RESUMO
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.
Assuntos
Trifosfato de Adenosina/metabolismo , Bicamadas Lipídicas/metabolismo , ATPases do Tipo-P/metabolismo , Adenosina Trifosfatases/metabolismo , Adsorção , Animais , Transporte Biológico , Cálcio/metabolismo , ATPases Transportadoras de Cálcio/metabolismo , ATPases Transportadoras de Cobre/metabolismo , Humanos , Hidrólise , Íons , Membranas Artificiais , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Retículo Sarcoplasmático/metabolismoRESUMO
The solid supported membrane (SSM) represents a convenient model system for a biological membrane with the advantage of being mechanically so stable that solutions can be rapidly exchanged at the surface. The SSM consists of a hybrid alkanethiol-phospholipid bilayer supported by a gold electrode. Proteoliposomes, membrane vesicles, or membrane fragments containing the transport protein of interest are adsorbed on the SSM surface and are subjected to a rapid substrate concentration jump. The substrate concentration jump activates the protein and the charge displacement concomitant with its transport activity is recorded as a current transient. Since this technique is well suited for the functional characterization of electrogenic membrane transporters, it is expected to become a promising platform technology for drug screening and development.