CaСl2 Salt Signaling in Primary Root Architecture and Lateral Root Emergence in Arabidopsis thaliana

Russian Journal of Plant Physiology - Plant root architecture modulates during developmental stages and adjusts with the environmental condition. The cytosolic calcium which is a ubiquitous...     

Changes in Root Exudates and Root Proteins in Groundnut–Pseudomonas sp. Interaction Contribute to Root Colonization by Bacteria and Defense Response of the Host

Journal of Plant Growth Regulation - Selection and application of rhizobacteria, for improved plant health will benefit from a complete understanding of the plant–bacteria interaction. Root...     

Are Redox Reactions Involved in Regulation of K+ Channels in the Plasma Membrane of Limnobium stoloniferum Root Hairs?

The effects of the impermeant electron acceptor hexacyanoferrate III (HCF III) and the potassium channel blocker tetraethylam-monium (TEA) on the current-voltage relationship and electrical potential across the plasma membrane of Limnobium stoloniferum root hairs was investigated using a modified sucrose gap technique. One millimolar HCF III immediately and reversibly depolarized the membrane by 27 mV, whereas the effect on the trans-membrane current was markedly delayed. After 6 min of treatment with this electron acceptor, outwardly rectifying current was inhibited by 50%, whereas the inwardly rectifying current was activated approximately 3-fold. Ten millimolar TEA blocked both outward (65%) and inward (52%) currents. Differential TEA-sensitive current was shown to be blocked (55%) by HCF III at -20 mV and was shown to be stimulated (230%) by this electron acceptor at -200 mV. The inward current at -200 mV was eliminated in the absence of K+ or after addition of 10 mM Cs+ and was not affected by addition of either 10mM Na+ or Li+, independent of the presence of HCF III. The addition of any alkali cation to the external medium decreased the outward current both in the presence and in the absence of HCF III. The membrane depolarization evoked by HCF III did not correlate with the corresponding modification of the inward current. HCF III is proposed to activate inwardly rectifying potassium channels and to inactivate outwardly rectifying potassium channels. It is concluded that the plasma membrane depolarization did not result from modulation of the potassium channels by HCF III and may originate from trans-plasma membrane electron transfer.     

Lactobacillic Acid Accumulation in the Plasma Membrane of Oenococcus oeni: A Response to Ethanol Stress?

It is known that ethanol strongly interferes with the development and activity of lactic acid bacteria in wine. In this work, it was observed that membrane composition was dependent of ethanol concentration and cell physiological state. The protein electrophoretic profile was modified in the membranes of Oenococcus oeni cultured in presence of 8 and 10% ethanol. Concerning the membrane lipid composition, it was observed that O. oeni maintained a high level of phospholipid biosynthesis via the relative increased biosynthesis of phosphoethanolamine and sphingomyelin in presence of ethanol. On the other hand, ethanol induced an increase in the membrane lactobacillic acid percentage at the expense of cis-vaccenic acid. This increased synthesis of lactobacillic acid appears as the more significant change induced by ethanol in O. oeni membrane. The increase of lactobacillic acid in the membrane of O. oeni clearly appears as a factor that provides protection against the toxic effect of ethanol, balancing the increase of membrane fluidity normally attributed to ethanol. The results presented in this paper constitute evidence that lactobacillic acid may have a part in the survival and or adaptive mechanisms developed by O. oeni under culture adverse conditions, allowing these bacteria to maintain their activity in the presence of ethanol, namely performing malolactic fermentation in wine.     

Detection of Oxidation Products of 5-Methyl-2′-Deoxycytidine in Arabidopsis DNA

Epigenetic regulations play important roles in plant development and adaptation to environmental stress. Recent studies from mammalian systems have demonstrated the involvement of ten-eleven translocation (Tet) family of dioxygenases in the generation of a series of oxidized derivatives of 5-methylcytosine (5-mC) in mammalian DNA. In addition, these oxidized 5-mC nucleobases have important roles in epigenetic remodeling and aberrant levels of 5-hydroxymethyl-2′-deoxycytidine (5-HmdC) were found to be associated with different types of human cancers. However, there is a lack of evidence supporting the presence of these modified bases in plant DNA. Here we reported the use of a reversed-phase HPLC coupled with tandem mass spectrometry method and stable isotope-labeled standards for assessing the levels of the oxidized 5-mC nucleosides along with two other oxidatively induced DNA modifications in genomic DNA of Arabidopsis. These included 5-HmdC, 5-formyl-2′-deoxycytidine (5-FodC), 5-carboxyl-2′-deoxycytidine (5-CadC), 5-hydroxymethyl-2′-deoxyuridine (5-HmdU), and the (5′S) diastereomer of 8,5′-cyclo-2′-deoxyguanosine (S-cdG). We found that, in Arabidopsis DNA, the levels of 5-HmdC, 5-FodC, and 5-CadC are approximately 0.8 modifications per 10⁶ nucleosides, with the frequency of 5-HmdC (per 5-mdC) being comparable to that of 5-HmdU (per thymidine). The relatively low levels of the 5-mdC oxidation products suggest that they arise likely from reactive oxygen species present in cells, which is in line with the lack of homologous Tet-family dioxygenase enzymes in Arabidopsis.     

Root System Dynamics of Miscanthus × giganteus and Panicum virgatum in Response to Rainfed and Irrigated Conditions in California

Miscanthus (Miscanthus × giganteus) and switchgrass (Panicum virgatum) are large perennial grass bioenergy crops in the USA and Europe. Despite much research into their agronomic potential, few studies have examined in situ root growth dynamics under irrigation and soil water deficits, particularly as they relate to shoot performance. We grew miscanthus and switchgrass in outdoor mesocosms under irrigated and rainfed conditions and assessed the spatial distribution and abundance of roots using minirhizotron images and whole root system sampling. Despite surviving an extended period of drought, shoot and root biomass, root length density, numbers of culms, and culm height were reduced in both species under rainfed (dry) conditions. However, rainfed switchgrass far outperformed rainfed miscanthus in all shoot and root growth metrics. The rainfed (drought) treatment reduced switchgrass and miscanthus whole plant biomass by 83 and 98 %, culm production by 67 and 90 %, and root length density by 67 and 94 % compared to irrigated plants, respectively. Root nitrogen concentration was higher for miscanthus (3-fold) and switchgrass (4-fold) in the rainfed treatment compared to irrigated plants and did not significantly differ between species. Unlike miscanthus, switchgrass grew roots continuously into regions of available soil moisture as surface soil layers grew increasingly dry, indicating a drought avoidance strategy. Our study suggests that switchgrass is more likely to tolerate drought by mining deep wet soils, while miscanthus relies on shallow rhizome production to tolerate dry soils.     

A Galacturonic Acid–Containing Xyloglucan Is Involved in Arabidopsis Root Hair Tip Growth

Root hairs provide a model system to study plant cell growth, yet little is known about the polysaccharide compositions of their walls or the role of these polysaccharides in wall expansion. We report that Arabidopsis thaliana root hair walls contain a previously unidentified xyloglucan that is composed of both neutral and galacturonic acid–containing subunits, the latter containing the β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→ and/or α-l-fucosyl-(1→2)-β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→) side chains. Arabidopsis mutants lacking root hairs have no acidic xyloglucan. A loss-of-function mutation in At1g63450, a root hair–specific gene encoding a family GT47 glycosyltransferase, results in the synthesis of xyloglucan that lacks galacturonic acid. The root hairs of this mutant are shorter than those of the wild type. This mutant phenotype and the absence of galacturonic acid in the root xyloglucan are complemented by At1g63450. The leaf and stem cell walls of wild-type Arabidopsis contain no acidic xyloglucan. However, overexpression of At1g63450 led to the synthesis of galacturonic acid–containing xyloglucan in these tissues. We propose that At1g63450 encodes XYLOGLUCAN-SPECIFIC GALACTURONOSYLTRANSFERASE1, which catalyzes the formation of the galactosyluronic acid-(1→2)-α-d-xylopyranosyl linkage and that the acidic xyloglucan is present only in root hair cell walls. The role of the acidic xyloglucan in root hair tip growth is discussed.     

Formation of Nuclear Bodies of Arabidopsis CRY2 in Response to Blue Light Is Associated with Its Blue Light–Dependent Degradation

Arabidopsis thaliana cryptochrome 2 (CRY2) mediates photoperiodic promotion of floral initiation and blue light inhibition of hypocotyl elongation. It has been hypothesized that photoexcitation derepresses CRY2 by disengaging its C-terminal domain from the N-terminal PHR domain. To test this hypothesis, we analyzed activities of CRY2 fused to green fluorescent protein (GFP) at either the N terminus (GFP-CRY2) or the C terminus (CRY2-GFP). While GFP-CRY2 exerts light-dependent biochemical and physiological activities similar to those of the endogenous CRY2, CRY2-GFP showed constitutive biochemical and physiological activities. CRY2-GFP is constitutively phosphorylated, it promotes deetiolation in both dark and light, and it activates floral initiation in both long-day and short-day photoperiods. These results are consistent with the hypothesis that photoexcited CRY2 disengages its C-terminal domain from the PHR domain to become active. Surprisingly, we found that CRY2-GFP, but not GFP-CRY2, formed distinct nuclear bodies in response to blue light. Compared with GFP-CRY2 or the endogenous CRY2, CRY2-GFP degradation was significantly retarded in response to blue light, suggesting that the nuclear bodies may result from accumulation of photoexcited CRY2-GFP waiting to be degraded. Consistent with this interpretation, we showed that both GFP-CRY2 and endogenous CRY2 formed nuclear bodies in the presence of the 26S-proteasome inhibitors that block blue light–dependent CRY2 degradation.     

Radiative Surface Waves in Layered Plasma–Dielectric Structures and Prospects of Their Application in Plasma Microwave Electronics

Plasma Physics Reports - Surface waves in layered systems consisting of material media with different frequency dispersions are considered: dielectric–plasma–vacuum,...     

Responses of Chrysanthemum Cells to Mechanical Stimulation Require Intact Microtubules and Plasma Membrane–Cell Wall Adhesion

Plant cells are highly susceptible and receptive to physical factors, both in nature and under experimental conditions. Exposure to mechanical forces dramatically results in morphological and microstructural alterations in their growth. In the present study, cells from chrysanthemum (Dendranthema morifolium) were subjected to constant pressure from an agarose matrix, which surrounded and immobilized the cells to form a cell-gel block. Cells in the mechanically loaded blocks elongated and divided, with an axis preferentially perpendicular to the direction of principal stress vectors. After a sucrose-induced plasmolysis, application of peptides containing an RGD motif, which interferes with plasma membrane-cell wall adhesion, reduced the oriented growth under stress conditions. Moreover, colchicines, but not cytochalasin B, abolished the effects of mechanical stress on cell morphology. Cellulose staining revealed that mechanical force reinforces the architecture of cell walls and application of mechanical force, and RGD peptides caused aggregative staining on the surface of plasmolyzed protoplasts. These results provide evidence that the oriented cell growth in response to compressive stress requires the maintenance of plasmalemma-cell wall adhesion and intact microtubules. Stress-triggered wall development in individual plant cells was also demonstrated.     

Biochemical and genetic characterization of 5-fluoro-2′-deoxyuridine-resistant mutants of Arabidopsis thaliana

Two independently isolated 5-fluoro-2-deoxyuridine (FUdR)-resistant mutant lines of Arabidopsis thaliana (L.) Heynh., FUD-1 and FUD-2, were identified by screening M2 populations of ethylmethane-sulfonatemutagenized seeds. The resistance was found to be due to single, recessive, nuclear gene mutations. Genetic complementation tests indicated that these two mutations were in the same gene locus, which was designated fur1, and mapped to linkage group four of Arabidopsis. Enzyme assays indicated that the mutants were not defective in thymidine-kinase activity. Greatly reduced concentrations of intracellular ³H were detected in fur1/fur1 plants compared with the wild type after incubation of wild-type and resistant plants in a medium with [³H]FUdR, indicating that either reduced uptake of FUdR or enhanced efflux of FUdR metabolites was the major reason for FUdR-resistance. fur1/fur1 plants also had significantly decreased uptake of thymidine and uridine compared with the wild type but no difference was found in the uptake of adenosine, guanosine, thymine, uracil or amino acids. It is suggested that the transport system affected in the fur1/fur1 mutants is one specific to pyrimidine nucleosides.Abbreviations BUdR 5-bromodeoxyuridine - FdUMP 5-fluoro-2-deoxyuridine monophosphate - FUdR 5-fluoro-2-deoxyuridine - FUR fluorouridine - TK thymidine kinase - TS thymidylate synthetase We thank Dr. George W. Haughn (Department of Biology, University of Saskatchewan) for providing Arabidopsis line W100 and Dr. George Mourad (Department of Biology, University of Saskatchewan) for help and advice. This work was supported by a Research Grant from the Natural Sciences and Engineering Research Council of Canada to J.K. K.W. is grateful for a University of Saskatchewan Graduate Scholarship.     

Combined Effect of Diazepam and GABAA-ergic Ligands on the Activity of Cl–-ATPase in Plasma Membrane of Bream Brain (Abramis brama L.)

We studied the combined effect of diazepam and GABA_A-ergic ligands on the activity of Cl^–-ATPase in plasma membrane of bream brain. The membrane fraction were preincubated and incubated with diazepam as well as with other GABA_A-ergic ligands at physiological pH (7.4), i.e. under the conditions when Cl^–-ATPase activity is undetectable. GABA (0.1–100 M) induced Cl^–-ATPase activity with the maximum effect at 10 M. Diazepam (0.1 M) enhanced the effect of low GABA concentrations (0.1–1 M) on Cl^–-ATPase activity but had no effect on the enzyme in the presence of high GABA concentrations (10–100 M). At the same time, GABA (1 M) enhanced the effect of low diazepam concentrations (0.1–1 M) on the enzyme activity but had no effect on it in the presence of high concentrations of the ligand. Blockers of GABA_A-ergic receptors, picrotoxin (50 M) and bicuculline (5 M), canceled the combined effect of diazepam and GABA on the enzyme activity. The obtained data demonstrate that the combined effect of diazepam and GABA_A-ergic ligands on Cl^–-ATPase activity at physiological pH is similar to the effect of these ligands on GABA_A/benzodiazepine/Cl^– channel.     

β-Amylase Is Predominantly Localized to Plastids in the Developing Tuberous Root of Sweet Potato

Starch degradation in cells is closely associated with cereal seed germination, photosynthesis in leaves, carbohydrate storage in tuber and tuberous roots, and fleshy fruit development. Based on previously reported in vitro assays, β amylase is considered as one of the key enzymes catalyzing starch breakdown, but up to date its role in starch breakdown in living cells remains unclear because the enzyme was shown often extrachloroplastic in living cells. Recently we have shown for the first time that β-amylase is predominantly immuno-localized to plastids in living cells of developing apple fruit. But it remains to know whether this model of β-amylase compartmentation is more widespread in plant living cells. The present experiment, conducted in tuberous root of sweet potato (Ipomea batatas Lam. cv. Xushu 18) and via immunogold electron-microscopy technique, showed that β amylase visualized by gold particles was predominantly localized in plastids especially at periphery of starch granules, but the gold particles were scarcely found in other subcellular compartments, indicating that the enzyme is subcellularly compartmented in the same zone as its starch substrates. The density of gold particles (β amylase) in plastids was increasing during growing season, but the predominantly plastid-distributed pattern of β amylase in cells was shown unchanged throughout the tuberous root development. These data prove that the enzyme is compartmented in its functional sites, and so provide evidence to support the possible widespread biological function of the enzyme in catalyzing starch breakdown in plant living cells or at least in living cells of plant storage organs.     

Beam–Plasma Discharge in Space and in a Lab

Plasma Physics Reports - The process and applications of a specific type of gaseous discharge—beam–plasma discharge (BPD)—are reviewed. A brief survey of the BPD theory is...     

αvβ3 Integrin Boosts the Innate Immune Response Elicited in Epithelial Cells through Plasma Membrane and Endosomal Toll-Like Receptors

We report that αvβ3 integrin strongly affects the innate immune response in epithelial cells. αvβ3 integrin greatly increased the response elicited via plasma membrane Toll-like receptors (TLRs) by herpes simplex virus or bacterial ligands. The endosomal TLR3, not the cytosolic sensor interferon gamma-inducible protein 16 (IFI16), was also boosted by αvβ3 integrin. The boosting was exerted specifically by αvβ3 integrin but not by αvβ6 or αvβ8 integrin. Current and previous work indicates that integrin-TLR cooperation occurs in epithelial and monocytic cells. The TLR response should be considered an integrin-TLR response.     

Class I α-Mannosidases Are Required for N-Glycan Processing and Root Development in Arabidopsis thaliana

In eukaryotes, class I α-mannosidases are involved in early N-glycan processing reactions and in N-glycan–dependent quality control in the endoplasmic reticulum (ER). To investigate the role of these enzymes in plants, we identified the ER-type α-mannosidase I (MNS3) and the two Golgi-α-mannosidase I proteins (MNS1 and MNS2) from Arabidopsis thaliana. All three MNS proteins were found to localize in punctate mobile structures reminiscent of Golgi bodies. Recombinant forms of the MNS proteins were able to process oligomannosidic N-glycans. While MNS3 efficiently cleaved off one selected α1,2-mannose residue from Man₉GlcNAc₂, MNS1/2 readily removed three α1,2-mannose residues from Man₈GlcNAc₂. Mutation in the MNS genes resulted in the formation of aberrant N-glycans in the mns3 single mutant and Man₈GlcNAc₂ accumulation in the mns1 mns2 double mutant. N-glycan analysis in the mns triple mutant revealed the almost exclusive presence of Man₉GlcNAc₂, demonstrating that these three MNS proteins play a key role in N-glycan processing. The mns triple mutants displayed short, radially swollen roots and altered cell walls. Pharmacological inhibition of class I α-mannosidases in wild-type seedlings resulted in a similar root phenotype. These findings show that class I α-mannosidases are essential for early N-glycan processing and play a role in root development and cell wall biosynthesis in Arabidopsis.N-glycosylation is a major co- and posttranslational modification of proteins in eukaryotic cells. The biosynthesis of protein N-linked glycans starts in the endoplasmic reticulum (ER) when the oligosaccharyltransferase complex catalyzes the transfer of the Glc₃Man₉GlcNAc₂ oligosaccharide from the lipid-linked precursor to Asn residues (N-X-S/T) of nascent polypeptide chains. Subsequent N-glycan processing involves a series of highly coordinated step-by-step enzymatic conversions occurring in the ER and Golgi apparatus (Kornfeld and Kornfeld, 1985). In the first trimming reactions, α-glucosidases I (GCSI) and GCSII cleave off three glucose residues from Glc₃Man₉GlcNAc₂ to generate Man₉GlcNAc₂ (Figure 1A). The next steps of the pathway are the removal of four α1,2-linked mannose residues to provide the Man₅GlcNAc₂ substrate for the formation of complex N-glycans in the Golgi apparatus. In mammals, these mannose trimming reactions are catalyzed by class I α-mannosidases (glycosyl hydrolase family 47 of the Carbohydrate Active Enzymes database; http://www.cazy.org/). These enzymes are inverting glycosyl hydrolases that are highly specific for α1,2-mannose residues, require Ca²⁺ for catalytic activity, and are sensitive to inhibition by pyranose analogs such as 1-deoxymannojirimycin and kifunensine (Lipari et al., 1995; Gonzalez et al., 1999). Class I α-mannosidases are conserved through eukaryotic evolution and do not share sequence homology with class II α-mannosidases, such as Golgi α-mannosidase II and the catabolic lysosomal and cytoplasmic α-mannosidases (Gonzalez et al., 1999; Herscovics, 2001).Open in a separate windowFigure 1.Cartoon of Important Oligosaccharide Structures.(A) Man₉GlcNAc₂ oligosaccharide (Man9): the substrate for ER-MNSI.(B) Man₈GlcNAc₂ isomer Man8.1 according to Tomiya et al. (1991): the product of ER-MNSI and substrate for Golgi-MNSI.(C) Man₅GlcNAc₂ (Man5.1): the product of the mannose trimming reactions.The linkage of the sugar residues is indicated.[See online article for color version of this figure.]The mammalian class I α-mannosidase family consists of three protein subgroups, which have been distinguished based on their sequence similarity and proposed function: ER-α1,2-mannosidases I (ER-MNSIs), Golgi-α-mannosidases I (Golgi-MNSIs), and ER degradation-enhancing α-mannosidase (EDEM)-like proteins (Mast and Moremen, 2006). In humans, there is a single ER-MNSI, which cleaves the terminal mannose residue from the b-branch of the Man₉GlcNAc₂ oligosaccharide to create the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B). Subsequently, Golgi-MNSI (three isoforms, Golgi-MNSIA, Golgi-MNSIB, and Golgi-MNSIC, are present in humans) catalyze the removal of the remaining three α1,2-linked mannose residues to generate Man₅GlcNAc₂ (Figure 1C). The three human EDEM proteins are not directly involved in N-glycan processing but play a role in ER-associated degradation of glycoproteins (Mast et al., 2005; Hirao et al., 2006; Olivari et al., 2006).The formation of the Man₈GlcNAc₂ isomer (Man8.1), which is catalyzed by ER-MNSI, is the last N-glycan processing step that is conserved in yeast and mammals. Apart from its N-glycan processing function, ER-MNSI plays a key role in ER-mediated quality control of glycoproteins in yeasts and mammals (Mast and Moremen, 2006; Lederkremer, 2009). It has been proposed that ER-MNSI cooperates with mammalian EDEM1 to 3 or the yeast α1,2-mannosidase HTM1 to generate the signal that marks misfolded glycoproteins for degradation through the ER-associated protein degradation (ERAD) pathway. This quality control process, which finally leads to retrotranslocation to the cytoplasm and hydrolysis by the 26S proteasome, serves to prevent the secretion of aberrantly folded cargo proteins and is required to maintain protein homeostasis in the ER. Initially it was proposed that the Man₈GlcNAc₂ isomer Man8.1 (Figure 1B) flags aberrantly folded glycoproteins for degradation; however, recent evidence suggests that further mannose trimming to Man₇GlcNAc₂ in yeast and Man_5-6GlcNAc₂ in mammals is required to trigger ERAD (Avezov et al., 2008; Clerc et al., 2009). In addition, these mannose cleavage reactions serve also to release glycoproteins from the calnexin/calreticulin quality control cycle (Caramelo and Parodi, 2008).Unlike for animals and yeast, much less is known about the biological function of plant class I α-mannosidases. Processing mannosidases have been purified and characterized from mung bean (Vigna radiata) seedlings and castor bean (Ricinus communis) cotyledons (Forsee, 1985; Szumilo et al., 1986; Kimura et al., 1991). These preparations were a mixture of different α-mannosidases, and no evidence for ER-MNSI-like activity was provided. A putative Golgi-α-mannosidase I has been cloned from soybean (Glycine max) (Nebenführ et al., 1999). A green fluorescent protein (GFP)-tagged fusion protein of the soybean enzyme has been shown to reside in the cis-stacks of the Golgi apparatus (Nebenführ et al., 1999; Saint-Jore-Dupas et al., 2006), but its role in N-glycan processing and its enzymatic properties have not been reported so far. Thus, the involvement of class I α-mannosidases in N-glycan processing as well as in glycoprotein quality control in plants is still unclear, and the existence of a plant ER-MNSI has so far been inferred only from the presence of Man₈GlcNAc₂ oligosaccharides on ER-resident glycoproteins (Pagny et al., 2000).Here, we report the molecular cloning and biochemical characterization of the enzymes accounting for ER-MNSI and Golgi-MNSI activities in Arabidopsis thaliana. We also demonstrate that disruption of these genes leads to severe cell expansion defects in roots as well as to distinct cell wall alterations. Hence, the identification of the Arabidopsis ER-type and Golgi class I α-mannosidases not only establishes the molecular basis for the missing steps in the plant N-glycan processing pathway but also provides unprecedented insights into the role of N-glycans in plant development.     

Functional Ryanodine Receptors in the Plasma Membrane of RINm5F Pancreatic ��-Cells

Ryanodine receptors (RyR) are Ca²⁺ channels that mediate Ca²⁺ release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca²⁺ entry that was independent of store-operated Ca²⁺ entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (γ_K = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine. Similar channels were detected in rat pancreatic β-cells. In RINm5F cells, the channels were blocked by cytosolic, but not extracellular, ruthenium red. Subcellular fractionation showed that type 3 IP₃ receptors (IP₃R3) were expressed predominantly in endoplasmic reticulum, whereas RyR2 were present also in plasma membrane fractions. Using RNAi selectively to reduce expression of RyR1, RyR2, or IP₃R3, we showed that RyR2 mediates both the Ca²⁺ entry and the plasma membrane currents evoked by agonists of RyR. We conclude that small numbers of RyR2 are selectively expressed in the plasma membrane of RINm5F pancreatic β-cells, where they mediate Ca²⁺ entry.Ryanodine receptors (RyR)³ and inositol 1,4,5-trisphosphate receptors (IP₃R) (1, 2) are the archetypal intracellular Ca²⁺ channels. Both are widely expressed, although RyR are more restricted in their expression than IP₃R (3, 4). In common with many cells, pancreatic β-cells and insulin-secreting cell lines express both IP₃R (predominantly IP₃R3) (5, 6) and RyR (predominantly RyR2) (7). Both RyR and IP₃R are expressed mostly within membranes of the endoplasmic (ER), where they mediate release of Ca²⁺. Functional RyR are also expressed in the secretory vesicles (8, 9) or, and perhaps more likely, in the endosomes of β-cells (10). Despite earlier suggestions (11), IP₃R are probably not present in the secretory vesicles of β-cells (8, 12, 13).All three subtypes of IP₃R are stimulated by IP₃ with Ca²⁺ (1), and the three subtypes of RyR are each directly regulated by Ca²⁺. However, RyR differ in whether their most important physiological stimulus is depolarization of the plasma membrane (RyR1), Ca²⁺ (RyR2) or additional intracellular messengers like cyclic ADP-ribose. The latter stimulates both Ca²⁺ release and insulin secretion in β-cells (8, 14). The activities of both families of intracellular Ca²⁺ channels are also modulated by many additional signals that act directly or via phosphorylation (15, 16). Although they commonly mediate release of Ca²⁺ from the ER, both IP₃R and RyR select rather poorly between Ca²⁺ and other cations (permeability ratio, P_Ca/P_K ∼7) (1, 17). This may allow electrogenic Ca²⁺ release from the ER to be rapidly compensated by uptake of K⁺ (18), and where RyR or IP₃R are expressed in other membranes it may allow them to affect membrane potential.Both Ca²⁺ entry and release of Ca²⁺ from intracellular stores contribute to the oscillatory increases in cytosolic Ca²⁺ concentration ([Ca²⁺]_i) that stimulate exocytosis of insulin-containing vesicles in pancreatic β-cells (7). Glucose rapidly equilibrates across the plasma membrane (PM) of β-cells and its oxidative metabolism by mitochondria increases the cytosolic ATP/ADP ratio, causing K_ATP channels to close (19). This allows an unidentified leak current to depolarize the PM (20) and activate voltage-gated Ca²⁺ channels, predominantly L-type Ca²⁺ channels (21). The resulting Ca²⁺ entry is amplified by Ca²⁺-induced Ca²⁺ release from intracellular stores (7), triggering exocytotic release of insulin-containing dense-core vesicles (22). The importance of this sequence is clear from the widespread use of sulfonylurea drugs, which close K_ATP channels, in the treatment of type 2 diabetes. Ca²⁺ uptake by mitochondria beneath the PM further stimulates ATP production, amplifying the initial response to glucose and perhaps thereby contributing to the sustained phase of insulin release (23). However, neither the increase in [Ca²⁺]_i nor the insulin release evoked by glucose or other nutrients is entirely dependent on Ca²⁺ entry (7, 24) or closure of K_ATP channels (25). This suggests that glucose metabolism may also more directly activate RyR (7, 26) and/or IP₃R (27) to cause release of Ca²⁺ from intracellular stores. A change in the ATP/ADP ratio is one means whereby nutrient metabolism may be linked to opening of intracellular Ca²⁺ channels because both RyR (28) and IP₃R (1) are stimulated by ATP.The other major physiological regulators of insulin release are the incretins: glucagon-like peptide-1 and glucose-dependent insulinotropic hormone (29). These hormones, released by cells in the small intestine, stimulate synthesis of cAMP in β-cells and thereby potentiate glucose-evoked insulin release (30). These pathways are also targets of drugs used successfully to treat type 2 diabetes (29). The responses of β-cells to cAMP involve both cAMP-dependent protein kinase and epacs (exchange factors activated by cAMP) (31, 32). The effects of the latter are, at least partly, due to release of Ca²⁺ from intracellular stores via RyR (33–35) and perhaps also via IP₃R (36). The interplays between Ca²⁺ and cAMP signaling generate oscillatory changes in the concentrations of both messengers (37). RyR and IP₃R are thus implicated in mediating responses to each of the major physiological regulators of insulin secretion: glucose and incretins.Here we report that in addition to expression in intracellular stores, which probably include both the ER and secretory vesicles and/or endosomes, functional RyR2 are also expressed in small numbers in the PM of RINm5F insulinoma cells and rat pancreatic β-cells.