Inhibition of SERCA Ca 2+ pumps by 2‐aminoethoxydiphenyl borate (2‐APB)

Abstract

2‐Aminoethoxydiphenyl Borate (2‐APB) has been extensively used recently as a membrane permeable modulator of inositol‐1,4,5‐trisphosphate‐sensitive Ca 2+ channels and store‐operated Ca 2+ entry. Here, we report that 2‐APB is also an inhibitor of sarco/endoplasmic reticulum Ca 2+ ‐ATPase (SERCA) Ca 2+ pumps, and additionally increases ion leakage across the phospholipid bilayer. Therefore, we advise caution in the interpretation of results when used in Ca 2+ signalling experiments. The inhibition of 2‐APB onthe SERCA Ca 2+ pumps is isoform‐dependent, with SERCA 2B being more sensitive than SERCA 1A (IC 50 values for inhibition being 325 and 725 µ m , respectively, measured at pH 7.2). The Ca 2+ ‐ATPase is also more potently inhibited at lower pH (IC 50 = 70 µ m for SERCA1A at pH 6). 2‐APB decreases the affinity for Ca 2+ 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 Ca 2+ ‐ATPases show that Tyr837 is critical for the inhibition of activity by 2‐APB. Molecular modeling studies of 2‐APB binding to the Ca 2+ 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 Ca 2+ to their binding sites.