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Functional characterisation of P-type ATPases using solid-supported membrane based electrophysiology

F. Tadini-Buoninsegni, Department of Chemistry “Ugo Schiff”, University of Florence, Via della Lastruccia 3, 50019 Sesto Fiorentino, Italy.

P-type ATPases are a large, ubiquitous and varied family of membrane proteins that are involved in many transport processes in virtually all living organisms (Bublitz et al., 2011). These membrane proteins couple the energy provided by ATP hydrolysis to the active transport of various ions across biological membranes. A specific feature of P-type ATPases is the formation of a phosphorylated intermediate state during their enzymatic cycle.

Solid supported membranes (SSM) have been employed for the functional characterisation of P-type ATPases, e.g. sarcoplasmic reticulum (SR) Ca2+-ATPase and Na+,K+-ATPase (Tadini-Buoninsegni et al., 2008). The SSM, consisting of a hybrid alkanethiol/phospholipid bilayer supported by a gold electrode, is a convenient model system for a biological membrane. An advantageous feature of the SSM is its high mechanical stability which allows fast solution exchange at the membrane surface (Schulz et al., 2008). Proteoliposomes or native membranes (vesicles or fragments) incorporating the ATPase are adsorbed on the SSM surface and are subjected to a rapid substrate concentration jump. The substrate concentration jump activates the ATPase and the charge displacement concomitant with the transport activity of the enzyme is recorded as a current transient via capacitive coupling (Tadini-Buoninsegni & Fendler, 2015).

Charge transfer in P-type ATPases was investigated by SSM-based electrophysiology in order to gain insights into the ion transport mechanism. In the case of SR Ca2+-ATPase, the SSM technique provided useful information for a detailed characterisation of the enzyme’s transport cycle, especially as concerns Ca2+ binding and Ca2+/H+ exchange (Tadini-Buoninsegni et al., 2006). More recently, SSM measurements were performed on bacterial and human Cu+-ATPases (Tadini-Buoninsegni et al., 2010; Mattle et al., 2015) to demonstrate electrogenic Cu+ ion displacement across the ATPase protein. While the SSM-based technique is well-suited for the investigation of fundamental questions concerning the transport mechanism of membrane transporters, it is also a promising platform technology for drug screening and development.

Bublitz M, Morth JP & Nissen P (2011). J Cell Sci 124, 2515-2519.

Mattle D, Zhang L, Sitsel O, Pedersen LT, Moncelli MR, Tadini-Buoninsegni F, Gourdon P, Rees DC, Nissen P & Meloni G (2015). EMBO Rep 16, 728-740.

Schulz P, Garcia-Celma JJ & Fendler K (2008). Methods 46, 97-103.

Tadini-Buoninsegni F, Bartolommei G, Moncelli MR, Guidelli R & Inesi G (2006). J Biol Chem 281, 37720-37727.

Tadini-Buoninsegni F, Bartolommei G, Moncelli MR & Fendler K (2008). Arch Biochem Biophys 476, 75-86.

Tadini-Buoninsegni F, Bartolommei G, Moncelli MR, Pilankatta R, Lewis D & Inesi G (2010). FEBS Lett 584, 4619-4622.

Tadini-Buoninsegni F & Fendler K (2015). In Pumps, Channels and Transporters: Methods of Functional Analysis, 1st edn, ed. Clarke RJ & Khalid MAA, pp. 147-177. John Wiley & Sons Inc., Hoboken, New Jersey, USA.

Supported by Ente Cassa di Risparmio di Firenze and Italian Ministry of Education, University and Research.