It is known that Ca 2؉ influx plays an important role in the modulation of inositol trisphosphate-generated Ca 2؉ oscillations, but controversy over the mechanisms underlying these effects exists. In addition, the effects of blocking membrane transport or reducing Ca 2؉ entry vary from one cell type to another; in some cell types oscillations persist in the absence of Ca 2؉ entry (although their frequency is affected), whereas in other cell types oscillations depend on Ca 2؉ entry. We present theoretical and experimental evidence that membrane transport can control oscillations by controlling the total amount of Ca 2؉ in the cell (the Ca 2؉ load). Our model predicts that the cell can be balanced at a point where small changes in the Ca 2؉ load can move the cell into or out of oscillatory regions, resulting in the appearance or disappearance of oscillations. Our theoretical predictions are verified by experimental results from HEK293 cells. We predict that the role of Ca 2؉ influx during an oscillation is to replenish the Ca 2؉ load of the cell. Despite this prediction, even during the peak of an oscillation the cell or the endoplasmic reticulum may not be measurably depleted of Ca 2؉ .I n response to an increased concentration of inositol trisphosphate (IP 3 ), oscillations in the concentration of free intracellular calcium (Ca 2ϩ ) occur in many cell types and are important for the control of many cellular functions (1-4). In nonexcitable cells, such as epithelial cells, these oscillations occur as the result of Ca 2ϩ flux into and out of the endoplasmic reticulum (ER). However, although the oscillations result from the cycling of Ca 2ϩ between the ER and the cytoplasm, the transport of Ca 2ϩ across the cell membrane can have a dramatic effect on these oscillations.Although Ca 2ϩ influx is known to be important, disagreement exists, first, over the mechanisms by which Ca 2ϩ influx is modulated during a Ca 2ϩ oscillation, and, second, over the role played by Ca 2ϩ influx. The capacitative entry hypothesis proposes that depletion of ER Ca 2ϩ causes enhanced entry of Ca 2ϩ across the plasma membrane (5-9). Often, although not necessarily, in this scenario, Ca 2ϩ entry is necessary for the refilling of the ER, and thus oscillation frequency is controlled by the refilling time (10, 11). Although capacitative entry is certainly an important factor during the response to a maximal stimulus, other investigators have pointed to a lack of direct evidence that it plays an important role during smaller stimuli that generate oscillatory behavior (12). They maintain that Ca 2ϩ influx, under these conditions, is controlled by a noncapacitative pathway involving arachidonic acid and that the purpose of the influx is to increase the likelihood that low levels of IP 3 will induce Ca 2ϩ release from internal stores (13). This controversy is complicated by the fact that, in some cell types, Ca 2ϩ oscillations persist in the absence of Ca 2ϩ influx (14-16), whereas in other cell types, oscillations depend absolutely on influx (17, ...

Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

22Word Count: 5145 23 Figure Count: 5 figures 24 25 26 42 platelets express members of the acetylcholine signaling pathway including CHRNA2, CHRNA7,43 CHRNB1, and ACHE. Platelets from mice lacking Chrna7 are hyperactive when stimulated by 44 thrombin and resistant to inhibition by acetylcholine. Furthermore, acetylcholinesterase 45 inhibitors prolonged bleeding in wild-type mice. Knockout mice lacking Chrna7 subunits of the 46 acetylcholine receptor display prolonged bleeding as well. 47 52 Abbreviations 53 NO nitric oxide 54 CHRNA7 cholinergic receptor neuronal nicotinic alpha polypeptide 7 55 GPIIbIIIA glycoprotein IIb IIIa 56 AChR acetylcholine receptors 57 AChE acetylcholinesterase 58 TRAP thrombin receptor activating peptide 6 59 PAR1 protease activated receptor 1 60 P2Y12 purinergic receptor P2Y 61 GPVI glycoprotein VI 62 NOS3 nitric oxide synthase isoform 3 63 L-NAME L-nitroarginine methyl ester