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Modification of Store-operated Channel Coupling and Inositol Trisphosphate Receptor Function by 2-Aminoethoxydiphenyl Borate in DT40 Lymphocytes

2002; Elsevier BV; Volume: 277; Issue: 9 Linguagem: Inglês

10.1074/jbc.m107755200

1083-351X

Hong-Tao Ma, Kartik Venkatachalam, Jan B. Parys, Donald L. Gill,

Ion Transport and Channel Regulation

Store-operated channels (SOCs) provide an important means for mediating longer-term Ca 2+ signals and replenishment of Ca 2+ 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 (InsP 3 R) modifier, 2-aminoethoxydiphenyl borate (2-APB), was a powerful activator of store-operated Ca 2+ entry between 1–10 μm. 2-APB activated authentic SOCs because the entry was totally selective for Ca 2+ (no detectable entry of Ba 2+ or Sr 2+ ions), and highly sensitive to La 3+ ions (IC 50 30–100 nm). To assess the role of InsP 3 Rs in this response, we used the DT40 triple InsP 3 R-knockout (ko) cell line, DT40InsP 3 R-ko, in which the absence of full-length InsP 3 Rs or InsP 3 R fragments was verified by Western analysis using antibodies cross-reacting with N-terminal epitopes of all three chicken InsP 3 R subtypes. The 2-APB-induced activation of SOCs was identical in the DT40InsP 3 R-ko, cells indicating InsP 3 Rs were not involved. With both wild type (wt) and ko DT40 cells, 2-APB had no effect on Ca 2+ 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 Ca 2+ release from stores in intact DT40wt cells but not in DT40InsP 3 R-ko cells, indicating an InsP 3 R-mediated effect. In contrast, 2-APB blocked InsP 3 Rs in permeabilized DT40wt cells, suggesting that the stimulatory action of 2-APB was restricted to functionally coupled InsP 3 Rs 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 InsP 3 R activation but did not alter InsP 3 R activation mediated by phospholipase C-coupled receptor stimulation. The results indicate that the robust bifunctional actions of 2-APB on both SOCs and InsP 3 Rs are dependent on the coupled state of these channels and suggest that 2-APB may target the coupling machinery involved in mediating store-operated Ca 2+ entry.