Protein Adsorption on Solid Supported Membranes: Monitoring the Transport Activity of P-Type ATPases

Abstract

P-type ATPases are a large family of membrane transporters that are found in all forms of life. These enzymes couple ATP hydrolysis to the transport of various ions or phospholipids across cellular membranes, thereby generating and maintaining crucial electrochemical potential gradients. P-type ATPases have been studied by a variety of methods that have provided a wealth of information about the structure, function, and regulation of this class of enzymes. Among the many techniques used to investigate P-type ATPases, the electrical method based on solid supported membranes (SSM) was employed to investigate the transport mechanism of various ion pumps. In particular, the SSM method allows the direct measurement of charge movements generated by the ATPase following adsorption of the membrane-bound enzyme on the SSM surface and chemical activation by a substrate concentration jump. This kind of measurement was useful to identify electrogenic partial reactions and localize ion translocation in the reaction cycle of the membrane transporter. In the present review, we discuss how the SSM method has contributed to investigate some key features of the transport mechanism of P-type ATPases, with a special focus on sarcoplasmic reticulum Ca2+-ATPase, mammalian Cu+-ATPases (ATP7A and ATP7B), and phospholipid flippase ATP8A2.

Stimulation of Ca2+‐ATPase Transport Activity by a Small‐Molecule Drug

“…As a general remark, the shape and time frame of the ATP‐induced electrical currents in the absence and presence of CDN1163 were similar to those reported in previous studies of the SERCA transport activity with the SSM method. [ 13 , 25 , 32 ] These observations suggest that the kinetics of Ca 2+ translocation by SERCA was not affected by CDN1163. Therefore, the increase of translocated charge observed in the presence of CDN1163 may be explained by different mechanisms: 1) A tentative explanation is that CDN1163 enhances ATP binding and/or Ca 2+ binding to SERCA.…”

“…[25,32,48] This flow of electrons corresponds to the measured capacitive current, which is correlated with the protein-generated current and is recorded as transient current signal. [25,43,48] Quite often, experiments are carried out under short-circuit conditions, i. e. at zero applied voltage relative to the reference electrode. [48] In ATP concentration jump experiments two buffered solutions were employed, the non-activating and the activating solutions.…”

“…This method has been used to investigate in detail the ion translocation mechanism of the SERCA enzyme. [25] The SSM represents a convenient model system for a bilayer lipid membrane, and consists of a hybrid alkanethiol/phospholipid bilayer supported by a gold electrode (Figure 2 ). [ 26 , 27 ] The hybrid bilayer is characterized by a high mechanical stability so that solutions can be rapidly exchange at the SSM surface.…”

The CopA2-Type P1B-Type ATPase CcoI Serves as Central Hub for cbb3-Type Cytochrome Oxidase Biogenesis

“…Cu transport by CopA-like P 1B -type ATPases has been extensively studied using different methods ( Gonzalez-Guerrero et al, 2010 ; Völlmecke et al, 2012 ; Drees et al, 2015 ; Abeyrathna et al, 2020 ; Tadini-Buoninsegni, 2020 ), but similar information on CcoI-type ATPases is limited. Cu transport by CopA1 and CopA2 of Pseudomonas aeruginosa has been determined by using E. coli expressed proteins and monitoring 64 Cu accumulation in membrane vesicles ( Gonzalez-Guerrero et al, 2010 ).…”

“…To determine the ability of CcoI to transport Cu(I), solid-supported membrane (SSM) electrophysiology was employed ( Schulz et al, 2008 ; Grewer et al, 2013 ; Wacker et al, 2014 ; Rycovska-Blume et al, 2015 ; Bazzone et al, 2017 ; Tadini-Buoninsegni, 2020 ). All measurements were performed on a SURFE 2 R-N1 instrument (Nanion Technologies, Munich, Germany).…”