Sarcolipin alters SERCA1a interdomain communication by impairing binding of both calcium and ATP

Abstract

Sarcolipin (SLN), a single-spanning membrane protein, is a regulator of the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA1a). Chemically synthesized SLN, palmitoylated or not (pSLN or SLN), and recombinant wild-type rabbit SERCA1a expressed in S. cerevisiae design experimental conditions that provide a deeper understanding of the functional role of SLN on the regulation of SERCA1a. Our data show that chemically synthesized SLN interacts with recombinant SERCA1a, with calcium-deprived E2 state as well as with calcium-bound E1 state. This interaction hampers the binding of calcium in agreement with published data. Unexpectedly, SLN has also an allosteric effect on SERCA1a transport activity by impairing the binding of ATP. Our results reveal that SLN significantly slows down the E2 to Ca2.E1 transition of SERCA1a while it affects neither phosphorylation nor dephosphorylation. Comparison with chemically synthesized SLN deprived of acylation demonstrates that palmitoylation is not necessary for either inhibition or association with SERCA1a. However, it has a small but statistically significant effect on SERCA1a phosphorylation when various ratios of SLN-SERCA1a or pSLN-SERCA1a are tested.

Sarcoplasmic Reticulum from Horse Gluteal Muscle Is Poised for Enhanced Calcium Transport

“…Although neither of these two biochemical assays provided information on molecular mechanism(s) that produce robust Ca 2+ transport by horse SR, both assays demonstrated the functional and structural similarities of SERCA orthologs from horse gluteus and rabbit muscles. 2+ Transport to ATP Hydrolysis Is Greater for Horse SR Vesicles Than Rabbit SR Vesicles SLN decreases the energetic efficiency of SERCA activity by partially uncoupling Ca 2+ transport from ATP hydrolysis, i.e., by decreasing the Ca 2+ /ATP coupling ratio below the maximum ratio of 2 Ca 2+ ions transported per ATP molecule hydrolyzed [18,[20][21][22][23]26]. Table 1 reports the Ca 2+ transport and ATP hydrolysis activities of horse and rabbit SR vesicles.…”

“…The general consensus is that SLN inhibits SERCA activity via multiple enzymatic mechanisms: by decreasing the maximal velocity (V max ), by decreasing the apparent Ca 2+ binding affinity (1/K Ca ), by decreasing ATP binding affinity (1/K ATP ), and by decreasing the number of Ca 2+ ions transported per ATP molecule hydrolyzed (coupling ratio) below the optimal Ca 2+ /ATP coupling ratio of two [17][18][19][20][21][22][23]. The SLN inhibition of SERCA activity is relieved in part by SLN phosphorylation or de-acylation [24][25][26].…”