Increases in the concentration of free ATP within the islet ␤-cell may couple elevations in blood glucose to insulin release by closing ATP-sensitive K ؉ (K ATP ) channels and activating Ca 2؉ influx. Here, we use recombinant targeted luciferases and photon counting imaging to monitor changes in free [ATP] Increases in extracellular glucose concentration stimulate the exocytosis of insulin from islet ␤-cells. This is probably achieved by an increase in glycolysis and flux through the citrate cycle (1), leading to elevated intracellular levels of likely coupling factors (2), including ATP. Closure of ATP-sensitive K ϩ channels (3-5) then leads to plasma membrane depolarization and the influx of Ca 2ϩ through voltage gated Ca 2ϩ channels. Increases in the total intracellular concentration of ATP have been measured in isolated islets (6) and cell lines (7) exposed to increases in extracellular glucose concentration. However, the measured changes are generally small and difficult to interpret because of the large depot of intragranular ATP, and the presence of non-␤-cells (8). Furthermore, such measurements give no indication of the concentration of unbound ATP. Unfortunately, measurements of free [ATP] in living cells, for example by 31 P NMR (9), cannot easily be extended to the islet micro-organ and do not provide sufficient sensitivity to detect changes at the cellular or subcellular level. This is an important question because differences in [ATP] at different intracellular sites have been predicted. In particular, locally high ATP consumption by the plasma membrane Na ϩ -K ϩ and Ca 2ϩ -ATPase, may mean that [ATP] is lower in this domain than in the bulk of the cell cytosol (1). Similarly, the electrogenic nature of the mitochondrial ATP/ADP translocase (10) is predicted to create differences in ATP/ADP ratio across the inner mitochondrial membrane (cytosolic high).The role of changes in free Ca 2ϩ ion concentration ([Ca 2ϩ ]) in regulating ␤-cell metabolism and ATP concentration is controversial. Increases in [Ca 2ϩ ] following plasma membrane depolarization act both to stimulate ATP requiring processes (i.e. secretory granule movement and exocytosis) (11) and possibly to enhance mitochondrial oxidative metabolism (12, 13). Recent measurements of total ATP content of whole islets have suggested that the former may dominate and that Ca 2ϩ influx may diminish glucose-induced increases in ATP/ADP ratio (14).The use of firefly luciferase, targeted to discrete intracellular domains, should provide an extremely sensitive method of monitoring free [ATP] dynamically and at the subcellular level. In previous studies, we have shown that photon counting imaging of total luciferase activity in single cells provides a convenient means to measure changes in gene expression in single cells (15,16). Luciferase has previously been employed to measure intracellular ATP concentration in single cardiac myocytes (17) and hepatocytes (18), but only after microinjection of the purified protein. In recent reports, Maechler...

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The role of dense core secretory vesicles in the control of cytosolic-free Ca2+ concentrations ([Ca2+]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 β-cells: (a) increases in [Ca2+]c cause a prompt increase in intravesicular-free Ca2+ concentration ([Ca2+]SV), which is mediated by a P-type Ca2+-ATPase distinct from the sarco(endo) plasmic reticulum Ca2+-ATPase, but which may be related to the PMR1/ATP2C1 family of Ca2+ pumps; (b) steady state Ca2+ 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 Ca2+; (c) inositol (1,4,5) trisphosphate has no impact on [Ca2+]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 [Ca2+]SV. Thus, secretory vesicles represent a dynamic Ca2+ 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 Ca2+-induced Ca2+ release from vesicles docked at the plasma membrane could participate in triggering exocytosis.

SUMMARY Using high-throughput screening we identified small molecules that suppress superoxide and/or H2O2 production during reverse electron transport through mitochondrial respiratory complex I (site IQ) without affecting oxidative phosphorylation (suppressors of site IQ electron leak, “S1QELs”). S1QELs diminished endogenous oxidative damage in primary astrocytes cultured at ambient or low oxygen tension, showing that site IQ is a normal contributor to mitochondrial superoxide-H2O2 production in cells. They diminished stem cell hyperplasia in Drosophila intestine in vivo and caspase activation in a cardiomyocyte cell model driven by endoplasmic reticulum stress, showing that superoxide-H2O2 production by site IQ is involved in cellular stress signaling. They protected against ischemia-reperfusion injury in perfused mouse heart, showing directly that superoxide-H2O2 production by site IQ is a major contributor to this pathology. S1QELs are tools for assessing the contribution of site IQ to cell physiology and pathology and have great potential as therapeutic leads.

Available drugs are unable to effectively rescue the folding defects in vitro and ameliorate the clinical-phenotype of cystic fibrosis (CF), caused by deletion of F508 (ΔF508 or F508del) and some point mutations in the CF transmembrane conductance regulator (CFTR), a plasma membrane (PM) anion channel. To overcome the corrector efficacy ceiling, here we show that compounds targeting distinct structural defects of CFTR can synergistically rescue mutants expression and function at the PM. High throughput cell-based screens and mechanistic analysis identified three small-molecule series that target defects at the nucleotide binding domain (NBD1), NBD2 and their membrane spanning domains (MSDs) interfaces. While individually these compounds marginally improve ΔF508-CFTR folding efficiency, function, and stability, their combinations lead to ~50–100% of wild type-level correction in immortalized and primary human airway epithelia, and in mouse nasal epithelia. Likewise, corrector combinations were effective for rare missense mutations in various CFTR domains, probably acting via structural allostery, suggesting a mechanistic framework for their broad application.