Modification of store-operated channel coupling and inositol trisphosphate receptor function by 2-aminoethoxydiphenyl borate in DT40 lymphocytes

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca²⁺ signals and replenishment of Ca²⁺ stores in a multitude of cell types. However, the coupling mechanism between endoplasmic reticulum stores to activate plasma membrane SOCs remains unknown. In DT40 chicken B lymphocytes, the permeant inositol trisphosphate receptor (insP₃R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca²⁺ entry between 1-10 μM. 2-APB activated authentic SOCs because the entry was totally selective for Ca²⁺ (no detectable entry of Ba²⁺ or Sr²⁺ ions), and highly sensitive to La³⁺ ions (IC₅₀ 30-100 nM). To assess the role of InsP₃Rs in this response, we used the DT40 triple InsP₃R-knockout (ko) cell line, DT40InsP₃R-ko, in which the absence of full-length InsP₃Rs or InsP₃R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP₃R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP₃R-ko, cells indicating InsP₃Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca²⁺ entry in store-replete cells, indicating that its action was restricted to SOCs in a store-coupled state. 2-APB induced a robust activation of Ca²⁺ release from stores in intact DT40wt cells but not in DT40InsP₃R-ko cells, indicating an InsP₃R-mediated effect. In contrast, 2-APB blocked InsP₃Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP₃Rs in intact cells. Uncoupling of ER/PM interactions in intact cells by calyculin A-induced cytoskeletal rearrangement prevented SOC activation by store-emptying and 2-APB; this treatment completely prevented 2-APB-induced InsP₃R activation but did not alter InsP₃R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP₃Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca²⁺ entry.