No abstract

Interactions between cyclic adenosine monophosphate (cAMP) and Ca2+ 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 (IP3R) to IP3. We show that PTH communicates with IP3R via “cAMP junctions” that allow local delivery of a supramaximal concentration of cAMP to IP3R, directly increasing their sensitivity to IP3. These junctions are robust binary switches that are digitally recruited by increasing concentrations of PTH. Human embryonic kidney cells express several isoforms of adenylyl cyclase (AC) and IP3R, but IP3R2 and AC6 are specifically associated, and inhibition of AC6 or IP3R2 expression by small interfering RNA selectively attenuates potentiation of Ca2+ signals by PTH. We define two modes of cAMP signaling: binary, where cAMP passes directly from AC6 to IP3R2; and analogue, where local gradients of cAMP concentration regulate cAMP effectors more remote from AC. Binary signaling requires localized delivery of cAMP, whereas analogue signaling is more dependent on localized cAMP degradation.

Inositol 1,4,5-trisphosphate receptors (IP3R) are ubiquitous intracellular Ca2+ channels. IP3binding to the IP3-binding core (IBC) near the N-terminal initiates conformational changes that lead to opening of a pore. The mechanisms are unresolved. We synthesized 2-O-modified IP3 analogues that are partial agonists of IP3R. These are like IP3 in their interactions with the IBC, but they are less effective than IP3 in rearranging the relationship between the IBC and N-terminal suppressor domain (SD), and they open the channel at slower rates. IP3R with a mutation in the SD occupying a position similar to the 2-O-substituent of the partial agonists has a reduced open probability that is similar for full and partial agonists. Bulky or charged substituents from either the ligand or SD therefore block obligatory coupling of the IBC and SD. Analysis of ΔG for ligand binding shows that IP3 is recognised by the IBC and conformational changes then propagate entirely via the SD to the pore.

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP 3 ), and pure cerebellar InsP 3 receptors were characterized by using a scintillation proximity assay.

Inositol 1,4,5-trisphosphate receptors (IP 3 Rs), by releasing Ca 2+ from the endoplasmic reticulum (ER) of animal cells, allow Ca 2+ to be redistributed from the ER to the cytosol or other organelles, and they initiate store-operated Ca 2+ entry (SOCE). For all three IP 3 R subtypes, binding of IP 3 primes them to bind Ca 2+ , which then triggers channel opening. We are now close to understanding the structural basis of IP 3 R activation. Ca 2+ -induced Ca 2+ release regulated by IP 3 allows IP 3 Rs to regeneratively propagate Ca 2+ signals. The smallest of these regenerative events is a Ca 2+ puff, which arises from the nearly simultaneous opening of a small cluster of IP 3 Rs. Ca 2+ puffs are the basic building blocks for all IP 3 -evoked Ca 2+ signals, but only some IP 3 clusters, namely those parked alongside the ER-plasma membrane junctions where SOCE occurs, are licensed to respond. The location of these licensed IP 3 Rs may allow them to selectively regulate SOCE.