title: Identification of Small Ankyrin 1 as a Novel Sarco(endo)plasmic Reticulum Ca2+-ATPase 1 (SERCA1) Regulatory Protein in Skeletal Muscle.
abstract: Small ankyrin 1 (sAnk1) is a 17-kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum in skeletal muscle. We report that sAnk1 shares homology in its TM amino acid sequence with sarcolipin, a small protein inhibitor of the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Here we investigate whether sAnk1 and SERCA1 interact. Our results indicate that sAnk1 interacts specifically with SERCA1 in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle, and in COS7 cells transfected to express these proteins. This interaction was demonstrated by co-immunoprecipitation and an anisotropy-based FRET method. Binding was reduced ~2-fold by the replacement of all of the TM amino acids of sAnk1 with leucines by mutagenesis. This suggests that, like sarcolipin, sAnk1 interacts with SERCA1 at least in part via its TM domain. Binding of the cytoplasmic domain of sAnk1 to SERCA1 was also detected in vitro. ATPase activity assays show that co-expression of sAnk1 with SERCA1 leads to a reduction of the apparent Ca(2+) affinity of SERCA1 but that the effect of sAnk1 is less than that of sarcolipin. The sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM and cytoplasmic domains to regulate SERCA1 activity and modulate sequestration of Ca(2+) in the sarcoplasmic reticulum lumen. The identification of sAnk1 as a novel regulator of SERCA1 has significant implications for muscle physiology and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca(2+) misregulation.
authors.affiliation: From the Department of Physiology and Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21230.
authors.forename: Patrick F
authors.initials: PF
authors.lastname: Desmond
authors.affiliation: From the Department of Physiology and.
authors.forename: Joaquin
authors.initials: J
authors.lastname: Muriel
authors.affiliation: From the Department of Physiology and.
authors.forename: Michele L
authors.initials: ML
authors.lastname: Markwardt
authors.affiliation: From the Department of Physiology and.
authors.forename: Megan A
authors.initials: MA
authors.lastname: Rizzo
authors.affiliation: From the Department of Physiology and Program in Biochemistry and Molecular Biology, University of Maryland, Baltimore, Maryland 21230 rbloch@umaryland.edu.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Bloch
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017487:Ankyrins / Q000737:chemistry* / Q000235:genetics; D019556:COS Cells; D002522:Chlorocebus aethiops; D047468:Immunoprecipitation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry; D018482:Muscle, Skeletal / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics
chemical_list: D017487:Ankyrins; D009124:Muscle Proteins; D011510:Proteolipids; C077258:sarcolipin; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases
keywords: SERCA; calcium ATPase; calcium regulation; membrane protein; sarcoplasmic reticulum (SR); skeletal muscle; small ankyrin 1; transmembrane domain