Dense core secretory vesicles revealed as a dynamic Ca(2+) store in neuroendocrine cells with a vesicle-associated membrane protein aequorin chimaera

The role of dense core secretory vesicles in the control of cytosolic-free Ca(2+) concentrations ([Ca(2+)](c)) in neuronal and neuroendocrine cells is enigmatic. By constructing a vesicle-associated membrane protein 2-synaptobrevin.aequorin chimera, we show that in clonal pancreatic islet beta-cells: (a) increases in [Ca(2+)](c) cause a prompt increase in intravesicular-free Ca(2+) concentration ([Ca(2+)]SV), which is mediated by a P-type Ca(2+)-ATPase distinct from the sarco(endo) plasmic reticulum Ca(2+)-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca(2+) pumps; (b) steady state Ca(2+) concentrations are 3-5-fold lower in secretory vesicles than in the endoplasmic reticulum (ER) or Golgi apparatus, suggesting the existence of tightly bound and more rapidly exchanging pools of Ca(2+); (c) inositol (1,4,5) trisphosphate has no impact on [Ca(2+)](SV) in intact or permeabilized cells; and (d) ryanodine receptor (RyR) activation with caffeine or 4-chloro-3-ethylphenol in intact cells, or cyclic ADPribose in permeabilized cells, causes a dramatic fall in [Ca(2+)](SV). Thus, secretory vesicles represent a dynamic Ca(2+) store in neuroendocrine cells, whose characteristics are in part distinct from the ER/Golgi apparatus. The presence of RyRs on secretory vesicles suggests that local Ca(2+)-induced Ca(2+) release from vesicles docked at the plasma membrane could participate in triggering exocytosis.     

NAADP: a new second messenger for glucose-induced Ca2+ responses in clonal pancreatic beta cells

Important questions remain concerning how elevated blood glucose levels are coupled to insulin secretion from pancreatic beta cells and how this process is impaired in type 2 diabetes. Glucose uptake and metabolism in beta cells cause the intracellular Ca(2+) concentration ([Ca(2+)](i)) to increase to a degree necessary and sufficient for triggering insulin release. Although both Ca(2+) influx and Ca(2+) release from internal stores are critical, the roles of inositol 1,4,5-trisphosphate (IP(3)) and cyclic adenosine dinucleotide phosphate ribose (cADPR) in regulating the latter have proven equivocal. Here we show that glucose also increases [Ca(2+)](i) via the novel Ca(2+)-mobilizing agent nicotinic acid adenine dinucleotide phosphate (NAADP) in the insulin-secreting beta-cell line MIN6. NAADP binds to specific, high-affinity membrane binding sites and at low concentrations elicits robust Ca(2+) responses in intact cells. Higher concentrations of NAADP inactivate NAADP receptors and attenuate the glucose-induced Ca(2+) increases. Importantly, glucose stimulation increases endogenous NAADP levels, providing strong evidence for recruitment of this pathway. In conclusion, our results support a model in which NAADP mediates glucose-induced Ca(2+) signaling in pancreatic beta cells and are the first demonstration in mammalian cells of the presence of endogenous NAADP levels that can be regulated by a physiological stimulus.     

5'-AMP-activated protein kinase controls insulin-containing secretory vesicle dynamics

Changes in 5'-AMP-activated protein kinase (AMPK) activity have recently been implicated in the control of insulin secretion by glucose (da Silva Xavier, G., Leclerc, I., Varadi, A., Tsuboi, T., Moule, S. K., and Rutter, G. A. (2003) Biochem. J. 371, 761-774). Here, we examine the possibility that activation of AMPK may regulate distal steps in insulin secretion, including vesicle movement and fusion with the plasma membrane. Vesicle dynamics were imaged in single pancreatic MIN6 beta-cells expressing lumen-targeted pH-insensitive yellow fluorescent protein, neuropeptide Y.Venus, or monomeric red fluorescent protein by total internal reflection fluorescence and Nipkow disc confocal microscopy. Overexpression of a truncated, constitutively active form of AMPK (AMPKalpha1, 1-312, T172D; AMPK CA), inhibited glucose-stimulated (30 versus 3.0 mM) vesicle movements, and decreased the number of vesicles docked or fusing at the plasma membrane, while having no effect on the kinetics of individual secretory events. Expression of the activated form of AMPK also prevented dispersal of the cortical actin network at high glucose concentrations. Monitored in permeabilized cells, where the effects of AMPK CA on glucose metabolism and ATP synthesis were bypassed, AMPK CA inhibited Ca2+ and ATP-induced insulin secretion, and decreased ATP-dependent vesicle movements. These findings suggest that components of the vesicle transport network, including vesicle-associated motor proteins, may be targets of AMPK in beta-cells, dephosphorylation of which is required for vesicle mobilization at elevated glucose concentrations.     

Cyclic adenosine diphosphate ribose activates ryanodine receptors, whereas NAADP activates two-pore domain channels

The mechanism by which cyclic adenosine diphosphate ribose (cADPR) and nicotinic acid adenine dinucleotide phosphate (NAADP) mobilize intracellular Ca(2+) stores remains controversial. It is open to question whether cADPR regulates ryanodine receptors (RyRs) directly, as originally proposed, or indirectly by promoting Ca(2+) uptake into the sarco/endoplasmic reticulum by sarco/endoplasmic reticulum Ca(2+)-ATPases. Conversely, although we have proposed that NAADP mobilizes endolysosomal Ca(2+) stores by activating two-pore domain channels (TPCs), others suggest that NAADP directly activates RyRs. We therefore assessed Ca(2+) signals evoked by intracellular dialysis from a patch pipette of cADPR and NAADP into HEK293 cells that stably overexpress either TPC1, TPC2, RyR1, or RyR3. No change in intracellular Ca(2+) concentration was triggered by cADPR in either wild-type HEK293 cells (which are devoid of RyRs) or in cells that stably overexpress TPC1 and TPC2, respectively. By contrast, a marked Ca(2+) transient was triggered by cADPR in HEK293 cells that stably expressed RyR1 and RyR3. The Ca(2+) transient was abolished following depletion of endoplasmic reticulum stores by thapsigargin and block of RyRs by dantrolene but not following depletion of acidic Ca(2+) stores by bafilomycin. By contrast, NAADP failed to evoke a Ca(2+) transient in HEK293 cells that expressed RyR1 or RyR3, but it induced robust Ca(2+) transients in cells that stably overexpressed TPC1 or TPC2 and in a manner that was blocked following depletion of acidic stores by bafilomycin. We conclude that cADPR triggers Ca(2+) release by activating RyRs but not TPCs, whereas NAADP activates TPCs but not RyRs.     

Synchronization of Ca(2+)-signals within insulin-secreting pseudoislets: effects of gap-junctional uncouplers

The secretory response of the intact islet is greater than the response of individual beta-cells in isolation, and functional coupling between cells is critical in insulin release. The changes in intracellular Ca(2+)([Ca(2+)](i)) which initiate insulin secretory responses are synchronized between groups of cells within the islet, and gap-junctions are thought to play a central role in coordinating signalling events. We have used the MIN6 insulin-secreting cell line, to examine whether uncoupling gap-junctions alters the synchronicity of nutrient- and non-nutrient-evoked Ca(2+)oscillations, or affects insulin secretion. MIN6 cells express mRNA species that can be amplified using PCR primers for connexin 36. A commonly used gap-junctional inhibitor, heptanol, inhibited glucose- and tolbutamide-induced Ca(2+)-oscillations to basal levels in MIN6 cell clusters at concentrations of 0.5 mM and greater, and it had similar effects in pseudoislets when used at 2.5 mM. Lower heptanol concentrations altered the frequency of Ca(2+)transients without affecting their synchronicity, in both monolayers and pseudoislets. Heptanol also had effects on insulin secretion from MIN6 pseudoislets such that 1 mM enhanced secretion while 2.5 mM was inhibitory. These data suggest that heptanol has multiple effects in pancreatic beta-cells, none of which appears to be related to uncoupling of synchronicity of Ca(2+)signalling between cells. A second gap-junction uncoupler, 18 alpha-glycyrrhetinic acid, also failed to uncouple synchronized Ca(2+)-oscillations, and it had no effect on insulin secretion. These data provide evidence that Ca(2+)signalling events occur simultaneously across the bulk mass of the pseudoislet, and suggest that gap-junctions are not required to coordinate the synchronicity of these events, nor is communication via gap junctions essential for integrated insulin secretory responses.     

Lysosomes co-localize with ryanodine receptor subtype 3 to form a trigger zone for calcium signalling by NAADP in rat pulmonary arterial smooth muscle

In arterial myocytes the Ca(2+) mobilizing messenger NAADP evokes spatially restricted Ca(2+) bursts from a lysosome-related store that are subsequently amplified into global Ca(2+) waves by Ca(2+)-induced Ca(2+)-release from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs). Lysosomes facilitate this process by forming clusters that co-localize with a subpopulation of RyRs on the SR. We determine here whether RyR subtypes 1, 2 or 3 selectively co-localize with lysosomal clusters in pulmonary arterial myocytes using affinity purified specific antibodies. The density of: (1) alphalgP120 labelling, a lysosome-specific protein, in the perinuclear region of the cell (within 1.5mum of the nucleus) was approximately 4-fold greater than in the sub-plasmalemmal (within 1.5mum of the plasma membrane) and approximately 2-fold greater than in the extra-perinuclear (remainder) regions; (2) RyR3 labelling within the perinuclear region was approximately 4- and approximately 14-fold greater than that in the extra-perinuclear and sub-plasmalemmal regions, and approximately 2-fold greater than that for either RyR1 or RyR2; (3) despite there being no difference in the overall densities of fluorescent labelling of lysosomes and RyR subtypes between cells, co-localization with alphalgp120 labelling within the perinuclear region was approximately 2-fold greater for RyR3 than for RyR2 or RyR1; (4) co-localization between alphalgp120 and each RyR subtype declined markedly outside the perinuclear region. Furthermore, selective block of RyR3 and RyR1 with dantrolene (30muM) abolished global Ca(2+) waves but not Ca(2+) bursts in response to intracellular dialysis of NAADP (10nM). We conclude that a subpopulation of lysosomes cluster in the perinuclear region of the cell and form junctions with SR containing a high density of RyR3 to comprise a trigger zone for Ca(2+) signalling by NAADP.     

Dantrolene prevents arrhythmogenic Ca2+ release in heart failure

In heart failure (HF), arrhythmogenic Ca(2+) release and chronic Ca(2+) depletion of the sarcoplasmic reticulum (SR) arise due to altered function of the ryanodine receptor (RyR) SR Ca(2+)-release channel. Dantrolene, a therapeutic agent used to treat malignant hyperthermia associated with mutations of the skeletal muscle type 1 RyR (RyR1), has recently been suggested to have effects on the cardiac type 2 RyR (RyR2). In this investigation, we tested the hypothesis that dantrolene exerts antiarrhythmic and inotropic effects on HF ventricular myocytes by examining multiple aspects of intracellular Ca(2+) handling. In normal rabbit myocytes, dantrolene (1 μM) had no effect on SR Ca(2+) load, postrest decay of SR Ca(2+) content, the threshold for spontaneous Ca(2+) wave initiation (i.e., the SR Ca(2+) content at which spontaneous waves initiate) and Ca(2+) spark frequency. In cardiomyocytes from failing rabbit hearts, SR Ca(2+) load and the wave initiation threshold were decreased compared with normal myocytes, Ca(2+) spark frequency was increased, and the postrest decay was potentiated. Using a novel approach of measuring cytosolic and intra-SR Ca(2+) concentration (using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR), we showed that treatment of HF cardiomyocytes with dantrolene rescued postrest decay and increased the wave initiation threshold. Additionally, dantrolene decreased Ca(2+) spark frequency while increasing the SR Ca(2+) content in HF myocytes. These data suggest that dantrolene exerts antiarrhythmic effects and preserves inotropy in HF cardiomyocytes by decreasing the incidence of diastolic Ca(2+) sparks, increasing the intra-SR Ca(2+) threshold at which spontaneous Ca(2+) waves occur, and decreasing the loss of Ca(2+) from the SR. Furthermore, the observation that dantrolene reduces arrhythmogenicity while at the same time preserves inotropy suggests that dantrolene is a potentially useful drug in the treatment of arrhythmia associated with HF.     

Imaging Ca2+ concentration changes at the secretory vesicle surface with a recombinant targeted cameleon.

Regulated exocytosis involves the Ca(2+)-triggered fusion of secretory vesicles with the plasma membrane, by activation of vesicle membrane Ca(2+)-binding proteins [1]. The Ca(2+)-binding sites of these proteins are likely to lie within 30 nm of the vesicle surface, a domain in which changes in Ca2+ concentration cannot be resolved by conventional fluorescence microscopy. A fluorescent indicator for Ca2+ called a yellow 'cameleon' (Ycam2) - comprising a fusion between a cyan-emitting mutant of the green fluorescent protein (GFP), calmodulin, the calmodulin-binding peptide M13 and an enhanced yellow-emitting GFP - which is targetable to specific intracellular locations, has been described [2]. Here, we generated a fusion between phogrin, a protein that is localised to secretory granule membranes [3], and Ycam2 (phogrin-Ycam2) to monitor changes in Ca2+ concentration ([Ca2+]) at the secretory vesicle surface ([Ca2+]gd) through alterations in fluorescence resonance energy transfer (FRET) between the linked cyan and yellow fluorescent proteins (CFP and YFP, respectively) in Ycam2. In both neuroendocrine PC12 and MIN6 pancreatic beta cells, apparent resting values of cytosolic [Ca2+] and [Ca2+](gd) were similar throughout the cell. In MIN6 cells following the activation of Ca2+ influx, the minority of vesicles that were within approximately 1 microm of the plasma membrane underwent increases in [Ca2+](gd) that were significantly greater than those experienced by deeper vesicles, and greater than the apparent cytosolic [Ca2+] change. The ability to image both global and compartmentalised [Ca2+] changes with recombinant targeted cameleons should extend the usefulness of these new Ca2+ probes.     

Direct detection of calmodulin tuning by ryanodine receptor channel targets using a Ca2+-sensitive acrylodan-labeled calmodulin

Calmodulin (CaM) activates the skeletal muscle ryanodine receptor (RyR1) at nanomolar Ca(2+) concentrations but inhibits it at micromolar Ca(2+) concentrations, indicating that binding of Ca(2+) to CaM may provide a molecular switch for modulating RyR1 channel activity. To directly examine the Ca(2+) sensitivity of RyR1-complexed CaM, we used an environment-sensitive acrylodan adduct of CaM. The resulting (ACR)CaM probe displayed high-affinity binding to, and Ca(2+)-dependent regulation of, RyR1 similar to that of unlabeled wild-type (WT) CaM. Upon addition of Ca(2+), (ACR)CaM exhibited a substantial (>50%) decrease in fluorescence (K(Ca) = 2.7 +/- 0.8 microM). A peptide derived from the RyR1 CaM binding domain (RyR1(3614)(-)(43)) caused an even more pronounced Ca(2+)-dependent fluorescence decrease, and a >or=10-fold leftward shift in its K(Ca) (0.2 +/- 0.1 microM). In the presence of intact RyR1 channels in SR vesicles, (ACR)CaM fluorescence spectra were distinct from those in the presence of RyR1(3614)(-)(43), although a Ca(2+)-dependent decrease in fluorescence was still observed. The K(Ca) for (ACR)CaM fluorescence in the presence of SR (0.8 +/- 0.4 microM) was greater than in the presence of RyR1(3614)(-)(43) but was consistent with functional determinations showing the conversion of (ACR)CaM from channel activator (apoCaM) to inhibitor (Ca(2+)CaM) at Ca(2+) concentrations between 0.3 and 1 microM. These results indicate that binding to RyR1 targets evokes significant changes in the CaM structure and Ca(2+) sensitivity (i.e., CaM tuning). However, changes resulting from binding of CaM to the full-length, tetrameric channels are clearly distinct from changes caused by the RyR1-derived peptide. We suggest that the Ca(2+) sensitivity of CaM when in complex with full-length channels may be tuned to respond to physiologically relevant changes in Ca(2+).     

Transmembrane redox sensor of ryanodine receptor complex

Inositol 1,4,5-trisphosphate receptors (IP(3)R) and ryanodine receptors (RyR) mediate the release of endoplasmic and sarcoplasmic reticulum (ER/SR) Ca(2+) stores and regulate Ca(2+) entry through voltage-dependent or ligand-gated channels of the plasma membrane. A prominent property of ER/SR Ca(2+) channels is exquisite sensitivity to sulfhydryl-modifying reagents. A plausible role for sulfhydryl chemistry in physiologic regulation of Ca(2+) release channels and the fidelity of Ca(2+) release from ER/SR is lacking. This study reveals the existence of a transmembrane redox sensor within the RyR1 channel complex that confers tight regulation of channel activity in response to changes in transmembrane redox potential produced by cytoplasmic and luminal glutathione. A transporter selective for glutathione is co-localized with RyR1 within the SR membrane to maintain local redox potential gradients consistent with redox regulation of ER/SR Ca(2+) release. Hyperreactive sulfhydryls previously shown to reside within the RyR1 complex (Liu, G., and Pessah, I. N. (1994) J. Biol. Chem. 269, 33028-33034) are an essential biochemical component of a transmembrane redox sensor. Transmembrane redox sensing may represent a fundamental mechanism by which ER/SR Ca(2+) channels respond to localized changes in transmembrane glutathione redox potential produced by physiologic and pathophysiologic modulators of Ca(2+) release from stores.     

Bile acids induce Ca2+ release from both the endoplasmic reticulum and acidic intracellular calcium stores through activation of inositol trisphosphate receptors and ryanodine receptors

Gallstones can cause acute pancreatitis, an often fatal disease in which the pancreas digests itself. This is probably because of biliary reflux into the pancreatic duct and subsequent bile acid action on the acinar cells. Because Ca(2+) toxicity is important for the cellular damage in pancreatitis, we have studied the mechanisms by which the bile acid taurolithocholic acid 3-sulfate (TLC-S) liberates Ca(2+). Using two-photon plasma membrane permeabilization and measurement of [Ca(2+)] inside intracellular stores at the cell base (dominated by ER) and near the apex (dominated by secretory granules), we have characterized the Ca(2+) release pathways. Inhibition of inositol trisphosphate receptors (IP(3)Rs), by caffeine and 2-APB, reduced Ca(2+) release from both the ER and an acidic pool in the granular area. Inhibition of ryanodine receptors (RyRs) by ruthenium red (RR) also reduced TLC-S induced liberation from both stores. Combined inhibition of IP(3)Rs and RyRs abolished Ca(2+) release. RyR activation depends on receptors for nicotinic acid adenine dinucleotide phosphate (NAADP), because inactivation by a high NAADP concentration inhibited release from both stores, whereas a cyclic ADPR-ribose antagonist had no effect. Bile acid-elicited intracellular Ca(2+) liberation from both the ER and the apical acidic stores depends on both RyRs and IP(3)Rs.     

CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic beta cells

CAPS1 and CAPS2 regulate dense-core vesicle release of transmitters and hormones in neuroendocrine cells, but their precise roles in the secretory process remain enigmatic. Here we show that CAPS2(-/-) and CAPS1(+/-);CAPS2(-/-) mice, despite having increased insulin sensitivity, are glucose intolerant and that this effect is attributable to a marked reduction of glucose-induced insulin secretion. This correlates with diminished Ca(2+)-dependent exocytosis, a reduction in the size of the morphologically docked pool, a decrease in the readily releasable pool of secretory vesicles, slowed granule priming, and suppression of second-phase (but not first-phase) insulin secretion. In beta cells of CAPS1(+/-);CAPS2(-/-) mice, the lowered insulin content and granule numbers were associated with an increase in lysosome numbers and lysosomal enzyme activity. We conclude that although CAPS proteins are not required for Ca(2+)-dependent exocytosis to proceed, they exert a modulatory effect on insulin granule priming, exocytosis, and stability.     

Fertilization and nicotinic acid adenine dinucleotide phosphate induce pH changes in acidic Ca(2+) stores in sea urchin eggs

The second messenger nicotinic acid adenine dinucleotide phosphate (NAADP) releases Ca(2+) from the acidic Ca(2+) stores of many organisms, including those of the sea urchin egg. We investigated whether the pH within the lumen of these acidic organelles changes in response to stimuli. Fertilization activates the egg by Ca(2+) release dependent upon NAADP, and accordingly, we report that fertilization also alters organellar pH in a spatio-temporally complex manner. Upon sperm fusion, vesicles deep in the egg center slowly acidify, whereas cortical vesicles undergo a rapid alkalinization. The cortical vesicle alkalinization is independent of exocytosis and cytosolic pH but coincides with the NAADP-dependent fertilization Ca(2+) wave. Microinjection of NAADP mimicked the fertilization cortical response, suggesting that it occurred within NAADP-sensitive acidic Ca(2+) stores. Our data show that NAADP and physiological stimuli alter the pH within intracellular organelles and suggest that NAADP signals through pH as well as Ca(2+).     

Ca2+ microdomains and the control of insulin secretion

Nutrient-induced increases in intracellular free Ca(2+) concentrations are the key trigger for insulin release from pancreatic islet beta-cells. These Ca(2+) changes are tightly regulated temporally, occurring as Ca(2+) influx-dependent baseline oscillations. We explore here the concept that locally high [Ca(2+)] concentrations (i.e. Ca(2+) microdomains) may control exocytosis via the recruitment of key effector proteins to sites of exocytosis. Importantly, recent advances in the development of organelle- and membrane-targeted green fluorescent protein (GFP-) or aequorin-based Ca(2+) indicators, as well as in rapid imaging techniques, are providing new insights into the potential role of these Ca(2+) microdomains in beta-cells. We summarise here some of the evidence indicating that Ca(2+) microdomains beneath the plasma membrane and at the surface of large dense core vesicles may be important in the normal regulation of insulin secretion, and may conceivably contribute to "ATP-sensitive K(+)-channel independent" effects of glucose. We also discuss evidence that, in contrast to certain non-excitable cells, direct transfer of Ca(2+) from the ER to mitochondria via localised physical contacts between these organelles is relatively less important for efficient mitochondrial Ca(2+) uptake in beta-cells. Finally, we discuss evidence from single cell imaging that increases in cytosolic Ca(2+) are not required for the upstroke of oscillations in mitochondrial redox state, but may underlie the reoxidation process.     

Azumolene inhibits a component of store-operated calcium entry coupled to the skeletal muscle ryanodine receptor

Dantrolene reduces the elevated myoplasmic Ca(2+) generated during malignant hyperthermia, a pharmacogenetic crisis triggered by volatile anesthetics. Although specific binding of dantrolene to the type 1 ryanodine receptor (RyR1), the Ca(2+) release channel of skeletal muscle sarcoplasmic reticulum, has been demonstrated, there is little evidence for direct dantrolene inhibition of RyR1 channel function. Recent studies suggest store-operated Ca(2+) entry (SOCE) contributes to skeletal muscle function, but the effect of dantrolene on this pathway has not been examined. Here we show that azumolene, an equipotent dantrolene analog, inhibits a component of SOCE coupled to activation of RyR1 by caffeine and ryanodine, whereas the SOCE component induced by thapsigargin is not affected. Our data suggest that azumolene distinguishes between two mechanisms of cellular signaling to SOCE in skeletal muscle, one that is coupled to and one independent from RyR1.     

Normalization of intracellular Ca(2+) induces a glucose-responsive state in glucose-unresponsive beta-cells

Although intracellular Ca(2+) in pancreatic beta-cells is the principal signal for insulin secretion, the effect of chronic elevation of the intracellular Ca(2+) concentration ([Ca(2+)](i)) on insulin secretion is poorly understood. We recently established two pancreatic beta-cell MIN6 cell lines that are glucose-responsive (MIN6-m9) and glucose-unresponsive (MIN6-m14). In the present study we have determined the cause of the glucose unresponsiveness in MIN6-m14. Initially, elevated [Ca(2+)](i) was observed in MIN6-m14, but normalization of the [Ca(2+)](i) by nifedipine, a Ca(2+) channel blocker, markedly improved the intracellular Ca(2+) response to glucose and the glucose-induced insulin secretion. The expression of subunits of ATP-sensitive K(+) channels and voltage-dependent Ca(2+) channels were increased at both mRNA and protein levels in MIN6-m14 treated with nifedipine. As a consequence, the functional expression of these channels at the cell surface, both of which are decreased in MIN6-m14 without nifedipine treatment, were increased significantly. Contrariwise, Bay K8644, a Ca(2+) channel agonist, caused severe impairment of glucose-induced insulin secretion in glucose-responsive MIN6-m9 due to decreased expression of the channel subunits. Chronically elevated [Ca(2+)](i), therefore, is responsible for the glucose unresponsiveness of MIN6-m14. The present study also suggests normalization of [Ca(2+)](i) in pancreatic beta-cells as a therapeutic strategy in treatment of impaired insulin secretion.     

Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells.

Synaptotagmin (Syt) I-deficient phaeochromocytoma (PC12) cell lines show normal Ca(2+)-dependent norepinephrine (NE) release (Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R., and Takahashi, M. (1992) Science 256, 1821-1823). To identify an alternative Ca(2+) sensor, we searched for other Syt isoforms in Syt I-deficient PC12 cells and identified Syt IX, an isoform closely related to Syt I, as an abundantly expressed dense-core vesicle protein. Here we show that Syt IX is required for the Ca(2+)-dependent release of NE from PC12 cells. Antibodies directed against the C2A domain of either Syt IX or Syt I inhibited Ca(2+)-dependent NE release in permeable PC12 cells indicating that both Syt proteins function in dense-core vesicle exocytosis. Our results support the idea that Syt family proteins that co-reside on secretory vesicles may function cooperatively and redundantly as potential Ca(2+) sensors for exocytosis.     

Calcium-induced calcium release contributes to somatic secretion of serotonin in leech Retzius neurons

We analyzed the contribution of calcium (Ca2+)-induced Ca2+ release to somatic secretion in serotonergic Retzius neurons of the leech. Somatic secretion was studied by the incorporation of fluorescent dye FM1-43 upon electrical stimulation with trains of 10 impulses and by electron microscopy. Quantification of secretion with FM1-43 was made in cultured neurons to improve optical resolution. Stimulation in the presence of FM1-43 produced a frequency-dependent number of fluorescent spots. While a 1-Hz train produced 19.5+/-5.0 spots/soma, a 10-Hz train produced 146.7+/-20.2 spots/soma. Incubation with caffeine (10 mM) to induce Ca2+ release from intracellular stores without electrical stimulation and external Ca2+, produced 168+/-21.7 spots/soma. This staining was reduced by 49% if neurons were preincubated with the Ca2+- ATPase inhibitor thapsigargin (200 nM). Moreover, in neurons stimulated at 10 Hz in the presence of ryanodine (100 microM) to block Ca2+-induced Ca2+ release, FM1-43 staining was reduced by 42%. In electron micrographs of neurons at rest or stimulated at 1 Hz in the ganglion, endoplasmic reticulum lay between clusters of dense core vesicles and the plasma membrane. In contrast, in neurons stimulated at 20 Hz, the vesicle clusters were apposed to the plasma membrane and flanked by the endoplasmic reticulum. These results suggest that Ca2+-induced Ca2+ release produces vesicle mobilization and fusion in the soma of Retzius neurons, and supports the idea that neuronal somatic secretion shares common mechanisms with secretion by excitable endocrine cells.     

Divergent effects of the malignant hyperthermia-susceptible Arg(615)-->Cys mutation on the Ca(2+) and Mg(2+) dependence of the RyR1

The sarcoplasmic reticulum (SR) Ca(2+) release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg(2+). This diminished Mg(2+) inhibition has been attributed to a lower Mg(2+) affinity of the inhibition (I) site. To determine whether alterations in the Ca(2+) and Mg(2+) affinity of the activation (A) site contribute to the altered Mg(2+) inhibition, we estimated the Ca(2+) and Mg(2+) affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca(2+) to half-maximally activate [(3)H]ryanodine binding (K(A,Ca): MHS = 0.17 +/- 0.01 microM; normal = 0.29 +/- 0.02 microM) and more Ca(2+) to half-maximally inhibit ryanodine binding (K(I,Ca): MHS = 519.3 +/- 48.7 microM; normal = 293.3 +/- 24.2 microM). The apparent Mg(2+) affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (K(A,Mg): MHS = 44.36 +/- 4.54 microM; normal = 21.59 +/- 1.66 microM; K(I,Mg): MHS = 660.8 +/- 53.0 microM; normal = 299.2 +/- 24.5 microM). Thus, the reduced Mg(2+) inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca(2+) over Mg(2+) and a reduced Mg(2+) affinity of the I-site.     

The skeletal muscle ryanodine receptor identified as a molecular target of [3H]azidodantrolene by photoaffinity labeling

Dantrolene is a skeletal muscle relaxant which acts by inhibiting intracellular Ca(2+) release from sarcoplasmic reticulum (SR). It is used primarily in the treatment of malignant hyperthermia (MH), a pharmacogenetic sensitivity to volatile anesthetics resulting in massive intracellular Ca(2+) release. Determination of the site and mechanism of action of dantrolene should contribute to the understanding of the regulation of intracellular Ca(2+) release in skeletal muscle. Photoaffinity labeling of porcine SR with [(3)H]azidodantrolene, a photoactivatable analogue of dantrolene, has identified a 160 kDa SR protein with immunologic cross-reactivity to skeletal muscle ryanodine receptor (RyR) as a possible target [Palnitkar et al. (1999) J. Med. Chem. 42, 1872-1880]. Here we demonstrate specific, AMP-PCP-enhanced, [(3)H]azidodantrolene photolabeling of both the RyR monomer and a 160 or 172 kDa protein in porcine and rabbit SR, respectively. The 160/172 kDa protein is shown to be the NH(2)-terminus of the RyR cleaved from the monomer by an endogenous protease activity consistent with that of n-calpain. MALDI-mass spectrometric analysis of the porcine 160 kDa protein identifies it as the 1400 amino acid NH(2)-terminal fragment of the skeletal muscle RyR reportedly generated by n-calpain [Shevchenko et al. (1998) J. Membr. Biol. 161, 33-34]. Immunoprecipitation of solubilized, [(3)H]azidodantrolene-photolabeled SR protein reveals that the cleaved 160/172 kDa protein remains associated with the C-terminal, 410 kDa portion of the RyR. [(3)H]Dantrolene binding to both the intact and the n-calpain-cleaved channel RyR is similarly enhanced by AMP-PCP. n-Calpain cleavage of the RyR does not affect [(3)H]dantrolene binding in the presence of AMP-PCP, but depresses drug binding in the absence of nucleotide. These results demonstrate that the NH(2)-terminus of the RyR is a molecular target for dantrolene, and suggest a regulatory role for both n-calpain activity and ATP in the interaction of dantrolene with the RyR in vivo.