ABSTRACT
Phospholamban (PLN) is a small integral membrane protein, which modulates the activity of the Sarcoplasmic Reticulum Ca(2+)-ATPase (SERCA) of cardiac myocytes. PLN, as a monomer, can directly interact and tune SERCA activity, but the physiological function of the pentameric form is not yet fully understood and still debated. In this work, we reconstituted PLN in Giant Unilamellar Vesicles (GUVs), a simple and reliable experimental model system to monitor the activity of proteins in membranes. By Laser Scanning Confocal Microscopy (LSCM) and Fluorescence Correlation Spectroscopy (FCS) we verified a spontaneous reconstitution of PLN into the phospholipid bilayer. In parallel experiments, we measured with the patch clamp technique canonical ion channel fluctuations, which highlight a preference for Cs(+) over K(+) and do not conduct Ca(2+). The results prove that PLN forms, presumably in its pentameric form, a cation selective ion channel.
Subject(s)
Calcium-Binding Proteins/chemistry , Ion Channels/chemistry , CationsABSTRACT
Current and voltage measurements were performed on Na,K-ATPase and sarcoplasmic reticulum (SR) Ca-ATPase. Measurements of current transients under short-circuit conditions and of voltage transients under open-circuit conditions were carried out by employing a solid supported membrane (SSM). Purified membrane fragments containing Na,K-ATPase or native SR vesicles were adsorbed on a SSM and were activated by performing substrate concentration jumps. Current and voltage transients were recorded in the external circuit. They are related to pump activity and can be attributed to electrogenic events in the reaction cycles of the two enzymes. While current transients of very small amplitude are difficult to detect, the corresponding voltage transients can be measured with higher accuracy because of a much more favorable signal-to-noise ratio. Therefore, voltage measurements are preferable for the investigation of slow processes generating low current signals, e.g., for the analysis of low turnover transporters.
Subject(s)
Electric Conductivity , Intracellular Membranes/enzymology , Membranes, Artificial , Sarcoplasmic Reticulum Calcium-Transporting ATPases/metabolism , Sarcoplasmic Reticulum/enzymology , Sodium-Potassium-Exchanging ATPase/metabolism , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Adsorption , Animals , RabbitsABSTRACT
A novel experimental technique used to investigate chlorophyll films on a hanging mercury drop electrode is described. Two different procedures are employed to prepare self-assembled chlorophyll monolayers and multilayers on the mercury electrode. Upon illuminating the chlorophyll a (Chl)-coated mercury electrode with an appropriate light source, the photocurrents generated by the Chl aggregates are measured under short-circuit conditions in the absence of photoartefacts. The preliminary results obtained by this novel technique are presented.
