Journal articles

Previous structure-activity relationship studies of adenophostin A, a potent IP(3) receptor agonist, led us to design the novel adenophostin A analogues 5a-c, conjugating an aromatic group at the 5'-position to develop useful IP(3) receptor ligands. The common key intermediate, a D-ribosyl alpha-D-glucoside 10alpha, was stereoselectively synthesized by a glycosidation with the 1-sulfinylglucoside donor 11, which was conformationally restricted by a 3,4-O-cyclic diketal protecting group.

Ryanodine receptors (RyR) are Ca(2+) channels that mediate Ca(2+) release from intracellular stores in response to diverse intracellular signals. In RINm5F insulinoma cells, caffeine, and 4-chloro-m-cresol (4CmC), agonists of RyR, stimulated Ca(2+) entry that was independent of store-operated Ca(2+) entry, and blocked by prior incubation with a concentration of ryanodine that inactivates RyR. Patch-clamp recording identified small numbers of large-conductance (gamma(K) = 169 pS) cation channels that were activated by caffeine, 4CmC or low concentrations of ryanodine.

In several cell types, including hepatocytes, submaximal concentrations of Ins(1,4,5)P3 stimulate an initial rapid mobilization of intracellular Ca2+ stores that is followed by either no further Ca2+ release or very much slower release. Further additions of Ins(1,4,5)P3 then evoke further Ca2+ mobilization. Such 'incremental' responses [Meyer & Stryer (1990) Proc. Natl. Acad. Sci. U.S.A.

Interactions between cyclic adenosine monophosphate (cAMP) and Ca(2+) are widespread, and for both intracellular messengers, their spatial organization is important. Parathyroid hormone (PTH) stimulates formation of cAMP and sensitizes inositol 1,4,5-trisphosphate receptors (IP(3)R) to IP(3). We show that PTH communicates with IP(3)R via "cAMP junctions" that allow local delivery of a supramaximal concentration of cAMP to IP(3)R, directly increasing their sensitivity to IP(3).

Inositol 1,4,5-trisphosphate (InsP3) functions as a second messenger to mobilize Ca2+ from intracellular reservoirs. The release mechanism displays all-or-none characteristics, that may account for other observations that the InsP3-induced mobilization of Ca2+ is quantal. Quantal release may depend on the sensitivity of the InsP3 receptor being regulated by the Ca2+ concentration in the lumen of the endoplasmic reticulum. We report here that the InsP3-sensitive store in hepatocytes discharges spontaneously when overloaded with Ca2+.

IP3Rs (inositol 1,4,5-trisphosphate receptors) are expressed in the membranes of non-mitochondrial organelles in most animal cells, but their presence and role within the plasma membrane are unclear. Whole-cell patch-clamp recording from DT40 cells expressing native or mutated IP3Rs has established that each cell expresses just two or three functional IP3Rs in its plasma membrane. Only approx. 50% of the Ca2+ entry evoked by stimulation of the B-cell receptor is mediated by store-operated Ca2+ entry, the remainder appears to be carried by the IP3Rs expressed in the plasma membrane.

Store-operated Ca2+ entry (SOCE) mediates much of the Ca2+ entry evoked by receptors that stimulate phospholipase C. However, for 20 years, the plasma membrane Ca2+ channel and the signal linking its activation to loss of Ca2+ from the endoplasmic reticulum (ER) have eluded detection. But the search might now be over. Two proteins, STIM1 (the ER Ca2+ sensor) and Orai1 (the Ca2+ channel), have recently been identified as the missing links in SOCE.

PTH (parathyroid hormone), acting via type 1 PTH receptors, is a major regulator of plasma [Ca(2+)]. The G-protein, G(s), is an essential component of the sequence linking PTH to plasma Ca(2+) regulation, but the relative importance of intracellular signals, including Ca(2+) and cAMP, that lie downstream of G(s) is not resolved.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses.