Journal articles

Most inositol 1,4,5-trisphosphate receptors (IP3R) are expressed in the endoplasmic reticulum ( ER), where their precise distribution underlies the spatially complex Ca2+ signals evoked by extracellular stimuli. The signals that target IP3R to the ER or, less commonly, to other membranes are unknown. We expressed yellow fluorescent protein-tagged fragments of type 1 IP3R alone or fused with a plasma membrane protein to establish the determinants of ER targeting in COS-7 cells.

CaM (calmodulin) has been implicated in the regulation of IP3R [IP3 (inositol 1,4,5-trisphosphate) receptors] and a recent report suggested that CaM tightly tethered to IP3R was essential for IP3R activation [Nadif Kasri, Torok, Galione, Garnham, Callewaert, Missiaen, Parys and De Smedt (2006) J. Biol. Chem. 281, 8332-8338]. In the present study, we confirm that a CaM-binding peptide derived from MLCK (myosin light chain kinase) inhibits IP3-evoked Ca2+ release via all three IP3R subtypes.

Functional assays of intracellular Ca2+ channels, such as the inositol 1,4,5-trisphosphate receptor (IP3R), have generally used 45Ca2+-flux assays, fluorescent indicators loaded within either the cytosol or the endoplasmic reticulum (ER) of single cells, or electrophysiological analyses. None of these methods is readily applicable to rapid, high-throughput quantitative analyses. Here we provide a detailed protocol for high-throughput functional analysis of native and recombinant IP3Rs.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca2+ channels that link extracellular stimuli to Ca2+ signals. Ca2+ release from intracellular stores is ‘quantal’: low IP3 concentrations rapidly release a fraction of the stores. Ca2+ release then slows or terminates without compromising responses to further IP3 additions. The mechanisms are unresolved. Here, we synthesise a high-affinity partial agonist of IP3Rs and use it to demonstrate that quantal responses do not require heterogenous Ca2+ stores.

Reactions that form sec-sec ethers are well known, but few lead to compounds with dense functionality around the O-linkage. Replacement of the α-glucopyranosyl unit of adenophostin A, a potent d-myo-inositol 1,4,5-trisphosphate (IP3R) agonist, with a d-chiro-inositol surrogate acting substantially as a pseudosugar, leads to "d-chiro-inositol adenophostin". At its core, this cyclitol-nucleoside trisphosphate comprises a nucleoside sugar linked via an axial d-chiro-inositol 1-hydroxyl-adenosine 3'-ribose ether linkage.

Synthetic Ca2+ indicators are widely used to report changes in free [Ca2+], usually in the cytosol but also within organelles. Mag-Fluo-4, loaded into the endoplasmic reticulum (ER) by incubating cells with Mag-Fluo-4 AM, has been used to measure changes in free [Ca2+] within the ER, where the free [Ca2+] is estimated to be between 100 µM and 1 mM. Many results are consistent with Mag-Fluo-4 reliably reporting changes in free [Ca2+] within the ER, but the results are difficult to reconcile with the affinity of Mag-Fluo-4 for Ca2+ measured in vitro (KDCa 22 µM).