Measurement of sugar transport in single living cells

A fluorometric method that allows repeatable measurement of sugar transport rates and parameters in single living cells is presented. Intracellular su gar concentrations were estimated in real time from changes in cell volume that occur secondary to permeation of sugars across the plasma membrane. In turn, the cell volume changes were estimated from variations of intracellu lar calcein fluorescence measured by confocal microscopy. Using HeLa cells, the assay allowed reproducible measurement of the uptake and exit of D-gal actose and 3-O-methyl-D-glucose. The rate of zero-trans uptake (i.e. at an intracellular concentration of zero) of galactose at an extracellular conce ntration of 200 mM was 0.34+/-0.05 mM/s (n=8). Apparent V-max and K-m for g alactose exit were 0.32+/-0.05 mM/s (n=9) and 30+/-7.2 mM (n=9), respective ly. The apparent affinity of infinite-trans (i.e. at a very high intracellu lar concentration) uptake of 3-O-methyl-D-glucose was 3.8+/-0.47 mM (n=8). Galactose uptake was 93+/-8% (n=8) inhibited in the presence of 50 mu M phl oretin, whereas galactose exit was 96+/-6% (n=5) trans-inhibited by 100 mM 4,6-ethylidine-D-glucose. This technique may help to characterize sugar tra nsport in freshly isolated cells, co-cultures and heterogeneous cell explan ts. It may also allow available cell microinjection technology to be used t o study the regulation of sugar transporters' intrinsic activity. In princi ple, similar approaches might also be applied in functional studies of othe r transporters for which non-metabolized substrates are available.