Reactivation of formyl peptide receptors triggers the neutrophil NADPH-oxidase but not a transient rise in intracellular calcium

In neutrophils, coupling of chemoattractants to their cell surface receptor at low temperature (相似文献   

Voltage-dependent calcium channels at the plasma membrane, but not vesicular channels, couple exocytosis to endocytosis

Although calcium influx triggers endocytosis at many synapses and non-neuronal secretory cells, the identity of the calcium channel is unclear. The plasma membrane voltage-dependent calcium channel (VDCC) is a candidate, and it was recently proposed that exocytosis transiently inserts vesicular calcium channels at the plasma membrane, thus triggering endocytosis and coupling it to exocytosis, a mechanism suggested to be conserved from sea urchin to human. Here, we report that the vesicular membrane, when inserted into the plasma membrane upon exocytosis, does not generate a calcium current or calcium increase at a mammalian nerve terminal. Instead, VDCCs at the plasma membrane, including the P/Q-type, provide the calcium influx to trigger rapid and slow endocytosis and, thus, couple endocytosis to exocytosis. These findings call for reconsideration of the vesicular calcium channel hypothesis. They are likely to apply to many synapses and non-neuronal cells in which VDCCs control exocytosis, and exocytosis is coupled to endocytosis.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs--fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca(2+) permeable ion channel using Ca(2+) indicators like fluo-3. These Single Channel Ca(2+) Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca(2+) sparks and Ca(2+) puffs caused by Ca(2+) release from intracellular stores (due to the opening of ryanodine receptors and IP(3) receptors, respectively). In contrast to intracellular Ca(2+) release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca(2+) handling in the vicinity of a channel with a known Ca(2+) influx, to obtain the Ca(2+) current passing through plasma membrane cation channels in near physiological solutions, to localize Ca(2+) permeable ion channels on the plasma membrane, and to estimate the Ca(2+) currents underlying those elementary events where the Ca(2+) currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca(2+) channels, and stretch-activated channels. For the L-type Ca(2+) channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca(2+) currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Imaging calcium entering the cytosol through a single opening of plasma membrane ion channels: SCCaFTs—fundamental calcium events

Recently, it has become possible to record the localized fluorescence transient associated with the opening of a single plasma membrane Ca²⁺ permeable ion channel using Ca²⁺ indicators like fluo-3. These Single Channel Ca²⁺ Fluorescence Transients (SCCaFTs) share some of the characteristics of such elementary events as Ca²⁺ sparks and Ca²⁺ puffs caused by Ca²⁺ release from intracellular stores (due to the opening of ryanodine receptors and IP₃ receptors, respectively). In contrast to intracellular Ca²⁺ release events, SCCaFTs can be observed while simultaneously recording the unitary channel currents using patch-clamp techniques to verify the channel openings. Imaging SCCaFTs provides a way to examine localized Ca²⁺ handling in the vicinity of a channel with a known Ca²⁺ influx, to obtain the Ca²⁺ current passing through plasma membrane cation channels in near physiological solutions, to localize Ca²⁺ permeable ion channels on the plasma membrane, and to estimate the Ca²⁺ currents underlying those elementary events where the Ca²⁺ currents cannot be recorded. Here we review studies of these fluorescence transients associated with caffeine-activated channels, L-type Ca²⁺ channels, and stretch-activated channels. For the L-type Ca²⁺ channel, SCCaFTs have been termed sparklets. In addition, we discuss how SCCaFTs have been used to estimate Ca²⁺ currents using the rate of rise of the fluorescence transient as well as the signal mass associated with the total fluorescence increase.     

Calcium-pH crosstalks in the human mast cell line HMC-1: intracellular alkalinization activates calcium extrusion through the plasma membrane Ca2+-ATPase

The human mast cell line (HMC-1) has been used to study the relationship between intracellular pH and cytosolic calcium (Ca2+) in mast cells. Thapsigargin (TG) caused store-operated Ca2+ entry, that is enhanced by the PKC activator PMA. NH4Cl-induced alkalinization showed an inhibitory effect on TG-sensitive stores depletion (not on TG-insensitive stores), and also on final cytosolic Ca2+ levels reached in response to both TG and the ionophore ionomycin. Loperamide, a positive modulator of store-operated channels, induced a slight Ca2+ entry by itself, and also increased TG-induced Ca2+ entry. This enhancement was not enough to reverse the inhibitory effect of NH4Cl-induced alkalinization. When comparing the effect of NH4Cl-induced alkalinization on Ca2+ levels, with those observed using Ca2+ channel blockers (namely Ni2+ and SKF-96365), cytosolic profiles for this ion are different, either in modified saline solution or in HCO3(-)-free medium. Thus, it seems unlikely that the inhibitory effect of NH4Cl-induced alkalinization on Ca2+ is taking place by blockage of Ca2+ entry. Furthermore, inhibition of the plasma membrane Ca2+-ATPase (an important mechanism for Ca2+ efflux) with sodium orthovanadate (SO) matches with the inhibition of the negative effect on Ca2+ levels elicited by NH4Cl. Data indicate that NH4Cl-induced alkalinization might be activating Ca2+ efflux from the cell, by stimulation of the plasma membrane Ca2+-ATPase, and also confirm our previous finding that Ca2+ is a secondary signal to activate HMC-1 cells.     

A complex peptide-sorting signal, but no mRNA signal, is required for the Sec-independent transport of Ist2 from the yeast ER to the plasma membrane

The yeast integral plasma membrane protein Ist2 belongs to a group of membrane proteins which are synthesized from localized mRNAs. The protein reaches the plasma membrane via the ER on a route operating independently of the classical secretory pathway. We have identified a complex peptide-sorting signal located at the extreme C-terminus. This sorting signal operates independently of targeting information in IST2 mRNA and sorting to the plasma membrane does not require She-mediated mRNA transport into daughter cells. Based on these results, we suggest a posttranslational mechanism, which leads to the concentration of Ist2--via multimerization--at ER sites, followed by direct transport to the plasma membrane. This novel mechanism operates downstream of IST2 mRNA localization.     

Microvillar channels: a unique plasma membrane compartment for concentrating lipoproteins on the surface of rat adrenal cortical cells

Electron microscopic studies of perfused rat adrenals indicate that plasma lipoproteins become concentrated in a specialized cell surface compartment called microvillar channels. Closely associated plasma membranes of sinusoidal microvilli of zona fasciculata cells form channels that normally are filled with electron dense particles the size of high density lipoproteins (HDL). In rats made acutely deficient in plasma lipoproteins (by treatment with 4-aminopyrazolo[3,4-d]pyrimidine (4-APP) for 1 day), particles within the microvillar channels are decreased in number. When adrenal glands of these rats are perfused with media lacking plasma lipoproteins, many but not all of these HDL-like particles are washed out. However, when these adrenals are perfused with large amounts (100-500 micrograms protein/ml) of HDL, microvillar channels become packed with electron dense particles similar to those found in vivo. These microvillar channels become wider and filled with larger particles when low density lipoproteins (LDL) are perfused through the adrenals. Autoradiograms of 125I-labeled HDL-perfused adrenals show silver grains specifically associated with the cell surface microvillar channels, and confirm the notion that the particles filling the channels are exogenously delivered HDL. Physiologic data from similarly perfused adrenals in a parallel study show that the channel-refilling process is directly related to selective (i.e., nonendocytic) cholesterol uptake and that this cholesterol uptake is associated with corticosterone production. Together, these data suggest the hypothesis that plasma lipoprotein cholesterol utilized for corticosteroid synthesis in rat adrenal fasciculata cells may be derived from lipoproteins trapped in surface-associated microvillar channels. Although the mechanism responsible for the cholesterol transfer is not yet defined, it is clearly distinct from the classical process of receptor-mediated endocytosis and catabolism of lipoprotein particles.     

The Homer-1 protein Ania-3 interacts with the plasma membrane calcium pump

The Homer family of scaffold proteins couples NMDA receptors to metabotropic glutamate receptors and links extracellular signals to calcium release from intracellular stores. Ania-3 is a member of the Homer family and is rapidly inducible in brain in response to diverse stimuli. Here, we report the identification of the plasma membrane Ca2+ ATPase (PMCA) as a novel Ania-3/Homer-associated protein. Ania-3/Homer interacts with the b-splice forms of all PMCAs (PMCA1b, 2b, 3b, and 4b) via their PDZ domain-binding COOH-terminal tail. Ectopically expressed Ania-3 colocalized with the PMCA at the plasma membrane of polarized MDCK epithelial cells, and endogenous Ania-3/Homer and PMCA2 are co-expressed in the soma and dendrites of primary rat hippocampal neurons. The interaction between Ania-3/Homer and PMCAs may represent a novel mechanism by which local calcium signaling and hence synaptic function can be modulated in neurons.     

The CD2 ligand LFA-3 activates T cells but depends on the expression and function of the antigen receptor

The T cell Ag receptor (CD3/Ti) and the sheep E receptor (CD2) expressed on the surface of human T cells are both capable of initiating intracellular signals necessary for T cell activation. CD3/Ti interacts with Ag to initiate cellular immune responses. Although the exact function of CD2 is unknown, lymphocyte function-associated Ag 3 (LFA-3), a 55- to 70-kDa receptor expressed on a broad spectrum of hemopoietic and nonhemopoietic cells, has recently been shown to be its natural ligand. We show here that although purified multimeric LFA-3 is not capable of initiating transmembrane signaling events on its own, the combination of LFA-3 and the anti-CD2 mAb CD2.1 induces intracellular calcium increases, phosphatidylinositol second messenger generation and lymphokine secretion in the T cell leukemic line Jurkat. In order to study the signaling requirements of CD2, we compared the ability of CD2 mAb and LFA-3 to initiate activation signals in Jurkat and in three Jurkat-derived mutants. A CD3-CD2+ mutant failed to increase calcium or exhibit phosphatidylinositol hydrolysis to either the combination of agonist CD2 mAb 9-1 and 9.6 or LFA-3 and CD2.1. Reconstitution of the Ag receptor by transfection of the Ti-beta-chain restored the expression of the CD3/Ti complex and the ability to respond to either combination of CD2 ligands. However, no response to CD2 ligands was detected in a CD3+CD2+ mutant selected for signaling defects to CD3/Ti ligands. Complementation of the CD3/Ti signaling defect by cell fusion also restored competency to respond to CD2 agonists. These results demonstrate that LFA-3 under appropriate conditions can activate T cells via the CD2 complex and that this activation requires not only the cell surface expression of the CD3/Ti complex but also a functional Ag receptor pathway.     

Calcium inhibition and calcium potentiation of Orai1, Orai2, and Orai3 calcium release-activated calcium channels

The recent discoveries of Stim1 and Orai proteins have shed light on the molecular makeup of both the endoplasmic reticulum Ca(2+) sensor and the calcium release-activated calcium (CRAC) channel, respectively. In this study, we investigated the regulation of CRAC channel function by extracellular Ca(2+) for channels composed primarily of Orai1, Orai2, and Orai3, by co-expressing these proteins together with Stim1, as well as the endogenous channels in HEK293 cells. As reported previously, Orai1 or Orai2 resulted in a substantial increase in CRAC current (I(crac)), but Orai3 failed to produce any detectable Ca(2+)-selective currents. However, sodium currents measured in the Orai3-expressing HEK293 cells were significantly larger in current density than Stim1-expressing cells. Moreover, upon switching to divalent free external solutions, Orai3 currents were considerably more stable than Orai1 or Orai2, indicating that Orai3 channels undergo a lesser degree of depotentiation. Additionally, the difference between depotentiation from Ca(2+) and Ba(2+) or Mg(2+) solutions was significantly less for Orai3 than for Orai1 or -2. Nonetheless, the Na(+) currents through Orai1, Orai2, and Orai3, as well as the endogenous store-operated Na(+) currents in HEK293 cells, were all inhibited by extracellular Ca(2+) with a half-maximal concentration of approximately 20 mum. We conclude that Orai1, -2, and -3 channels are similarly inhibited by extracellular Ca(2+), indicating similar affinities for Ca(2+) within the selectivity filter. Orai3 channels appeared to differ from Orai1 and -2 in being somewhat resistant to the process of Ca(2+) depotentiation.     

A plasma membrane-targeted cytosolic domain of STIM1 selectively activates ARC channels,an arachidonate-regulated store-independent Orai channel

The Orai family of calcium channels includes the store-operated CRAC channels and store-independent, arachidonic acid (AA)-regulated ARC channels. Both depend on STIM1 for their activation but, whereas CRAC channel activation involves sensing the depletion of intracellular calcium stores via a luminal N terminal EF-hand of STIM1 in the endoplasmic reticulum (ER) membrane, ARC channels are exclusively activated by the pool of STIM1 that constitutively resides in the plasma membrane (PM). Here, the EF-hand is extracellular and unlikely to ever lose its bound calcium, suggesting that STIM1-dependent activation of ARC channels is very different from that of CRAC channels. We now show that attachment of the cytosolic portion of STIM1 to the inner face of the PM via an N terminal Lck-domain sequence is sufficient to enable normal AA-dependent activation of ARC channels, while failing to allow activation of store-operated CRAC channels. Introduction of a point mutation within the Lck-domain resulted in the loss of both PM localization and ARC channel activation. Reversing the orientation of the PM-anchored STIM1 C terminus via a C-terminal CAAX-box fails to support either CRAC or ARC channel activation. Finally, the Lck-anchored STIM1 C-terminal domain also enabled the exclusive activation of the ARC channels following physiological agonist addition. These data demonstrate that simple tethering of the cytosolic C-terminal domain of STIM1 to the inner face of the PM is sufficient to allow the full, normal and exclusive activation of ARC channels, and that the N-terminal regions of STIM1 (including the EF-hand domain) play no significant role in this activation.     

Early signaling through the Arabidopsis pattern recognition receptors FLS2 and EFR involves Ca2+‐associated opening of plasma membrane anion channels

The perception of microbes by plants involves highly conserved molecular signatures that are absent from the host and that are collectively referred to as microbe‐associated molecular patterns (MAMPs). The Arabidopsis pattern recognition receptors FLAGELLIN‐SENSING 2 (FLS2) and EF‐Tu receptor (EFR) represent genetically well studied paradigms that mediate defense against bacterial pathogens. Stimulation of these receptors through their cognate ligands, bacterial flagellin or bacterial elongation factor Tu, leads to a defense response and ultimately to increased resistance. However, little is known about the early signaling pathway of these receptors. Here, we characterize this early response in situ, using an electrophysiological approach. In line with a release of negatively charged molecules, voltage recordings of microelectrode‐impaled mesophyll cells and root hairs of Col‐0 Arabidopsis plants revealed rapid, dose‐dependent membrane potential depolarizations in response to either flg22 or elf18. Using ion‐selective microelectrodes, pronounced anion currents were recorded upon application of flg22 and elf18, indicating that the signaling cascades initiated by each of the two receptors converge on the same plasma membrane ion channels. Combined calcium imaging and electrophysiological measurements revealed that the depolarization was superimposed by an increase in cytosolic calcium that was indispensable for depolarization. NADPH oxidase mutants were still depolarized upon elicitor stimulation, suggesting a reactive oxygen species‐independent membrane potential response. Furthermore, electrical signaling in response to either flg22 or elf 18 critically depends on the activity of the FLS2‐associated receptor‐like kinase BAK1, suggesting that activation of FLS2 and EFR lead to BAK1‐dependent, calcium‐associated plasma membrane anion channel opening as an initial step in the pathogen defense pathway.     

The CNGCb and CNGCd genes from Physcomitrella patens moss encode for thermosensory calcium channels responding to fluidity changes in the plasma membrane

Land plants need precise thermosensors to timely establish molecular defenses in anticipation of upcoming noxious heat waves. The plasma membrane-embedded cyclic nucleotide-gated Ca²⁺ channels (CNGCs) can translate mild variations of membrane fluidity into an effective heat shock response, leading to the accumulation of heat shock proteins (HSP) that prevent heat damages in labile proteins and membranes. Here, we deleted by targeted mutagenesis the CNGCd gene in two Physcomitrella patens transgenic moss lines containing either the heat-inducible HSP-GUS reporter cassette or the constitutive UBI-Aequorin cassette. The stable CNGCd knockout mutation caused a hyper-thermosensitive moss phenotype, in which the heat-induced entry of apoplastic Ca²⁺ and the cytosolic accumulation of GUS were triggered at lower temperatures than in wild type. The combined effects of an artificial membrane fluidizer and elevated temperatures suggested that the gene products of CNGCd and CNGCb are paralogous subunits of Ca²⁺channels acting as a sensitive proteolipid thermocouple. Depending on the rate of temperature increase, the duration and intensity of the heat priming preconditions, terrestrial plants may thus acquire an array of HSP-based thermotolerance mechanisms against upcoming, otherwise lethal, extreme heat waves.     

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana.

Voltage-dependent Ca2+ channels in the plasma membrane and the vacuolar membrane of Arabidopsis thaliana have been studied at the single-channel level using the patch-clamp technique. The Ca2+ channel in the plasma membrane opened for extracellular Ca2+ influx. The Ca2+ channel in the vacuolar membrane opened for cytoplasmic Ca2+ influx.     

Cyclic adenosine monophosphate regulates calcium channels in the plasma membrane of Arabidopsis leaf guard and mesophyll cells

The effect of cAMP on Ca(2+)-permeable channels from Arabidopsis thaliana leaf guard cell and mesophyll cell protoplasts was studied using the patch clamp technique. In the whole cell configuration, dibutyryl cAMP was found to increase a hyperpolarization-activated Ba(2+) conductance (I(Ba)). The increase of I(Ba) was blocked by the addition of GdCl(3). In excised outside-out patches, the addition of dibutyryl cAMP consistently activated a channel with particularly fast gating kinetics. Current/voltage analyses indicated a single channel conductance of approximately 13 picosiemens. In patches where we measured some channel activity prior to cAMP application, the data suggest that cAMP enhances channel activity without affecting the single channel conductance. The cAMP activation of these channels was reversible upon washout. The results obtained with excised patches indicate that the cAMP-activated I(Ba) seen in the whole cell configuration could be explained by a direct effect of cAMP on the Ca(2+) channel itself or a close entity to the channel. This work represents the first demonstration using patch clamp analysis of the presence in plant cell membranes of an ion channel directly activated by cAMP.