Identification of Small Ankyrin 1 as a novel SERCA1 regulatory protein in Skeletal Muscle

Abstract

Small Ankyrin 1 (sAnk1) is a ~20 kDa transmembrane (TM) protein that binds to the cytoskeletal protein, obscurin, and stabilizes the network sarcoplasmic reticulum (nSR) in skeletal muscle. Previous reports from out lab show that sAnk1 knock down results in loss of network SR integrity, along with a decrease in SR Ca2+ load and Ca2+ re-uptake rates. Upon closer examination of the sAnk1 transmembrane domain, we discovered that sAnk1 shares sequence similarity with sarcolipin (SLN), a small protein that inhibits activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA). The goal of the current study was to determine if sAnk1 interacts with SERCA1 or SLN directly in skeletal muscle, and elucidate the consequences such interactions pose on SERCA1 activity. Our results indicate that sAnk1 interacts specifically with SERCA1 in SR 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 (AFRET). Binding was significantly reduced by the replacement of all the TM amino acids of sAnk1 to leucines by mutagenesis. This suggests that, like SLN, sAnk1 interacts with SERCA1 via its TM domain. Assays of ATPase activity show that co-expression of sAnk1 with SERCA1 leads to a reduction of SERCA1's apparent Ca2+ affinity, but that sAnk1's effect is less than that of SLN. Interestingly, the sAnk1 TM mutant has no effect on SERCA1 activity. Our results suggest that sAnk1 interacts with SERCA1 through its TM domain to regulate SERCA1 activity and thereby modulate the sequestration of Ca2+ in the lumen of the ER and SR. Additionally, we determined that sAnk1 can also interact with SLN using the same analytical methods. Unexpectedly, ATPase assays in which all three proteins were co-expressed showed that sAnk1 was able to limit SLN's ability to inhibit SERCA1 activity. Furthermore, coIP and AFRET experiments demonstrate that SLN promotes the interaction between SERCA1 and sAnk1. The identification of sAnk1 as a novel regulator of SERCA1 activity has significant implications for the physiology of muscle and the development of therapeutic approaches to treat heart failure and muscular dystrophies linked to Ca2+ misregulation.