Abstract
The effect of cholesterol was investigated on the OCTN1 transport activity measured as [14C]-tetraethylamonium or [3H]-acetylcholine uptake in proteoliposomes reconstituted with native transporter extracted from HeLa cells or the human recombinant OCTN1 over-expressed in E. coli. Removal of cholesterol from the native transporter by MβCD before reconstitution led to impairment of transport activity. A similar activity impairment was observed after treatment of proteoliposomes harboring the recombinant (cholesterol-free) protein by MβCD, suggesting that the lipid mixture used for reconstitution contained some cholesterol. An enzymatic assay revealed the presence of 10 µg cholesterol/mg total lipids corresponding to 1% cholesterol in the phospholipid mixture used for the proteoliposome preparation. On the other way around, the activity of the recombinant OCTN1 was stimulated by adding the cholesterol analogue, CHS to the proteoliposome preparation. Optimal transport activity was detected in the presence of 83 µg CHS/ mg total lipids for both [14C]-tetraethylamonium or [3H]-acetylcholine uptake. Kinetic analysis of transport demonstrated that the stimulation of transport activity by CHS consisted in an increase of the Vmax of transport with no changes of the Km. Altogether, the data suggests a direct interaction of cholesterol with the protein. A further support to this interpretation was given by a docking analysis indicating the interaction of cholesterol with some protein sites corresponding to CARC-CRAC motifs. The observed direct interaction of cholesterol with OCTN1 points to a possible direct influence of cholesterol on tumor cells or on acetylcholine transport in neuronal and non-neuronal cells via OCTN1.
Highlights
OCTN1 belongs to a small sub-family of organic cation transporters that includes only three members, OCTN1, 2 and 3 [1,2]
To further investigate the function of this transporter on the single protein, the human OCTN1 was over-expressed in E. coli and its transport activity was assayed in proteoliposomes harboring the recombinant transporter
To evaluate the possible effect of cholesterol on the native human OCTN1, the protein extracted from HeLa cells was exploited
Summary
OCTN1 belongs to a small sub-family of organic cation transporters that includes only three members, OCTN1, 2 and 3 [1,2]. From OCTN2, which is a well-acknowledged carnitine transporter, human OCTN1 presents still uncertainties concerning the substrate specificity and, the physiological role It is expressed in several districts [6] including peritoneum, where the transporter should be involved in control of inflammatory processes that are critical in this district [7]. Transport function of OCTN1 was firstly characterized in intact cells transfected with OCTN1 cDNA [1,8,9,10], in OCTN1 cRNA injected Xenopus oocytes [9] or in membrane vesicles obtained by OCTN1 expressing cells [11] It was defined as an organic cation transporter on the basis of specificity towards the prototype cation TEA, with a low affinity for carnitine. We have used two approaches, one based on cholesterol sequestration from the native membranes the other based on cholesterol addition to the artificial phospholipid bilayer of proteoliposomes
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
