Mechanism and role of furosemide-sensitive K+ transport in L cells: A genetic approach

Summary As part of a genetic study of the mechanisms for cation transport in cultured mammalian cells, two mouse fibroblastic cell lines have been compared with respect to unidirectional⁴²K⁺ influx. The cell lines areLM(TK^–) andLTK-5, a mutant selected fromLM(TK^–) by the ability to grow in medium containing 0.2mm K⁺. In both cell lines, the overall influx can be resolved into three components: (i) a ouabain- and vanadate-sensitive component (ⁱM_K^f), presumably the Na/K pump, which is a saturable function of extracellular K⁺ with aK_1/2 of 1.3mm; (ii) a furosemide-sensitive component (ⁱM_k^fx), also a saturable function of extracellular K⁺, with aK_1/2 of 6mm; and (iii) a diffusional component (ⁱM_k^d); which is a linear function of extracellular K⁺.By several independent criteria,ⁱM_k^o andⁱM_k^f appear to be distinct transport processes. First, as indicated above, they can be separated with the use of inhibitors. In addition, they can be separated genetically, since theLTK-5 mutant shows a threefold elevation inⁱM_k^f with no change inⁱM_k^o. And finally, extracellular Na⁺ has no effect onⁱM_k^o, but stimulatesⁱM_k^f, a result consistent with the notion thatⁱM_k^f influx occurs by Na–K cotransport.Further experiments were directed towards understanding the nature of theLTK-5 mutation and the physiological role ofⁱM_k^f. LTK-5 differs from the parental cell line, not only in having an increasedⁱM_k^f, but also in having a large cell volume, a slow maximal growth rate, and an ability to grow at 0.2mm K⁺. The most straightforward interpretation — that the increasedⁱM_k^f is itself responsible—is unlikely since the addition of furosemide to the growth medium had no effect upon the growth rate or cell volume of the mutant at either normal or low extracellular K⁺ concentrations. It did, however, render the parent capable of growth at 0.2mm K⁺. Possible interpretations are discussed.