Imaging plasma membrane proteins in large membrane patches of Xenopus oocytes.
We describe the preparation of a Xenopus oocyte plasma membrane patch attached to a cover-slip with its intracellular face exposed to the bath solution. The proteins attached to the plasma membrane were visualized by confocal microscopy after fluorescence labelling. Since cortical microfilament elements were detected in these plasma membrane preparations we termed the patches plasma membrane-cortex patches. The way these patches are formed and the low concentration of proteins needed for cytochemical detection make the membrane-cortex patches similar to electrophysiological membrane patches and therefore allow the cytochemical study of ion channels to be correlated with electrophysiological experiments. Furthermore, the described patch is similar to manually isolated plasma membranes used for biochemical analysis by sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Cytochemical analysis of membrane-cortex patches also enables the detection of the two-dimensional pattern of organization of membrane proteins (clustered or non-clustered forms). In addition, patch preparations enable cytochemical study of the relative localization of membrane proteins. The methodology enables integration of electrophysiological, biochemical and cytochemical studies of ion channels, giving a comprehensive perspective on ion channel function.
Singer-Lahat
D
D
Department of Physiology and Pharmacology, Sackler School of Medicine, Tel Aviv University, Ramat Aviv, Israel.
Dascal
N
N
Mittelman
L
L
Peleg
S
S
Lotan
I
I
D000199:Actins / Q000032:analysis; D000818:Animals; D000954:Antigens, Surface / Q000032:analysis; D015220:Calcium Channels / Q000032:analysis; D002462:Cell Membrane / Q000737:chemistry*; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure; D005260:Female; D016650:Fluorescein-5-isothiocyanate; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D008565:Membrane Proteins / Q000032:analysis*; D008856:Microscopy, Fluorescence; D009419:Nerve Tissue Proteins / Q000032:analysis; D009865:Oocytes / Q000648:ultrastructure*; D010590:Phalloidine; D015221:Potassium Channels / Q000032:analysis / Q000235:genetics; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger / Q000032:analysis; D050827:Syntaxin 1; D014982:Xenopus laevis
D000199:Actins; D000954:Antigens, Surface; D015220:Calcium Channels; D005456:Fluorescent Dyes; D051676:G Protein-Coupled Inwardly-Rectifying Potassium Channels; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D012333:RNA, Messenger; D050827:Syntaxin 1; D010590:Phalloidine; D015638:Cytochalasin D; D016650:Fluorescein-5-isothiocyanate