A transient osmotic permeabilization method for the introduction of impermeant molecules into functional brain membrane vesicles.
Mouse brain membrane vesicles (microsacs) were transiently permeabilized by hypo-osmotic shock. This permeabilization method resulted in the encapsulation of both [14C]sucrose and exogenous alkaline phosphatase. The efficiency of this method was estimated by [14C]sucrose encapsulation experiments to be approximately 81%. External membrane binding experiments with the lectin [3H]concanavalin A demonstrate that the microsacs were not inverted by permeabilization. Following permeabilization, the functional integrity of a ligand-gated ion channel, the GABAA receptor complex, was investigated. Muscimol-stimulated 36Cl-uptake experiments show that this receptor retains its functional properties including blockade by the receptor antagonist bicuculline and potentiation by the allosteric modulators flunitrazepam and pentobarbital. The osmotic permeabilization technique described here provides several advantages over other permeabilization methods. These advantages include a high trapping efficiency, the encapsulation of not only small solutes but large membrane impermeant compounds such as enzymes and the functional preservation of at least one transmembrane protein. Furthermore, this method does not require specialized equipment and does not result in large, permanent holes in the plasma membrane.
Leidenheimer
N J
NJ
Denver Veterans Affairs Medical Center, CO.
Harris
R A
RA
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002214:Capsules; D002250:Carbon Radioisotopes; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D018118:Chloride Channels; D002712:Chlorides / Q000378:metabolism*; D002713:Chlorine; D003208:Concanavalin A / Q000378:metabolism*; D005445:Flunitrazepam / Q000494:pharmacology; D007038:Hypotonic Solutions; D008565:Membrane Proteins / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D008813:Mice, Inbred ICR; D009118:Muscimol / Q000494:pharmacology; D011724:Pyridazines / Q000378:metabolism; D011865:Radioisotope Dilution Technique; D011868:Radioisotopes; D011952:Receptors, Concanavalin A / Q000378:metabolism; D011963:Receptors, GABA-A / Q000187:drug effects / Q000502:physiology*; D013395:Sucrose / Q000378:metabolism*; D014316:Tritium
D002214:Capsules; D002250:Carbon Radioisotopes; D018118:Chloride Channels; D002712:Chlorides; D007038:Hypotonic Solutions; D008565:Membrane Proteins; D011724:Pyridazines; D011868:Radioisotopes; D011952:Receptors, Concanavalin A; D011963:Receptors, GABA-A; D014316:Tritium; D003208:Concanavalin A; D009118:Muscimol; D002713:Chlorine; D013395:Sucrose; D005445:Flunitrazepam; C049853:gabazine; D000469:Alkaline Phosphatase