Microinjection of antisense oligonucleotides directed to rab3a mRNA increased the secretory activity observed at low (0.2–4 µ M ) [Ca²⁺], but did not change the maximal activity observed at 10 µ M free [Ca²⁺]. Moreover, after a train of depolarizing stimuli, the secretory activity of antisense-injected cells dialyzed with 10 µ M [Ca²⁺] was increased significantly compared with that of control cells. This result suggests that the activity of either Rab3a or its partners might change upon stimulation. We conclude that Rab3a, together with its partners, participates in the Ca²⁺ dependence of exocytosis and that its activity is modulated further in a stimulus-dependent manner. These findings should provide some clues to elucidate the role of Rab3a in synaptic plasticity.     

Ca2+ and Phospholipid-Dependent Protein Kinase Activity and Phosphorylation of Endogenous Proteins in Bovine Adrenal Medulla

Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca²⁺/ phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo-daltons) showed an absolute requirement for the presence of both Ca²⁺ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca²⁺/phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca²⁺/phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca²⁺/phos-pholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca²⁺/phospholipid-dependent protein kinase in chromaffin cell function.