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 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 microm. 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.
Ma
Hong-Tao
HT
Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 108 North Greene Street, Baltimore, MD 21201, USA.
Venkatachalam
Kartik
K
Parys
Jan B
JB
Gill
Donald L
DL
D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism; D015153:Blotting, Western; D001896:Boron Compounds / Q000378:metabolism*; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002645:Chickens; D006224:Cricetinae; D003599:Cytoskeleton / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015544:Inositol 1,4,5-Trisphosphate / Q000378:metabolism*; D008387:Marine Toxins; D009130:Muscle, Smooth / Q000166:cytology; D010080:Oxazoles / Q000494:pharmacology; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors; D010738:Type C Phospholipases / Q000378:metabolism
D001896:Boron Compounds; D004791:Enzyme Inhibitors; D008387:Marine Toxins; D010080:Oxazoles; C059041:calyculin A; D015544:Inositol 1,4,5-Trisphosphate; C109986:2-aminoethoxydiphenyl borate; D010738:Type C Phospholipases; D002118:Calcium