RAPID SMELLING OF A CHO-K1 ASPARTATE GLUTAMATE TRANSPORT MUTANT IN HYPOOSMOTIC MEDIUM/

Two Chinese hamster ovary cell (CHO-K1) mutants selected for defective glutamate transport via system X(AG)(-) are also highly permeable to small neutral molecules. Light microscopy demonstrated that exposure o f one of these mutants, Ed-A1, to hypo-osmotic medium led to extremely rapid swelling, presumably due to increased water flux. When placed i n 20% saline, Ed-A1 cells swelled to three times their original volume within 15 sec, a sixfold larger increase than parental CHO-K1. In spi te of this rapid volume increase, mutant and wildtype cells remained v iable for 20 min in dilute saline. A regulatory volume decrease in Ed- A1, and the continual swelling of CHO-K1, resulted in the two cells ac hieving equal size after 5 min in 20% saline. The time course of these volume changes permitted analysis of large numbers of cells by a hydr odynamic technique, steric field flow fractionation (FFF). Steric FFF demonstrated the expected inhibition of osmotic swelling of human eryt hrocytes by the mercurial, p-choloromercuribenzenesulfonic acid (PCMBS ). However, PCMBS increased the apparent swelling rate of Ed-A1 and CH O-K1, suggesting that an aquaporin-like molecule is not responsible fo r any significant fraction of the water fluxes into either line. PCMBS also strongly inhibited aspartate transport by system X(AG)(-). By ta king advantage of their different swelling rates in hypotonic medium, steric FFF can separate mixtures of CHO-K1 and Ed-A1.