Interactions between small ankyrin 1 and sarcolipin coordinately regulate activity of the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA1)

Patrick F Desmond,Amanda Labuza,Joaquin Muriel,Michele L Markwardt,Allison E Mancini,Megan A Rizzo,Robert J Bloch

doi:10.1074/jbc.m117.783613

Patrick F Desmond, Amanda Labuza + Show 5 more

Open Access

https://doi.org/10.1074/jbc.m117.783613

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Abstract

SERCA1, the sarco(endo)plasmic reticulum Ca2+-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca2+ levels in the myoplasm. We recently reported that small ankyrin 1 (sAnk1) interacts with the sarco(endo)plasmic reticulum Ca2+-ATPase in skeletal muscle (SERCA1) to inhibit its activity. We also showed that this interaction is mediated at least in part through sAnk1's transmembrane domain in a manner similar to that of sarcolipin (SLN). Earlier studies have shown that SLN and phospholamban, the other well studied small SERCA-regulatory proteins, oligomerize either alone or together. As sAnk1 is coexpressed with SLN in muscle, we sought to determine whether these two proteins interact with one another when coexpressed exogenously in COS7 cells. Coimmunoprecipitation (coIP) and anisotropy-based FRET (AFRET) assays confirmed this interaction. Our results indicated that sAnk1 and SLN can associate in the sarcoplasmic reticulum membrane and after exogenous expression in COS7 cells in vitro but that their association did not require endogenous SERCA2. Significantly, SLN promoted the interaction between sAnk1 and SERCA1 when the three proteins were coexpressed, and both coIP and AFRET experiments suggested the formation of a complex consisting of all three proteins. Ca2+-ATPase assays showed that sAnk1 ablated SLN's inhibition of SERCA1 activity. These results suggest that sAnk1 interacts with SLN both directly and in complex with SERCA1 and reduces SLN's inhibitory effect on SERCA1 activity.

Highlights

SERCA1, the sarco(endo)plasmic reticulum Ca2؉-ATPase of skeletal muscle, is essential for muscle relaxation and maintenance of low resting Ca2؉ levels in the myoplasm
CoIP was used to study the ability of small ankyrin 1 (sAnk1) to interact with SLN in SR vesicles prepared from rabbit skeletal muscle tissue
Discussion sAnk1 and SLN share several features that suggest that they could interact in the membrane of the nSR and regulate SERCA1 activity in unique ways

Summary

Results

Interaction of sAnk and SLN coIP was used to study the ability of sAnk to interact with SLN in SR vesicles prepared from rabbit skeletal muscle tissue. To learn if sAnk and SLN interact directly, and in complex with SERCA, we used coIP of the FLAG and mCherry constructs of SLN and sAnk to determine whether the endogenous SERCA2b expressed in COS7 cells associated with the sAnk11⁄7SLN complex. We first performed coIP experiments with antibodies to SERCA1 in extracts of COS7 cells transfected to express SERCA1 and sAnk1-FLAG or FLAG-SLN, or all three proteins. This result indicates that the SLN-mVen-N1⁄7sAnk1mVen-C complexes remain intact, but their relative molecular orientation becomes shifted after association with CFPSERCA1 This is consistent with the formation of a three-way complex containing sAnk, SLN, and SERCA1. As reported previously [1], sAnk1-FLAG reduced SERCA1’s apparent Ca2ϩ affinity, measured in pCa units, in COS7 and HEK293 cells (⌬KCa2ϩ ϭ Ϫ0.18 and Ϫ0.21, pCa2ϩ units, respectively; Fig. 8 and Tables 1 and 2). These findings suggest that sAnk reduces SLN-mediated SERCA1 inhibition and may ablate it completely

Discussion

ATPase assays

Experimental procedures