TAILIEUCHUNG - Báo cáo Y học: Inhibition of SERCA Ca2+ pumps by 2-aminoethoxydiphenyl borate (2-APB) 2-APB reduces both Ca2+ binding and phosphoryl transfer from ATP, by interfering with the pathway leading to the Ca2+-binding sites

2-Aminoethoxydiphenyl Borate (2-APB) has been extensively used recently as a membrane permeable modulator of inositol-1,4,5-trisphosphate-sensitive Ca2+ channels and store-operated Ca2+ entry. Here, we report that 2-APB is also an inhibitor of sarco/endoplasmic reticulum Ca2+ATPase (SERCA) Ca2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca2+ signalling experiments. The inhibition of 2-APB on the SERCA Ca2+ pumps is isoform-dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 lM, respectively, measured at pH ) | Eur. J. Biochem. 269 3678-3687 2002 FEBS 2002 doi Inhibition of SERCA Ca2 pumps by 2-aminoethoxydiphenyl borate 2-APB 2-APB reduces both Ca2 binding and phosphoryl transfer from ATP by interfering with the pathway leading to the Ca2 -binding sites Jonathan G. Bilmen Laura L. Wootton Rita E. Godfrey Oliver S. Smart and Francesco Michelangeli School of Biosciences University of Birmingham Edgbaston Birmingham UK 2-Aminoethoxydiphenyl Borate 2-APB has been extensively used recently as a membrane permeable modulator of inositol-1 4 5-trisphosphate-sensitive Ca2 channels and store-operated Ca2 entry. Here we report that 2-APB is also an inhibitor of sarco endoplasmic reticulum Ca2 -ATPase SERCA Ca2 pumps and additionally increases ion leakage across the phospholipid bilayer. Therefore we advise caution in the interpretation of results when used in Ca2 signalling experiments. The inhibition of 2-APB on the SERCA Ca2 pumps is isoform-dependent with SERCA 2B being more sensitive than SERCA 1A IC50 values for inhibition being 325 and 725 M respectively measured at pH . The Ca2 -ATPase is also more potently inhibited at lower pH IC50 70 M for SERCA 1A at pH 6 . 2-APB decreases the affinity for Ca2 binding to the ATPase by more than 20-fold and also inhibits phosphoryl transfer from ATP by 35 without inhibiting nucleotide binding. Activity studies performed using mutant Ca2 -ATPases show that Tyr837 is critical for the inhibition of activity by 2-APB. Molecular modeling studies of 2-APB binding to the Ca2 ATPase identified two potential binding sites close to this residue near or between transmembrane helices M3 M4 M5 and M7. The binding of 2-APB to these sites could influence the movement of the loop between M6 and M7 L6-7 and reduce access of Ca2 to their binding sites. Keywords 2-APB Ca2 -ATPase Inhibition SERCA. Ca2 plays a very important role in a number of signalling pathways both within and between cells. The modulation of its .

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