The impact of calcium signalling on so many areas of cell biology reflects the crucial role of calcium signals in the control of diverse cellular functions. Despite the precision with which spatial and temporal details of calcium signals have been resolved, a fundamental aspect of the generation of calcium signals -- the activation of 'store-operated channels' (SOCs) -- remains a molecular and mechanistic mystery. Here we review new insights into the exchange of signals between the endoplasmic reticulum (ER) and plasma membrane that result in activation of calcium entry channels mediating crucial long-term calcium signals.

The factors that organize the internal membranes of cells are still poorly understood. We have been addressing this question using striated muscle cells, which have regular arrays of membranes that associate with the contractile apparatus in stereotypic patterns. Here we examine links between contractile structures and the sarcoplasmic reticulum (SR) established by small ankyrin 1 (sAnk1), a approximately 17.5-kDa integral protein of network SR. We used yeast two-hybrid to identify obscurin, a giant Rho-GEF protein, as the major cytoplasmic ligand for sAnk1. The binding of obscurin to the cytoplasmic sequence of sAnk1 is mediated by a sequence of obscurin that is C-terminal to its last Ig-like domain. Binding was confirmed in two in vitro assays. In one, GST-obscurin, bound to glutathione-matrix, specifically adsorbed native sAnk1 from muscle homogenates. In the second, MBP-obscurin bound recombinant GST-sAnk1 in nitrocellulose blots. Kinetic studies using surface plasmon resonance yielded a K(D) = 130 nM. On subcellular fractionation, obscurin was concentrated in the myofibrillar fraction, consistent with its identification as sarcomeric protein. Nevertheless, obscurin, like sAnk1, concentrated around Z-disks and M-lines of striated muscle. Our findings suggest that obscurin binds sAnk1, and are the first to document a specific and direct interaction between proteins of the sarcomere and the SR.

We report here that PLC-gamma isoforms are required for agonist-induced Ca2+ entry (ACE). Overexpressed wild-type PLC-gamma1 or a lipase-inactive mutant PLC-gamma1 each augmented ACE in PC12 cells, while a deletion mutant lacking the region containing the SH3 domain of PLC-gamma1 was ineffective. RNA interference to deplete either PLC-gamma1 or PLC-gamma2 in PC12 and A7r5 cells inhibited ACE. In DT40 B lymphocytes expressing only PLC-gamma2, overexpressed muscarinic M5 receptors (M5R) activated ACE. Using DT40 PLC-gamma2 knockout cells, M5R stimulation of ER Ca2+ store release was unaffected, but ACE was abolished. Normal ACE was restored by transient expression of PLC-gamma2 or a lipase-inactive PLC-gamma2 mutant. The results indicate a lipase-independent role of PLC-gamma in the physiological agonist-induced activation of Ca2+ entry.

Many ion channels are regulated by lipids, but prominent motifs for lipid binding have not been identified in most ion channels. Recently, we reported that phospholipase Cgamma1 (PLC-gamma1) binds to and regulates TRPC3 channels, components of agonist-induced Ca2+ entry into cells. This interaction requires a domain in PLC-gamma1 that includes a partial pleckstrin homology (PH) domain-a consensus lipid-binding and protein-binding sequence. We have developed a gestalt algorithm to detect hitherto 'invisible' PH and PH-like domains, and now report that the partial PH domain of PLC-gamma1 interacts with a complementary partial PH-like domain in TRPC3 to elicit lipid binding and cell-surface expression of TRPC3. Our findings imply a far greater abundance of PH domains than previously appreciated, and suggest that intermolecular PH-like domains represent a widespread signalling mode.

The elusive coupling between endoplasmic reticulum (ER) Ca2+ stores and plasma membrane (PM) "store-operated" Ca2+ entry channels was probed through a novel combination of cytoskeletal modifications. Whereas coupling was unaffected by disassembly of the actin cytoskeleton, in situ redistribution of F-actin into a tight cortical layer subjacent to the PM displaced cortical ER and prevented coupling between ER and PM Ca2+ entry channels, while not affecting inositol 1,4,5-trisphosphate-mediated store release. Importantly, disassembly of the induced cortical actin layer allowed ER to regain access to the PM and reestablish coupling of Ca2+ entry channels to Ca2+ store depletion. Coupling is concluded to be mediated by a physical "secretion-like" mechanism involving close but reversible interactions between the ER and the PM.

Heat-shock proteins (HSPs) are abundant, inducible proteins best known for their ability to maintain the conformation of proteins and to refold damaged proteins. Some HSPs, especially HSP90, can be antiapoptotic and the targets of anticancer drugs. Inositol hexakisphosphate kinase-2 (IP6K2), one of a family of enzymes generating the inositol pyrophosphate IP7 [diphosphoinositol pentakisphosphate (5-PP-IP5)], mediates apoptosis. Increased IP6K2 activity sensitizes cancer cells to stressors, whereas its depletion blocks cell death. We now show that HSP90 physiologically binds IP6K2 and inhibits its catalytic activity. Drugs and selective mutations that abolish HSP90 -IP6K2 binding elicit activation of IP6K2, leading to cell death. Thus, the prosurvival actions of HSP90 reflect the inhibition of IP6K2, suggesting that selectively blocking this interaction could provide effective and safer modes of chemotherapy.apoptosis ͉ cisplatin ͉ novobiocin ͉ inositol polyphosphate

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TFII-I is a transcription factor and a target of phosphorylation by Bruton's tyrosine kinase. In humans, deletions spanning the TFII-I locus are associated with a cognitive defect, the Williams-Beuren cognitive profile. We report an unanticipated role of TFII-I outside the nucleus as a negative regulator of agonist-induced calcium entry (ACE) that suppresses surface accumulation of TRPC3 (transient receptor potential C3) channels. Inhibition of ACE by TFII-I requires phosphotyrosine residues that engage the SH2 (Src-homology 2) domains of phospholipase C-g (PLC-g) and an interrupted, pleckstrin homology (PH)-like domain that binds the split PH domain of PLC-g. Our observations suggest a model in which TFII-I suppresses ACE by competing with TRPC3 for binding to PLC-g.