Preparation of giant myelin vesicles and proteoliposomes to register ionic channels.
Myelin vesicles, reconstituted liposomes with proteolipid protein (PLP), the main protein component of myelin, and electrophysiological patch-clamp are potentially powerful tools to study the role of myelin in functional ionic channels. However, technical difficulties in the vesiculation of myelin and the small size of the vesicles obtained do not permit the application of micropipettes for current recordings. From a suspension of purified myelin we have prepared oligolamellar vesicles (mean diameter of 144 nm) using the so-called French pressure system. From this preparation we obtained giant myelin vesicles approximately 10 microns in mean diameter, using a dehydration-rehydration procedure. Qualitative analysis of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed no significant loss of any component in these vesicles due to pressure, in comparison with non-vesiculated myelin. A way of preparing giant liposomes of approximately 80-100 microns and proteoliposomes of approximately 30 microns in mean diameter, using the same dehydration-rehydration procedure, is also reported. Reconstitution of purified PLP in giant liposomes was confirmed by fluorescent labeling of PLP and by fluorescence microscopy. The current recordings from these vesicles prove the validity of these methods and provide significant evidence of the existence of ionic channels in myelin membranes and the possibility that PLP functions as a channel. The physiological significance and characterization of these channels remain yet unresolved. These results have a special significance for elucidating the molecular role of myelin in the regulation of neural activity and in the brain ion microenvironment.
Regueiro
P
P
Neurochemistry Laboratory, Instituto Santiago Ramón y Cajal, C.S.I.C., Madrid, Spain.
Monreal
J
J
Díaz
R S
RS
Sierra
F
F
D000818:Animals; D001059:Apoproteins / Q000302:isolation & purification / Q000378:metabolism; D001921:Brain / Q000502:physiology*; D002417:Cattle; D005614:Freeze Fracturing; D007473:Ion Channels / Q000502:physiology; D007700:Kinetics; D008081:Liposomes; D008564:Membrane Potentials; D008854:Microscopy, Electron; D018991:Myelin Proteolipid Protein / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D009186:Myelin Sheath / Q000502:physiology* / Q000648:ultrastructure; D018408:Patch-Clamp Techniques; D011510:Proteolipids / Q000378:metabolism* / Q000648:ultrastructure
D001059:Apoproteins; D007473:Ion Channels; D008081:Liposomes; D018991:Myelin Proteolipid Protein; D011510:Proteolipids; C020485:proteoliposomes