A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles.
In this paper, we describe a simple and highly sensitive manual assay for isotope fluxes through ion-conducting pathways, particularly cation-specific channels, in heterogenous populations of small membrane vesicles. We measure uptake of tracer of the ion of interest, against a large chemical gradient of the same ion. As a result of the imposed chemical gradient, a transient electrical diffusion potential is set up across the membranes of those vesicles which are highly permeable to the ion of interest. The isotope tends to equilibrate with the diffusion potential and is therefore concentrated selectively and transiently into those vesicle containing the channels. Furthermore, when performed in this way, the time course of tracer equilibration occurs over several minutes, rather than the sub-second range expected for tracer equilibration into channel-containing vesicles in the absence of an opposing chemical gradient of the permeant ion. The use of the procedure is demonstrated for three Na-conducting channels: gramicidin D incorporated into phospholipid vesicles, amiloride-blockable Na channels in toad bladder microsomes, and veratridine-activated tetrodotoxin-blockable Na channels in rat brain synaptic membranes. For all three cases, it proved simple to measure a specific 22Na uptake, in a minute time range, using very low concentrations of the channel-containing vesicles. By comparison with isotope flux measurements performed without an opposing Na gradient, the power of the present assay derives from both the very large gain in sensitivity and the convenient time course.
D000584:Amiloride / Q000494:pharmacology; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D001921:Brain / Q000378:metabolism*; D002024:Bufo marinus; D006096:Gramicidin; D007473:Ion Channels / Q000378:metabolism*; D007700:Kinetics; D008081:Liposomes; D008722:Methods; D008861:Microsomes / Q000378:metabolism*; D010713:Phosphatidylcholines; D051381:Rats; D012964:Sodium / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism*; D013574:Synaptosomes / Q000378:metabolism; D013779:Tetrodotoxin / Q000494:pharmacology; D001743:Urinary Bladder / Q000378:metabolism*
D007473:Ion Channels; D008081:Liposomes; D010713:Phosphatidylcholines; D006096:Gramicidin; D013779:Tetrodotoxin; D000584:Amiloride; D012964:Sodium