Characterization of novel store-operated calcium entry effectors

Abstract

2-Aminoethyl diphenylborinate (2-APB) is a well-known effector of the store-operated Ca(2+) entry of several cell types such as immune cells, platelets and smooth muscle cells. 2-APB has a dual effect: potentiation at 1-5μM and inhibition at >30μM. Unfortunately, it is also able to modify the activity of other Ca(2+) transporters and, thus, cannot be used as a therapeutic tool to control the leukocyte activity in diseases like inflammation. Previously, we have shown that SOCE potentiation by 2-APB depends on the presence of the central boron-oxygen core (BOC) and that the phenyl groups determine the sensitivity of the molecule to inhibit and/or potentiate the SOCE. We hypothesized that by modifying the two phenyl groups of 2-APB, we could identify more efficient and specific analogues. In fact, the addition of methoxyl groups to one phenyl group greatly decreased the potentiation ability without any significant effect on the inhibition. Surprisingly, when the free rotation of the two phenyl groups was blocked by a new hydrocarbon bridge, the BOC was no longer able to potentiate. Furthermore, larger aryl groups than phenyl also impaired the activity of the BOC. Thus, the potentiation site in the Ca(2+) channel is not accessible by the BOC when the lateral groups are too large or unable to freely rotate. However, these molecules are potent inhibitors of store-operated calcium entry with affinities below 1μM, and they can block the activation of the Jurkat T cells. Thus, it is possible to characterize 2-APB analogues with different properties that could be the first step in the discovery of new immunomodulators. This article is part of a special issue entitled "Calcium Signaling in Health and Disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

Potentiation of the store-operated calcium entry (SOCE) induces phytohemagglutinin-activated Jurkat T cell apoptosis

“…In a recently published work [47], we showed that the use of 2-APB analogues can help to understand which part of the molecule is requested for the potentiation or the inhibition capacity. Thus, the two phenyl groups seem to play a key role in the sensitivity to the inhibition process, the ethanolamine group plays a role only in the inhibition, and the central BOC in the potentiation.…”

“…However, when the free rotation of the two phenyl groups is blocked or when they are replaced by larger groups like benzothienyl groups, Ca 2+ influx potentiation is impaired [34]. A drawback was that all the new potentiating molecules we identified were still inhibitory at high concentrations [33].…”

“…Some 2-APB analogues have been developed with higher ability to block the SOCE, but they still inhibit Ca 2+ release [30,33,47].…”

Characterization of neutralizing antibodies reacting with the 213-224 amino-acid segment of human galectin-9

“…[Ca 2+ ] cyt was recorded in Jurkat cells by a fluorimetric ratio technique [ 33 ]. The fluorescent indicator Indo-1 (4 μM; Invitrogen/Molecular Probes) was loaded by incubating the cells at room temperature under gentle agitation.…”

“…Background and autofluorescence were subtracted from the values measured at 405 and 480 nm. Intracellular Ca 2+ concentrations were calculated following the method already described by O. Dellis and collaborators [ 33 ]. Traces were given without SEM for clarity (SEM values were usually < 40 nM).…”