Abstract
Phospholamban (PLN) is a small integral membrane protein, which binds and inhibits in a yet unknown fashion the Ca2+-ATPase (SERCA) in the sarcoplasmic reticulum. When reconstituted in planar lipid bilayers PLN exhibits ion channel activity with a low unitary conductance. From the effect of non-electrolyte polymers on this unitary conductance we estimate a narrow pore with a diameter of ca. 2.2 Å for this channel. This value is similar to that reported for the central pore in the structure of the PLN pentamer. Hence the PLN pentamer, which is in equilibrium with the monomer, is the most likely channel forming structure. Reconstituted PLN mutants, which either stabilize (K27A and R9C) or destabilize (I47A) the PLN pentamer and also phosphorylated PLN still generate the same unitary conductance of the wt/non-phosphorylated PLN. However the open probability of the phosphorylated PLN and of the R9C mutant is significantly lower than that of the respective wt/non-phosphorylated control. In the context of data on PLN/SERCA interaction and on Ca2+ accumulation in the sarcoplasmic reticulum the present results are consistent with the view that PLN channel activity could participate in the balancing of charge during Ca2+ uptake. A reduced total conductance of the K+ transporting PLN by phosphorylation or by the R9C mutation may stimulate Ca2+ uptake in the same way as an inhibition of K+ channels in the SR membrane. The R9C-PLN mutation, a putative cause of dilated cardiomyopathy, might hence affect SERCA activity also via its inherent low open probability.
Highlights
The contraction and relaxation of the heart is controlled by a periodic increase and decrease of the Ca2+ concentration in the cytosol [1]
SERCA is regulated by phospholamban (PLN), an integral membrane protein of only 52 amino acids (AA), which is composed of three domains: domain IA, a helical cytoplasmic domain (AAs 1–16), domain IB, a semi-flexible loop (AAs 17–21) and domain II, a helical hydrophobic transmembrane domain (AAs 22–52) [3,4]
For this purpose we compared single channel recordings of phosphorylated and non-phosphorylated PLN. We found that both forms of the protein exhibit the same prevailing unitary conductance of 18 pS; the I/V relations are quasi identical (Figure 3A)
Summary
The contraction and relaxation of the heart is controlled by a periodic increase and decrease of the Ca2+ concentration in the cytosol [1]. During relaxation of the cardiac myocytes the sarcoendoplasmic CaATPase (SERCA) maintains a low cytosolic calcium concentration by pumping Ca2+ into the lumen of the sarcoplasmic reticulum (SR) in exchange with lumenal H+ [2]. PLN occurs as a monomer (6 KDa) and as a pentamer (30 KDa) and both oligomeric forms are in an equilibrium [5]. While it is clear that monomeric PLN is sufficient for SERCA inhibition, the functional relevance of the oligomeric states is not yet fully understood. Structural details of the PLN pentamer and in particular the question whether it forms a central ion conducting pore, are not yet clarified. A so called ‘‘bellflower’’ [9] and a ‘‘pinwheel’’ structure [4] of the pentamer are discussed as structural models in the literature [4,9,10,11]; some structural data support [9,10,11] and others reject [4] the idea that a central pore in the pentamer can serve as pore for ionic currents
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