(Questions) on phloem biology. 1. Electropotential waves, Ca fluxes and cellular cascades along the propagation pathway

This review explores the relationships between electrical long-distance signalling, Ca²⁺ influx coincident with propagation of electropotential waves, and cellular responses to Ca²⁺ influx including the consequences for sieve-tube conductivity and mass flow. Ca²⁺ influx is inherent to electropotential waves and appears to constitute the key link between rapid physical signals and resultant chemical cascades in sieve tubes and adjacent cells. Members of several channel groups are likely involved the regulation of Ca²⁺ levels in sieve elements. Among them are hyperpolarization-activated, depolarization-activated, and mechanosensitive Ca²⁺ channels located in the plasma membrane and Ca²⁺ dependent Ca²⁺ channels that reside in ER-membranes of sieve elements. These channels collectively determine intracellular Ca²⁺ levels in sieve elements and their neighbour cells. The latter cells react to Ca²⁺ elevation by inducing diverse functional responses dependent on the cell type. If the Ca²⁺ concentration in sieve elements surpasses a threshold level, dual sieve-plate occlusion by proteins and callose deposition is triggered. Occlusion is reversed when Ca²⁺ levels subside. Electrical messages may regulate the degree of sieve plate hydraulic conductivity in intact plants by partial sieve-plate occlusion that has a major impact on volume flow through sieve tubes. Furthermore, complete but temporary occlusion of sieve tubes may modify mass flow patterns in intact plants.     

Sieve Element Ca2+ Channels as Relay Stations between Remote Stimuli and Sieve Tube Occlusion in Vicia faba

Damage induces remote occlusion of sieve tubes in Vicia faba by forisome dispersion, triggered during the passage of an electropotential wave (EPW). This study addresses the role of Ca²⁺ channels and cytosolic Ca²⁺ elevation as a link between EPWs and forisome dispersion. Ca²⁺ channel antagonists affect the initial phase of the EPW as well as the prolonged plateau phase. Resting levels of sieve tube Ca²⁺ of ∼50 nM were independently estimated using Ca²⁺-selective electrodes and a Ca²⁺-sensitive dye. Transient changes in cytosolic Ca²⁺ were observed in phloem tissue in response to remote stimuli and showed profiles similar to those of EPWs. The measured elevation of Ca²⁺ in sieve tubes was below the threshold necessary for forisome dispersion. Therefore, forisomes need to be associated with Ca²⁺ release sites. We found an association between forisomes and endoplasmic reticulum (ER) at sieve plates and pore-plasmodesma units where high-affinity binding of a fluorescent Ca²⁺ channel blocker mapped an increased density of Ca²⁺ channels. In conclusion, propagation of EPWs in response to remote stimuli is linked to forisome dispersion through transiently high levels of parietal Ca²⁺, release of which depends on both plasma membrane and ER Ca²⁺ channels.     

Plant- and stimulus-specific variations in remote-controlled sieve-tube occlusion

Phloem injury triggers local sieve-plate occlusion including callose-mediated constriction and protein plugging of sieve pores. In intact plants, reversible sieve-plate occlusion is induced by electric potential waves (EPWs)—accompanied by Ca²⁺-influx—as result of distant burning. Here, we present additional results which pertain to (a) the variability of EPW-profiles in relation to forisome conformation in intact Vicia faba plants and (b) the differential occlusion reactions to burning and cutting in various plant species. A correlation between stimulus perception and mode of phloem loading is discussed.Key words: callose, electrical potential waves, forisome, membrane potential, phloem transport, sieve-element occlusion, wound potentials     

Ca2+-mediated remote control of reversible sieve tube occlusion in Vicia faba

According to an established concept, injury of the phloem triggers local sieve plate occlusion including callose-mediated constriction and, possibly, protein plugging of the sieve pores. Sieve plate occlusion can also be achieved by distant stimuli, depends on the passage of electropotential waves (EPWs), and is reversible in intact plants. The time-course of the wound response was studied in sieve elements of main veins of intact Vicia faba plants using confocal and multiphoton microscopy. Only 15-45 s after burning a leaf tip, forisomes (giant protein bodies specific for legume sieve tubes) suddenly dispersed, as observed at 3-4 cm from the stimulus site. The dispersion was reversible; the forisomes had fully re-contracted 7-15 min after burning. Meanwhile, callose appeared at the sieve pores in response to the heat shock. Callose production reached a maximum after approximately 20 min and was also reversible; callose degraded over the subsequent 1-2 h. The heat induction of both modes of occlusion coincided with the passage of an EPW visualized by electrophysiology or the potential-sensitive dye RH-414. In contrast to burning, cutting of the leaf tip induced neither an EPW nor callose deposition. The data are consistent with a remote-controlled occlusion of sieve plates depending on the longitudinal propagation of an EPW releasing Ca(2+) into the sieve element lumen. It is hypothesized that forisome plugs and callose constriction are removed once the cytosolic calcium level has returned to the initial level in those sieve tubes.     

Ca2+ Release through Ryanodine Receptors in the Sarcoplasmic Reticulum and Ca2+ Sequestration by the Mitochondria in Smooth Muscle Cells

The contribution of the Ca²⁺-induced Ca²⁺ release (CICR) mechanism in excitation-contraction (E-C) coupling and the tightness of the coupling between Ca²⁺ influx and Ca²⁺ release are still controversial in smooth muscle cells (SMC). In SMC isolated from the guinea-pig vas deferens or urinary bladder, a depolarizing stimulus initially induced spot-like increases in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ), called “Ca²⁺ hot spots,” at several superficial areas in the cell. When a weak stimulus (a small or a short depolarizing step) was applied, only a few Ca²⁺ hot spots appeared transiently in the superficial area but did not spread into other regions, to trigger global [Ca²⁺] _i rise. Such depolarization-evoked local Ca2+ transients were distinctive from spontaneous Ca²⁺ sparks, since the former were susceptible to Ca²⁺ blockers, ryanodine, and inhibitors of the Ca²⁺ pump in the sarcoplasmic reticulum (SR), suggesting pivotal roles of Ca²⁺ influx through voltage-dependent Ca²⁺ channels (VDCC) and Ca²⁺ release from the SR through ryanodine receptors (RyR) for the activation of Ca²⁺ spots. Frequently discharging Ca²⁺ spark sites (FDS) under resting conditions were located exactly in the same areas as Ca²⁺ hot spots evoked by depolarization, indicating the existence of distinct local junction sites for tight coupling between VDCC in the plasmalemma and RyR in the SR. Co-localization of clusters of RyR and large-conductance Ca²⁺-activated K⁺ (BK) channels was also suggested. The fast and tight coupling for CICR in these junctional sites was triggered also by an action potential, whereas a slower spread of Ca²⁺ wave to the whole-cell areas suggests the loose coupling in propagating CICR to other cell areas. It can therefore be postulated that CICR may occur in two steps upon depolarization; the initial CICR in distinct junctional sites shows tight coupling between Ca²⁺ influx and release, and the following CICR may propagate slow Ca²⁺ waves to other areas. Ryanodine receptors form a multiprotein complex with molecules such as calsequestrin, junctin, triadin, junctophilins, and FK506-binding proteins, which directly or indirectly regulate the RyR activity and the tight coupling. Moreover, an evoked Ca²⁺ spot may enhance Ca²⁺ uptake by neighboring mitochondria and their ATP production to increase energy supply to the Ca²⁺ pump of the SR in the microdomain.     

Store-operated Ca2+ entry is not required for fertilization-induced Ca2+ signaling in mouse eggs

Repetitive oscillations in cytoplasmic Ca²⁺ due to periodic Ca²⁺ release from the endoplasmic reticulum (ER) drive mammalian embryo development following fertilization. Influx of extracellular Ca²⁺ to support the refilling of ER stores is required for sustained Ca²⁺ oscillations, but the mechanisms underlying this Ca²⁺ influx are controversial. Although store-operated Ca²⁺ entry (SOCE) is an appealing candidate mechanism, several groups have arrived at contradictory conclusions regarding the importance of SOCE in oocytes and eggs. To definitively address this question, Ca²⁺ influx was assessed in oocytes and eggs lacking the major components of SOCE, the ER Ca²⁺ sensor STIM proteins, and the plasma membrane Ca²⁺ channel ORAI1. We generated oocyte-specific conditional knockout (cKO) mice for Stim1 and Stim2, and also generated Stim1/2 double cKO mice. Females lacking one or both STIM proteins were fertile and their ovulated eggs displayed normal patterns of Ca²⁺ oscillations following fertilization. In addition, no impairment was observed in ER Ca²⁺ stores or Ca²⁺ influx following store depletion. Similar studies were performed on eggs from mice globally lacking ORAI1; no abnormalities were observed. Furthermore, spontaneous Ca²⁺ influx was normal in oocytes from Stim1/2 cKO and ORAI1-null mice. Finally, we tested if TRPM7-like channels could support spontaneous Ca²⁺ influx, and found that it was largely prevented by NS8593, a TRPM7-specific inhibitor. Fertilization-induced Ca²⁺ oscillations were also impaired by NS8593. Combined, these data robustly show that SOCE is not required to support appropriate Ca²⁺ signaling in mouse oocytes and eggs, and that TRPM7-like channels may contribute to Ca²⁺ influx that was previously attributed to SOCE.     

Magnetic field-induced Ca2+ intake by mesenchymal stem cells is mediated by intracellular Zn2+ and accompanied by a Zn2+ influx

Chronic exposure to magnetic fields (MFs) has a diverse range of effects on biological systems but definitive molecular mechanisms of the interaction remain largely unknown. One of the most frequently reported effects of MF exposure is an elevated concentration of intracellular Ca²⁺ through disputed pathways. Other prominent effects include increased oxidative stress and upregulation of neural markers through EGFR activation in stem cells. Further characterization of cascades triggered by MF exposure is hindered by the phenotype diversity of biological models used in the literature. In an attempt to reveal more mechanistic data in this field, we combined the most commonly used biological model and MF parameters with the most commonly reported effects of MFs.Based on clues from the pathways previously defined as sensitive to MFs (EGFR and Zn²⁺-binding enzymes), the roles of different types of channels (voltage gated Ca²⁺ channels, NMDA receptors, TRP channels) were inquired in the effects of 50 Hz MFs on bone marrow-derived mesenchymal stem cells. We report that, an influx of Zn²⁺ accompanies MF-induced Ca²⁺ intake, which is only attenuated by the broad-range inhibitor of TRP channels and store-operated Ca²⁺ entry (SOCE), 2-Aminoethoxydiphenyl borate (2-APB) among other blockers (memantine, nifedipine, ethosuximide and gabapentin). Interestingly, cation influx completely disappears when intracellular Zn²⁺ is chelated. Our results rule out voltage gated Ca²⁺ channels as a gateway to MF-induced Ca²⁺ intake and suggest Zn²⁺-related channels as a new focus in the field.     

Store-independent Orai1-mediated Ca2+ entry and cancer

Ca²⁺ channels play an important role in the development of different types of cancer, and considerable progress has been made to understand the pathophysiological mechanisms underlying the role of Ca²⁺ influx in the development of different cancer hallmarks. Orai1 is among the most ubiquitous and multifunctional Ca²⁺ channels. Orai1 mediates the highly Ca²⁺-selective Ca²⁺ release-activated current (I_CRAC) and participates in the less Ca²⁺-selective store-operated current (I_SOC), along with STIM1 or STIM1 and TRPC1, respectively. Furthermore, Orai1 contributes to a variety of store-independent Ca²⁺ influx mechanisms, including the arachidonate-regulated Ca²⁺ current, together with Orai3 and the plasma membrane resident pool of STIM1, as well as the constitutive Ca²⁺ influx processes activated by the secretory pathway Ca²⁺-ATPase-2 (SPCA2) or supported by physical and functional interaction with the small conductance Ca²⁺-activated K⁺ channel 3 (SK3) or the voltage-dependent K_v10.1 channel. This review summarizes the current knowledge concerning the store-independent mechanisms of Ca²⁺ influx activation through Orai1 channels and their role in the development of different cancer features.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

Enhanced ER Ca2+ store filling by overexpression of SERCA2b promotes IP3-evoked puffs

Liberation of Ca²⁺ from the endoplasmic reticulum (ER) through inositol trisphosphate receptors (IP₃R) is modulated by the ER Ca²⁺ content, and overexpression of SERCA2b to accelerate Ca²⁺ sequestration into the ER has been shown to potentiate the frequency and amplitude of IP₃-evoked Ca²⁺ waves in Xenopus oocytes. Here, we examined the effects of SERCA overexpression on the elementary IP₃-evoked puffs to elucidate whether ER [Ca²⁺] may modulate IP₃R function via luminal regulatory sites in addition to simply determining the size of the available store and electrochemical driving force for Ca²⁺ release. SERCA2b and Ca²⁺ permeable nicotinic plasmalemmal channels were expressed in oocytes, and hyperpolarizing pulses were delivered to induce Ca²⁺ influx and thereby load ER stores. Puffs evoked by photoreleased IP₃ were significantly potentiated in terms of numbers of responding sites, frequency and amplitude following transient Ca²⁺ influx in SERCA-overexpressing cells, whereas little change was evident with SERCA overexpression alone or following Ca²⁺ influx in control cells not overexpressing SERCA. Intriguingly, we observed the appearance of a new population of puffs that arose after long latencies and had prolonged durations supporting the notion of luminal regulation of IP₃R gating kinetics.     

Modeling the Contributions of Ca2+ Flows to Spontaneous Ca2+ Oscillations and Cortical Spreading Depression-Triggered Ca2+ Waves in Astrocyte Networks

Astrocytes participate in brain functions through Ca²⁺ signals, including Ca²⁺ waves and Ca²⁺ oscillations. Currently the mechanisms of Ca²⁺ signals in astrocytes are not fully clear. Here, we present a computational model to specify the relative contributions of different Ca²⁺ flows between the extracellular space, the cytoplasm and the endoplasmic reticulum of astrocytes to the generation of spontaneous Ca²⁺ oscillations (CASs) and cortical spreading depression (CSD)-triggered Ca²⁺ waves (CSDCWs) in a one-dimensional astrocyte network. This model shows that CASs depend primarily on Ca²⁺ released from internal stores of astrocytes, and CSDCWs depend mainly on voltage-gated Ca²⁺ influx. It predicts that voltage-gated Ca²⁺ influx is able to generate Ca²⁺ waves during the process of CSD even after depleting internal Ca²⁺ stores. Furthermore, the model investigates the interactions between CASs and CSDCWs and shows that the pass of CSDCWs suppresses CASs, whereas CASs do not prevent the generation of CSDCWs. This work quantitatively analyzes the generation of astrocytic Ca²⁺ signals and indicates different mechanisms underlying CSDCWs and non-CSDCWs. Research on the different types of Ca²⁺ signals might help to understand the ways by which astrocytes participate in information processing in brain functions.     

Regulation of CRAC channels by Ca2+-dependent inactivation

CRAC channels are a major route for Ca²⁺ influx in eukaryotic cells. The channels show prominent Ca²⁺-dependent inactivation through two spatially and temporally distinct mechanisms: fast inactivation, which develops over milliseconds and is triggered by Ca²⁺ near the mouth of the channel and slow inactivation, which arises over tens of seconds and requires a rise in global cytosolic Ca²⁺. Slow inactivation is controlled physiologically by Ca²⁺ uptake into mitochondria through the MCU. Site-directed mutagenesis studies on STIM1 and Orai1 have led to new molecular insight into how fast inactivation occurs. This review describes properties and molecular mechanisms that contribute to these important Ca²⁺-dependent inhibitory pathways.     

Ca2+-dependent and Ca2+-independent somatic release from trigeminal neurons

Besides the nerve endings, the soma of trigeminal neurons also respond to membrane depolarizations with the release of neurotransmitters and neuromodulators in the extracellular space within the ganglion, a process potentially important for the cross-communication between neighboring sensory neurons. In this study, we addressed the dependence of somatic release on Ca²⁺ influx in trigeminal neurons and the involvement of the different types of voltage-gated Ca²⁺ (Cav) channels in the process. Similar to the closely related dorsal root ganglion neurons, we found two kinetically distinct components of somatic release, a faster component stimulated by voltage but independent of the Ca²⁺ influx, and a slower component triggered by Ca²⁺ influx. The Ca²⁺-dependent component was inhibited 80% by ω-conotoxin-MVIIC, an inhibitor of both N- and P/Q-type Cav channels, and 55% by the P/Q-type selective inhibitor ω-agatoxin-IVA. The selective L-type Ca²⁺ channel inhibitor nimodipine was instead without effect. These results suggest a major involvement of N- and P/Q-, but not L-type Cav channels in the somatic release of trigeminal neurons. Thus antinociceptive Cav channel antagonists acting on the N- and P/Q-type channels may exert their function by also modulating the somatic release and cross-communication between sensory neurons.     

AMPA induced Ca2+ influx in motor neurons occurs through voltage gated Ca2+ channel and Ca2+ permeable AMPA receptor

The rise in intracellular Ca²⁺ mediated by AMPA subtype of glutamate receptors has been implicated in the pathogenesis of motor neuron disease, but the exact route of Ca²⁺ entry into motor neurons is not clearly known. In the present study, we examined the role of voltage gated calcium channels (VGCCs) in AMPA induced Ca²⁺ influx and subsequent intracellular signaling events responsible for motor neuron degeneration. AMPA stimulation caused sodium influx in spinal neurons that would depolarize the plasma membrane. The AMPA induced [Ca²⁺]_i rise in motor neurons as well as other spinal neurons was drastically reduced when extracellular sodium was replaced with NMDG, suggesting the involvement of voltage gated calcium channels. AMPA mediated rise in [Ca²⁺]_i was significantly inhibited by L-type VGCC blocker nifedipine, whereas ω-agatoxin-IVA and ω-conotoxin-GVIA, specific blockers of P/Q type and N-type VGCC were not effective. 1-Napthyl-acetyl spermine (NAS), an antagonist of Ca²⁺ permeable AMPA receptors partially inhibited the AMPA induced [Ca²⁺]_i rise but selectively in motor neurons. Measurement of AMPA induced currents in whole cell voltage clamp mode suggests that a moderate amount of Ca²⁺ influx occurs through Ca²⁺ permeable AMPA receptors in a subpopulation of motor neurons. The AMPA induced mitochondrial calcium loading [Ca²⁺]_m, mitochondrial depolarization and neurotoxicity were also significantly reduced in presence of nifedipine. Activation of VGCCs by depolarizing concentration of KCl (30 mM) in extracellular medium increased the [Ca²⁺]_i but no change was observed in mitochondrial Ca²⁺ and membrane potential. Our results demonstrate that a subpopulation of motor neurons express Ca²⁺ permeable AMPA receptors, however the larger part of Ca²⁺ influx occurs through L-type VGCCs subsequent to AMPA receptor activation and consequent mitochondrial dysfunction is the trigger for motor neuron degeneration. Nifedipine is an effective protective agent against AMPA induced mitochondrial stress and degeneration of motor neurons.     

Modulation of Endoplasmic Reticulum Ca2+ Store Filling by Cyclic ADP-ribose Promotes Inositol Trisphosphate (IP3)-evoked Ca2+ Signals

In addition to its well established function in activating Ca²⁺ release from the endoplasmic reticulum (ER) through ryanodine receptors (RyR), the second messenger cyclic ADP-ribose (cADPR) also accelerates the activity of SERCA pumps, which sequester Ca²⁺ into the ER. Here, we demonstrate a potential physiological role for cADPR in modulating cellular Ca²⁺ signals via changes in ER Ca²⁺ store content, by imaging Ca²⁺ liberation through inositol trisphosphate receptors (IP₃R) in Xenopus oocytes, which lack RyR. Oocytes were injected with the non-metabolizable analog 3-deaza-cADPR, and cytosolic [Ca²⁺] was transiently elevated by applying voltage-clamp pulses to induce Ca²⁺ influx through expressed plasmalemmal nicotinic channels. We observed a subsequent potentiation of global Ca²⁺ signals evoked by strong photorelease of IP₃, and increased numbers of local Ca²⁺ puffs evoked by weaker photorelease. These effects were not evident with cADPR alone or following cytosolic Ca²⁺ elevation alone, indicating that they did not arise through direct actions of cADPR or Ca²⁺ on the IP₃R, but likely resulted from enhanced ER store filling. Moreover, the appearance of a new population of puffs with longer latencies, prolonged durations, and attenuated amplitudes suggests that luminal ER Ca²⁺ may modulate IP₃R function, in addition to simply determining the size of the available store and the electrochemical driving force for release.     

TRPC5 Is a Ca2+-activated Channel Functionally Coupled to Ca2+-selective Ion Channels

TRPC5 forms non-selective cation channels. Here we studied the role of internal Ca²⁺ in the activation of murine TRPC5 heterologously expressed in human embryonic kidney cells. Cell dialysis with various Ca²⁺ concentrations (Ca²⁺_i) revealed a dose-dependent activation of TRPC5 channels by internal Ca²⁺ with EC₅₀ of 635.1 and 358.2 nm at negative and positive membrane potentials, respectively. Stepwise increases of Ca²⁺_i induced by photolysis of caged Ca²⁺ showed that the Ca²⁺ activation of TRPC5 channels follows a rapid exponential time course with a time constant of 8.6 ± 0.2 ms at Ca²⁺_i below 10 μm, suggesting that the action of internal Ca²⁺ is a primary mechanism in the activation of TRPC5 channels. A second slow activation phase with a time to peak of 1.4 ± 0.1 s was also observed at Ca²⁺_i above 10 μm. In support of a Ca²⁺-activation mechanism, the thapsigargin-induced release of Ca²⁺ from internal stores activated TRPC5 channels transiently, and the subsequent Ca²⁺ entry produced a sustained TRPC5 activation, which in turn supported a long-lasting membrane depolarization. By co-expressing STIM1 plus ORAI1 or the α₁C and β₂ subunits of L-type Ca²⁺ channels, we found that Ca²⁺ entry through either calcium-release-activated-calcium or voltage-dependent Ca²⁺ channels is sufficient for TRPC5 channel activation. The Ca²⁺ entry activated TRPC5 channels under buffering of internal Ca²⁺ with EGTA but not with BAPTA. Our data support the hypothesis that TRPC5 forms Ca²⁺-activated cation channels that are functionally coupled to Ca²⁺-selective ion channels through local Ca²⁺ increases beneath the plasma membrane.     

Formyl-peptide receptor like 1: a potent mediator of the Ca2+ release-activated Ca2+ current ICRAC

In electrically non-excitable cells, one major source of Ca²⁺ influx is through the store-operated (or Ca²⁺ release-activated Ca²⁺) channel by which the process of emptying the intracellular Ca²⁺ stores results in the activation of Ca²⁺ channels in the plasma membrane. Using both whole-cell patch-clamp and Ca²⁺ imaging technique, we describe the electrophysiology mechanism underlying formyl-peptide receptor like 1 (FPRL1) linked to intracellular Ca²⁺ mobilization. The FPRL1 agonists induced Ca²⁺ release from the endoplasmic reticulum and subsequently evoked I_CRAC-like currents displaying fast inactivation in K562 erythroleukemia cells which expresses FPRL1, but had almost no effect in K562 cells treated with FPRL1 RNA-interference and HEK293 cells which showed no FPRL1 expression. The currents were impaired after either complete store depletion by the sarco/endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, or after inhibition of PLC by U73122. Our results present the first evidence that FPRL1 is a potent mediator in the activation of CRAC channels.     

The identification of a caffeine-induced Ca2+ influx pathway in rat primary sensory neurons

Caffeine-induced Ca²⁺ transients (CICTs) in rabbit nodose ganglion neurons (NGNs) are produced by two distinct mechanisms: release from intracellular stores via ryanodine receptors and Ca²⁺ influx across the plasma membrane, due to activation of an unknown receptor. In isolated rat NGNs, we used single-cell microfluorimetry to measure changes in intracellular Ca²⁺ and to test whether TRPV1 receptors underlie the Ca²⁺ influx pathway. Caffeine (10 mM) evoked CICTs in all NGNs tested (n = 47) averaging 365 ± 32 nM. CICTs were partially dependent upon a Ca²⁺ influx pathway that ranged between 33% and 98% of the total Ca²⁺ transient. Application of two selective TRPV1 antagonists significantly attenuated CICTs. The peak average amplitudes of CICTs in Ca²⁺-free Locke solution and Ca²⁺-free Locke solution with IRTX or with BCTC were not significantly different from one another (n = 5 and 7, respectively). These observations suggest that caffeine can induce Ca²⁺ influx by activating TRPV1 channels.     

Ca2+ influx-dependent refilling of intracellular Ca2+ stores determines the frequency of Ca2+ oscillations in fertilized mouse eggs

On mammalian fertilization, long-lasting Ca²⁺ oscillations are induced in the egg by the fusing spermatozoon. While each transient Ca²⁺ increase in Ca²⁺ concentration ([Ca²⁺]) in the cytosol is due to Ca²⁺ release from the endoplasmic reticulum (ER), Ca²⁺ influx from outside is required for Ca²⁺ oscillations to persist. In this study, we investigated how Ca²⁺ influx is interrelated to the cycle of Ca²⁺ release and uptake by the intracellular Ca²⁺ stores during Ca²⁺ oscillations in fertilized mouse eggs. In addition to monitoring cytosolic [Ca²⁺] with fura-2, the influx rate was evaluated using Mn²⁺ quenching technique, and the change in [Ca²⁺] in the ER lumen was visualized with a targeted fluorescent probe. We found that the influx was stimulated after each transient Ca²⁺ release and then diminished gradually to the basal level, and demonstrated that the ER Ca²⁺ stores once depleted by Ca²⁺ release were gradually refilled until the next Ca²⁺ transient to be initiated. Experiments altering extracellular [Ca²⁺] in the middle of Ca²⁺ oscillations revealed the dependence of both the refilling rate and the oscillation frequency on the rate of Ca²⁺ influx, indicating the crucial role of Ca²⁺ influx in determining the intervals of Ca²⁺ transients. As for the influx pathway supporting Ca²⁺ oscillations to persist, STIM1/Orai1-mediated store-operated Ca²⁺ entry (SOCE) may not significantly contribute, since neither known SOCE blockers nor the expression of protein fragments that interfere the interaction between STIM1 and Orai1 inhibited the oscillation frequency or the influx rate.     

Suppression of Ca2+ syntillas increases spontaneous exocytosis in mouse adrenal chromaffin cells

A central concept in the physiology of neurosecretion is that a rise in cytosolic [Ca²⁺] in the vicinity of plasmalemmal Ca²⁺ channels due to Ca²⁺ influx elicits exocytosis. Here, we examine the effect on spontaneous exocytosis of a rise in focal cytosolic [Ca²⁺] in the vicinity of ryanodine receptors (RYRs) due to release from internal stores in the form of Ca²⁺ syntillas. Ca²⁺ syntillas are focal cytosolic transients mediated by RYRs, which we first found in hypothalamic magnocellular neuronal terminals. (scintilla, Latin for spark; found in nerve terminals, normally synaptic structures.) We have also observed Ca²⁺ syntillas in mouse adrenal chromaffin cells. Here, we examine the effect of Ca²⁺ syntillas on exocytosis in chromaffin cells. In such a study on elicited exocytosis, there are two sources of Ca²⁺: one due to influx from the cell exterior through voltage-gated Ca²⁺ channels, and that due to release from intracellular stores. To eliminate complications arising from Ca²⁺ influx, we have examined spontaneous exocytosis where influx is not activated. We report here that decreasing syntillas leads to an increase in spontaneous exocytosis measured amperometrically. Two independent lines of experimentation each lead to this conclusion. In one case, release from stores was blocked by ryanodine; in another, stores were partially emptied using thapsigargin plus caffeine, after which syntillas were decreased. We conclude that Ca²⁺ syntillas act to inhibit spontaneous exocytosis, and we propose a simple model to account quantitatively for this action of syntillas.