Absorption filtration. A tool for the measurement of ion tracer flux in native membranes and reconstituted lipid vesicles.
A rapid, simple and reproducible method has been developed for the measurement of ion tracer flux with both native membrane vesicles and reconstituted lipid vesicle systems. Following the absorption of vesicles onto glass fiber filters, tracer flux is performed directly upon the deposited samples. In contrast to the more conventional vacuum and gel filtration techniques, absorption filtration exhibits comprehensive data retrieval whereby the removal of extravesicular ions, the retention of intravesicular ions and the amount of ions fluxed can be accurately analyzed. Both influx and efflux assays have been designed to measure the carbamylcholine-induced flux of 22Na+ which is characteristic of acetylcholine receptor-enriched membranes from Torpedo californica electroplax. The flux signal-to-background noise ratio is maximized in the efflux assay, since agonist activation is performed subsequent to the exhaustive removal of extravesicular tracer. An interesting feature of the influx assay is that the agonist-induced uptake of 22Na+ can be repeated with the original vesicles which additionally maximizes the flux signal. With either approach, the inactivation of ionophoric activity due to prolonged exposure to agonists ('desensitization') can be reversed upon removal of agonist without dilution of the deposited samples. Due to the large array of glass fiber filters and ion-exchange disks, the absorption filtration technique should be able to accommodate the transport and binding of soluble molecules to a variety of intact cells, membranes and reconstituted lipid vesicles.
D000042:Absorption; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism; D004557:Electric Organ / Q000378:metabolism; D007473:Ion Channels / Q000187:drug effects / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D011950:Receptors, Cholinergic / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*; D014101:Torpedo
D007473:Ion Channels; D008081:Liposomes; D011950:Receptors, Cholinergic; D002217:Carbachol; D012964:Sodium