Abstract
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.
Highlights
Formed in this way, the time cofutrrsaecer equilibra- doplasmic reticulum, etc
Tion occurs over several minutes,rather than thesub- that bounded by the membranes containing the channels of second range expected for tracer equilibration into interest, can oftebne expected to be a relativelysmall fraction channel-containing vesicles in the absenocfean oppos- of the total vesicle volume
In this paper,we describe a simple, sensitive, and convenient flux assay for selective ionic channels in membrane vesicles and demonstrate its use to measure “Na fluxes through gramicidin incorporated intophospholipid vesicles, amiloride
Summary
Liposomes-80 mg of crude soybean lecithin were suspended in 2 ml of media containing 25 mM imidazole (pH 7), 5 mM EDTA, and 150mM NaCl. The bladders were excised and immersed in ice-cold homogenizing buffer containing 55 mM NaCl, 87.5 mM sucrose, 12.5 mM imidazole The preparationof microsomes is based on a procedure developed by Palmer and Edelman.' The epithelial cells were scraped from the supporting tissue with a glass microscope slide and washed twice in the above homogenization buffer by centrifugation a t 800 X g for 10 min. Thecombined fraction was centrifuged for 60 min at 27,000 X g, yielding a clear supernatant and pellet. This pellet, "the microsomal fraction," was suspended in the homogenizing buffer to a finalconcentration of 0.5-1.5 mg of protein/ ml and used for transport measurements within 4 h. The membranes were sonicated for 20 s in a bath sonicator prior to being used
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