Binding of a Monoclonal Antibody to the Phospholamban Cytoplasmic Domain Interferes with the Channel Activity of Phospholamban Reconstituted in a Tethered Bilayer Lipid Membrane Serena Smeazzetto, † Alessio Sacconi, † Adrian L. Schwan, ‡ Giancarlo Margheri, § and Francesco Tadini-Buoninsegni* ,† † Department of Chemistry “Ugo Schiff”, University of Florence, 50019 Sesto Fiorentino, Italy ‡ Department of Chemistry, University of Guelph, Guelph, ON Canada, N1G 2W1 § Institute for Complex Systems, National Research Council, 50019 Sesto Fiorentino, Italy * S Supporting Information ABSTRACT: Phospholamban (PLN), a membrane protein present in the sarcoplasmic reticulum of cardiac myocytes, is a crucial regulator of cardiac function. It is known that PLN appears as a monomer and as a pentamer. However, the role of the PLN pentamer and its ability to generate an ion channel are a matter of debate. To address this issue we employed an experimental approach that combines electrochemical impe- dance spectroscopy and surface plasmon resonance measure- ments. In particular, we investigated the channel activity of wild- type PLN reconstituted in a tethered bilayer lipid membrane (tBLM) on a gold surface. Our results indicate that reconstituted PLN can generate ion-conducting channels in a tBLM. Experiments with a PLN monoclonal antibody support an oriented incorporation of PLN in the tBLM. We show that the binding of the antibody to the PLN cytoplasmic domain interferes with PLN channel activity. ■ INTRODUCTION Phospholamban (PLN) is a 52 amino acid integral membrane protein present in the sarcoplasmic reticulum (SR) of cardiac myocytes. 1 PLN is involved in the contractility of cardiac muscle by regulating SR Ca-ATPase (SERCA). The activity of SERCA is inhibited by unphosphorylated PLN whereas PLN phosphorylation releases SERCA inhibition and allows Ca 2+ pumping. 2 PLN is composed of a helical cytoplasmic domain at the N-terminus, a semiflexible loop, and a transmembrane domain at the C-terminus which consists of a single-span highly hydrophobic α-helix. 1 PLN appears as a monomer (6 kDa) and as a pentamer (30 kDa), and the two forms are in equilibrium. 2 In particular, the role of the PLN pentamer is still a matter of active debate. 3-7 Some structural data support 8,9 and others reject 10 the hypothesis that the PLN pentamer can form an ion channel. It was shown that PLN, when incorporated into planar lipid bilayers, 4,11,12 exhibits ion channel activity. The recon- stituted PLN was found to have moderate selectivity between monovalent cations and no appreciable Ca 2+ permeability. 12 Moreover, experiments based on the polymeric nonelectrolytes method, which allows us to estimate the pore size, 13-15 indicate that the radii for the narrowest part and the wider part of the PLN channel are 2.2 and 6.2 Å, respectively. 4 These values are in agreement with the radii found in the NMR structure and by the molecular modeling of pentameric PLN. 8,9,16,17 A recent study on the incorporation of phosphorylated PLN in mercury- supported biomimetic membranes shows that phosphorylated PLN does not permeabilize lipid bilayers toward ions at physiological pH but exerts a permeabilizing action toward monovalent cations following a small decrease in pH. 18 In this work we employed surface plasmon resonance (SPR) and electrochemical impedance spectroscopy (EIS) to characterize wild-type PLN reconstituted in a tethered bilayer lipid membrane (tBLM). The tBLM is an experimental model of a biological membrane that is used to study channel-forming peptides and proteins 14,19-24 because it combines high stability with excellent biomembrane-mimicking features. 19 The tBLM consists of a lipid bilayer tethered to a gold surface via a hydrophilic spacer, which is terminated with a thiol or disulfide group that forms a covalent bond with the gold surface. 25 By combining optical and electrochemical methods we were able to investigate the channel activity and conductive properties of PLN reconstituted in a tBLM. ■ EXPERIMENTAL SECTION PLN Expression and Purification. The gene encoding human wild-type PLN was expressed as being fused with the maltose-binding protein. The fusion protein was first isolated and then cleaved overnight at room temperature with a TEV protease. 26 Subsequently, Received: April 29, 2014 Revised: July 9, 2014 Article pubs.acs.org/Langmuir © XXXX American Chemical Society A dx.doi.org/10.1021/la501660u | Langmuir XXXX, XXX, XXX-XXX