Sarcolipin Inhibits Polymerization of Phospholamban to Induce Superinhibition of Sarco(endo)plasmic Reticulum Ca2+-ATPases (SERCAs)

Michio Asahi,Kazimierz Kurzydlowski,Michihiko Tada,David H Maclennan

doi:10.1074/jbc.c200269200

Michio Asahi, Kazimierz Kurzydlowski + Show 2 more

Open Access

https://doi.org/10.1074/jbc.c200269200

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Abstract

Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca(2+)-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 Ca(2+) dependence of Ca(2+) transport activity in isolated microsomal fractions showed that NF-SLN can reduce the apparent Ca(2+) affinity of both SERCA1a (DeltaK(Ca) = -0.22 +/- 0.01 pCa units) and SERCA2a (DeltaK(Ca) = -0.37 +/- 0.04 pCa units). When SERCA1a or SERCA2a were co-expressed with both NF-SLN and PLN, inhibition was synergistic, reducing DeltaK(Ca) 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 Ca(2+) dissociates both PLN and NF-SLN from their complexes with both SERCA1a and SERCA2a, but NF-SLN induced resistance to Ca(2+) 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.

Highlights

From the ‡Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada and the §Department of Medicine and Pathophysiology, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan
Regulatory Effects of NF-SLN, PLN, or a Combination of NF-SLN plus PLN on SERCA Function— The effect of NF-SLN on the Ca2ϩ affinity of SERCA1a is illustrated in Fig. 1A where the Ca2ϩ dependence of Ca2ϩ transport is diagrammed for SERCA1a alone and for SERCA1a co-expressed with either NF-SLN or PLN or both
Since PLN and SLN are co-expressed in the heart, albeit at different ratios in different species [15, 18], it was of interest to determine whether they interact as regulators of SERCA2a activity

Summary

Accelerated Publication

Sarcolipin Inhibits Polymerization of Phospholamban to Induce Superinhibition of Sarco(endo)plasmic Reticulum Ca2؉-ATPases (SERCAs)*. Sarcolipin (SLN), a regulator of the sarco(endo)plasmic reticulum Ca2؉-ATPase of fast-twitch skeletal muscle (SERCA1a), is expressed in cardiac and slowtwitch skeletal muscles where phospholamban (PLN) and SERCA2a are expressed. PLN is a 52-amino acid transmembrane protein that interacts with SERCA molecules to lower their apparent affinity for Ca2ϩ and inhibit their activity at low, but not at high, Ca2ϩ concentrations [3]. PLN is not expressed in fast-twitch skeletal muscle, both PLN and SLN are expressed in cardiac and slow-twitch skeletal muscle of humans where SERCA2a is highly expressed [15]. Since SERCA2a and SLN are co-expressed in cardiac muscle, it was of interest to determine whether SLN might interact with and regulate the activity of SERCA2a. We deduce that an increase in the PLN monomer content can lead to superinhibition of SERCA2a activity through a mechanism in which mass action plays a significant but perhaps not the only role

EXPERIMENTAL PROCEDURES

Depolymerization of PLN by SLN

RESULTS

DISCUSSION