title: 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.
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 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 micro m, respectively, measured at pH 7.2). The Ca2+-ATPase is also more potently inhibited at lower pH (IC50 = 70 micro m 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.
authors.affiliation: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, UK.
authors.forename: Jonathan G
authors.initials: JG
authors.lastname: Bilmen
authors.forename: Laura L
authors.initials: LL
authors.lastname: Wootton
authors.forename: Rita E
authors.initials: RE
authors.lastname: Godfrey
authors.forename: Oliver S
authors.initials: OS
authors.lastname: Smart
authors.forename: Francesco
authors.initials: F
authors.lastname: Michelangeli
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001896:Boron Compounds / Q000378:metabolism / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005453:Fluorescence; D007700:Kinetics; D009154:Mutation; D010766:Phosphorylation; D011487:Protein Conformation; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D014364:Tryptophan / Q000737:chemistry
chemical_list: D001896:Boron Compounds; D004791:Enzyme Inhibitors; D014364:Tryptophan; D000255:Adenosine Triphosphate; C109986:2-aminoethoxydiphenyl borate; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D002118:Calcium