Sarcolipin inhibits polymerization of phospholamban to induce superinhibition of sarco(endo)plasmic reticulum Ca2+-ATPases (SERCAs)

Abstract

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca2+-ATPase of fast-twitch skeletal muscle (SERCA1a), is also expressed in cardiac and slow-twitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. Co-expression in HEK-293 cells of SLN tagged N-terminally with a FLAG epitope (NF-SLN), PLN, and SERCAs followed by measurement of the Ca2+ dependence of Ca2+ transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca2+ affinity of both SERCA1a (ΔKCa -0.22 ± 0.01 pCa units) and SERCA2a (ΔKCa = -0.37 ± 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing ΔKCa by about - 1.0 pCa units. Co-immunoprecipitation showed that NF-SLN increased the binding of PLN to SERCA, whereas PLN did not increase the binding of NF-SLN to SERCA. Elevated Ca2+ dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca2+ dissociation of the PLN-SERCA complex. Co-immunoprecipitation of PLN and NF-SLN without SERCA showed that NF-SLN binds directly to PLN and that NF-SLN inhibits the formation of PLN pentamers. Thus the ability of NF-SLN to elevate the content of PLN monomers can account, at least in part, for the superinhibitory effects of NF-SLN in the presence of PLN.