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Author(s): Damian B. van Rossum [1, 6]; Randen L. Patterson [4, 6]; Sumit Sharma [1]; Roxanne K. Barrow [1]; Michael Kornberg [1]; Donald L. Gill [5]; Solomon H. Snyder (corresponding [...]

It has been considered that [Ca.sup.2+] release is the causal trigger for [Ca.sup.2+] entry after receptor activation. In DT40 B cells devoid of inositol 1,4,5-trisphosphate receptors (I[P.sub.3]R), the lack of [Ca.sup.2+] entry in response to receptor activation is attributed to the absence of [Ca.sup.2+] release. We reveal in this article that I[P.sub.3]R recognition of I[P.sub.3] determines agonist-induced [Ca.sup.2+] entry (ACE), independent of its [Ca.sup.2+] release activity. In DT40 I[P.sub.3][R.sup.-/-] cells, endogenous ACE can be rescued with type 1 I[P.sub.3]R mutants (both a [DELTA]C-terminal truncation mutant and a D2550A pore mutant), which are defective in [Ca.sup.2+] release channel activity. Thus, in response to B cell receptor activation, ACE is restored in an I[P.sub.3]R-dependent manner without [Ca.sup.2+] store release. Conversely, ACE cannot be rescued with mutant I[P.sub.3]Rs lacking I[P.sub.3] binding (both the [DELTA]90-110 and R265Q I[P.sub.3]-binding site mutants). We conclude that an I[P.sub.3]-dependent conformational change in the I[P.sub.3]R, not endoplasmic reticulum [Ca.sup.2+] pool release, triggers ACE.

RACK1 is not a G protein but closely resembles the heterotrimeric G[beta]-subunit. RACK1 serves as a scaffold, linking protein kinase C to its substrates. We demonstrate that RACK1 physiologically binds inositol 1,4,5-trisphosphate receptors and regulates [Ca.sup.2+] release by enhancing inositol 1,4,5-trisphosphate receptor binding affinity for inositol 1,4,5-trisphosphate. Overexpression of RACK1 or depletion of RACK1 by interference RNA markedly augments or diminishes [Ca.sup.2+] release, respectively, without affecting [Ca.sup.2+] entry. These findings establish RACK1 as a physiologic mediator of agonist-induced [Ca.sup.2+] release.

NADH regulates the release of calcium from the endoplasmic reticulum by modulation of inositol 1,4,5-trisphosphate receptors (I[P.sub.3]R), accounting for the augmented calcium release of hypoxic cells. We report selective binding of I[P.sub.3]R to GAPDH, whose activity leads to the local generation of NADH to regulate intracellular calcium signaling. This interaction requires cysteines 992 and 995 of I[P.sub.3]R and C150 of GAPDH. Addition of native GAPDH and NA[D.sup.+] to WT I[P.sub.3]R stimulates calcium release, whereas no stimulation occurs with C992S/995S I[P.sub.3]R that cannot bind GAPDH. Thus, the I[P.sub.3]R/ GAPDH interaction likely enables cellular energy dynamics to impact calcium signaling. calcium | hypoxia | metabolism

NADH regulates the release of calcium from the endoplasmic reticulum by modulation of inositol 1,4,5-trisphosphate receptors (IP3R), accounting for the augmented calcium release of hypoxic cells. We report selective binding of IP3R to GAPDH, whose activity leads to the local generation of NADH to regulate intracellular calcium signaling. This interaction requires cysteines 992 and 995 of IP3R and C150 of GAPDH. Addition of native GAPDH and NAD+ to WT IP3R stimulates calcium release, whereas no stimulation occurs with C992S/995S IP3R that cannot bind GAPDH. Thus, the IP3R/ GAPDH interaction likely enables cellular energy dynamics to impact calcium signaling. [ABSTRACT FROM AUTHOR]

We report the cloning and characterization of DANGER, a novel protein which physiologically binds to inositol 1,4,5-trisphosphate receptors (IP3R). DANGER is a membrane-associated protein predicted to contain a partial MAB-21 domain. It is expressed in a wide variety of neuronal cell lineages where it localizes to membranes in the cell periphery together with IP3R. DANGER interacts with IP3R in vitro and co-immunoprecipitates with IP3R from cellular preparations. DANGER robustly enhances Ca2+-mediated inhibition of IP3R Ca2+ release without affecting IP3 binding in microsomal assays and inhibits gating in single-channel recordings of IP3R. DANGER appears to allosterically modulate the sensitivity of IP3R to Ca2+ inhibition, which likely alters IP3R-mediated Ca2+ dynamics in cells where DANGER and IP3R are co-expressed. [ABSTRACT FROM AUTHOR]