Inhibition of SERCA Ca2+ pumps by 2‐aminoethoxydiphenyl borate (2‐APB)

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Abstract
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 onthe SERCA Ca2+ pumps is isoform‐dependent, with SERCA 2B being more sensitive than SERCA 1A (IC50 values for inhibition being 325 and 725 µm, respectively, measured at pH 7.2). The Ca2+‐ATPase is also more potently inhibited at lower pH (IC50 = 70 µm for SERCA1A 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.

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