Enhanced Adsorption of Ca-ATPase Containing Vesicles on a Negatively Charged Solid-Supported-Membrane for the Investigation of Membrane Transporters Alessio Sacconi, † Maria Rosa Moncelli, † Giancarlo Margheri, ‡ and Francesco Tadini-Buoninsegni* ,† † Department of Chemistry “Ugo Schiff”, University of Florence, via della Lastruccia 3, 50019 Sesto Fiorentino, Italy ‡ Institute for Complex Systems, National Research Council, Via Madonna del Piano 10, 50019 Sesto Fiorentino, Italy * S Supporting Information ABSTRACT: A convenient model system for a biological membrane is a solid-supported membrane (SSM), which consists of a gold-supported alkanethiol|phospholipid bilayer. In combination with a concentration jump method, SSMs have been used for the investigation of several membrane transporters. Vesicles incorporating sarcoplasmic reticulum Ca-ATPase (SERCA) were adsorbed on a negatively charged SSM (octadecanethiol|phosphatidylserine bilayer). The current signal generated by the adsorbed vesicles following an ATP concentration jump was compared to that produced by SERCA-containing vesicles adsorbed on a conventional SSM (octadecanethiol|phosphatidylcholine bilayer). A significantly higher current amplitude was recorded on the serine-based SSM. The adsorption of SERCA-incorporating vesicles on the SSM was then characterized by surface plasmon resonance (SPR). The SPR measurements clearly indicate that in the presence of Ca 2+ and Mg 2+ , the amount of adsorbed vesicles on the serine-based SSM is about twice that obtained using the conventional SSM, thereby demonstrating that the higher current amplitude recorded on the negatively charged SSM is correlated with a greater quantity of adsorbed vesicles. The enhanced adsorption of membrane vesicles on the PS-based SSM may be useful to study membrane preparations with a low concentration of transport protein generating small current signals, as in the case of various recombinantly expressed proteins. ■ INTRODUCTION A convenient model system for a biological membrane is a solid-supported membrane (SSM), which has been employed for the functional characterization of several membrane transport proteins. 1,2 The SSM consists of a hybrid alkanethiol|phospholipid bilayer supported by a gold electrode. In particular, the SSM is formed by an octadecanethiol (Oct) monolayer covalently bound to the gold surface via the sulphydryl group and a second phosphatidylcholine monolayer on top of the thiol layer. In combination with a concentration jump method, 3 SSMs have successfully been used to study electrogenic transport by ion pumps, 3-6 secondary active transporters, 7,8 and ion channels. 9-12 In the concentration jump method, membrane preparations (membrane fragments or vesicles) containing the protein of interest are adsorbed on a SSM, which is usually formed by a diphytanoyl phosphatidylcholine (PC) monolayer on top of the gold-supported Oct film. 1-3 However, it has not been demonstrated whether this is definitely the SSM sensor that adsorbs the higher amount of membrane vesicles. Aiming to fabricate a SSM with higher affinity to vesicles with incorporated membrane transporters, a possible strategy would be that of using a lipid surface of different composition and charge (i.e., different lipid head groups). It is known that negatively charged lipid headgroups can strongly interact with bivalent cations 13-17 (e.g., Ca 2+ and Mg 2+ ), which were reported to promote the adsorption process of small unilamellar phospholipid vesicles on a gold-supported organo- phosphate monolayer. 18 However, the adsorption of protein- containing membrane vesicles on a negatively charged SSM has not yet been investigated. For this purpose, in this work we characterized a new SSM based on diphytanoyl phosphatidyl- serine (PS), which has the same hydrophobic moiety as that of PC, that is the model molecule to be compared, but a final negatively charged serine headgroup. 19 By employing the concentration jump method, we compared the current signal produced by vesicles containing sarcoplasmic reticulum Ca- ATPase (SERCA) adsorbed on our new SSM (Oct|PS bilayer) with that obtained with SERCA-incorporating vesicles adsorbed on the conventional SSM (Oct|PC bilayer). A SERCA- generated current transient with much higher amplitude was recorded on the PS-based SSM. To explain this result, the Received: September 5, 2013 Revised: October 15, 2013 Published: October 16, 2013 Article pubs.acs.org/Langmuir © 2013 American Chemical Society 13883 dx.doi.org/10.1021/la4034386 | Langmuir 2013, 29, 13883-13889