title: In vitro selection and characterization of DNA aptamers specific for phospholamban.
abstract: Calcium transport across the membrane of the sarcoplasmic reticulum (SR) plays an important role in the regulation of heart muscle contraction and relaxation. The sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA) 2a is responsible for Ca(2+) up-take by this organelle and is inhibited in a reversible manner by phospholamban, another SR membrane protein. Thus, alleviation of phospholamban-mediated inhibition of SERCA2a is a potential therapeutic option for heart failure and cardiomyopathy. We have now applied the systematic evolution of ligands by exponential enrichment protocol to a library of single-stranded DNA molecules containing a randomized 40-nucleotide sequence to isolate aptamers that bind phospholamban. One of the obtained aptamers, designated Apt-9, was found to specifically bind to the cytoplasmic region of phospholamban in vitro with high affinity (dissociation constant, approximately 20 nM). Apt-9 increased the Ca(2+)-dependent ATPase activity of cardiac SR vesicles but not that of SR vesicles from skeletal muscle in a concentration-dependent manner. It also shifted the Ca(2+) concentration-response curve for this ATPase activity to the left. These effects of Apt-9 were not mimicked by an oligonucleotide with a scrambled version of the Apt-9 sequence. Thus, our results indicate that Apt-9 activates SERCA2a by alleviating the inhibitory effect of phospholamban on this ATPase, and they suggest that phospholamban-specific aptamers warrant further investigation as potential therapeutic agents for heart failure and cardiomyopathy.
authors.affiliation: Department of Pharmacology, Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi 755-8505, Japan.
authors.forename: Yoshie
authors.initials: Y
authors.lastname: Tanaka
authors.forename: Takeshi
authors.initials: T
authors.lastname: Honda
authors.forename: Kenji
authors.initials: K
authors.lastname: Matsuura
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Kimura
authors.forename: Makoto
authors.initials: M
authors.lastname: Inui
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D001710:Biotin / Q000737:chemistry; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004247:DNA / Q000096:biosynthesis / Q000235:genetics; D006321:Heart / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D008024:Ligands; D018482:Muscle, Skeletal / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D052156:SELEX Aptamer Technique; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000378:metabolism; D013379:Substrate Specificity; D000099211:Phospholamban
chemical_list: D052157:Aptamers, Nucleotide; D001710:Biotin; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D004247:DNA; D008024:Ligands; D011994:Recombinant Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban