A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogenous populations of membrane vesicles.

摘要: 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 tracer the ion interest, against large chemical gradient same ion. As result imposed gradient, transient electrical diffusion potential is set up across membranes those vesicles which are permeable to interest. The tends equilibrate with therefore concentrated selectively transiently into vesicle containing channels. Furthermore, when performed way, time course equilibration occurs over several minutes, rather than sub-second range expected channel-containing absence an opposing permeant use procedure demonstrated three Na-conducting channels: gramicidin D incorporated phospholipid vesicles, amiloride-blockable Na channels toad bladder microsomes, veratridine-activated tetrodotoxin-blockable rat brain synaptic membranes. For all cases, it proved specific 22Na uptake, minute range, using very low concentrations By comparison flux measurements without power present derives from both gain sensitivity convenient course.